JP2003500094A - Method and apparatus for multiphoton photoactivation and improved detection of molecular agents - Google Patents

Abstract

SUMMARY OF THE INVENTION A method and apparatus for diagnosing or imaging a specific volume of a substance comprises providing sufficient light to enhance multiphoton excitation of at least one photoactive agent contained on or within the specific volume of the substance. Processing a specific volume of the substance, wherein at least one photoactive agent is adapted to be activated within the specific volume of the substance to generate an optical signal, wherein such optical signal is Captured in electronic form for diagnostic and imaging of specific volumes.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION This invention relates to methods and methods for achieving selective photoactivation of one or more molecular agents with a high degree of spatial control and improving the detection of diagnostic signals generated thereby. It relates to the device. The method disclosed in the present invention for achieving selective photoactivation is characterized by a special non-linear optical excitation for the promotion of agents from one molecular energy state to another with high spatial and molecular properties. Take advantage of nature. The special features of the method of the invention are applicable for the diagnosis of a wide variety of endogenous and exogenous, or activation of imaging agents, which are especially unique in diagnosing human and animal diseases. Provide advantages.

In particular, the non-linear multiphoton excitation method of the present invention utilizes infrared radiation in proximity to infrared radiation that is absorbed and scattered to a lesser extent during radiation in the currently used method to provide deep tissue Or facilitates the controlled activation of diagnostic or imaging agents within other specimens. The improved signal incoding and processing methods and the combination of these non-linear excitation methods have greatly increased the sensitivity in detecting diagnostic signals. Moreover, certain aspects of the invention have direct applicability in speculum and other fields.

BACKGROUND OF THE INVENTION In many fields, especially in the fields of medical diagnostics and imaging, because of the way in which selective remote activation control of diverse molecular agents is possible while producing some ancillary effects. There is an urgent need for. Preferred improvements in activation include (1) enhanced spatial or temporal control over the location and depth of activation, (2) unfavorable activity of other co-located or adjacent molecular agents or structures. Including reduced activation, (3) selective increase of preferred molecular agents over activation of undesired molecular agents, and (4) improved sensitivity in detecting resulting diagnostic signals. A variety of linear and non-linear optical methods have been developed to provide some improvements over several agents under very specific conditions. However, in general, the performance and application of these methods has been objectionable. As a result, there is a need for improved photoactivation and detection methods that provide improved performance in such photoactivation applications and control of detection, as well as selectively probe a variety of molecular diagnostic agents. .

The application of optical radiation to probe or convert molecular agents has long been known. For example, linear single-photon optical excitation is widely used in photodynamic therapy (PDT) as a means for activating molecular therapeutic agents and for remotely interrogating diagnostic dyes and other materials. A generalized Jablonski diagram for such activation is shown in Figure 1a.

In FIG. 1, single photon excitation (10) refers to a higher quantum mechanical permissive state provided by the photoactive agent from the lower quantum mechanical permissive state Sm through the interaction of the agent with the single photon P _1. It occurs when excited by Sn. The reaction relaxation R ₁ is a transient excited state in the form of photochemical conversion or characterization of other energy or activated molecular agents useful for radial emission or detection of light.
May arise from Sn. Furthermore, a similar reaction relaxation process R ₂ can result from the second persistently activated state Tm, the latter giving rise to the subsequent intersystem crossing IX. Unfortunately, the performance of such excitation methods is not as successful as desired. For example, the typically used high-energy optical radiation P ₁ can often cause illness or other unwanted side effects such as UV optical scattering and absorption or visible active optical It may be less than the desired penetration depth due to radiation.

[0006]   In addition, various multiphoton optical excitation methods have different photoactivation selectivity for specific applications.
Achieves special improvements in processing many of the limitations contained by single-photon excitation.
Used in numerous laboratory applications as an effort to For example, the excitation of simultaneous two-photons
The origin is a means for stimulating fluorescent emission from molecules present in the optically dense medium.
Used. The generalized mechanism for such excitation is shown in Figure 1b.
, Simultaneous photon excitation (12) is a two photon P
₁′ And P_TwoSome energy provided by '' interactions coupled with its agent
Lugie E₁Occurs when excited by absorption of. If two photon P₁′ And P_Two'of
If the energies are the same, the process of excitation is called "degenerate". Also
If the energies differ, the process is called "non-degenerate".
It Coincident two-photon interactions often occur at about 10 femtoseconds (fs) or
It can be explained that it is adjusted by a temporary virtual state V having a shorter life.

If there is no interaction between the two photons and the agent during this lifetime, no excitation occurs and the agent does not reach state Sn. If the agent promotes to the quantum mechanically allowed higher state Sn, its photochemical and photomaterial properties will be the same as those that emerge from single photon excitation (10 in Figure 1a). Two-photon excitation has been described for use in microscopy as a vitro probe of cell membrane properties.

Very rarely, coincidental three-photon excitation is used in a laboratory setting to probe the spectroscopy of various molecules including ammonia, benzene, butadiene and nitric oxide, while co-occurring. 4-photon excitation of appears in applications that are more limited in the study of molecules such as NO ₂ and butadiene. The general theory of such multiphoton spectroscopy (number of photons ≦ 4) has already been explained (eg Andrews and Ghoul, J. Ch.
em. Phys. 75 (1981) 530-538)). Recently, Lakowicz and coworkers studied diverse fluorescence properties in the dense state and described the use of degenerate simultaneous three-photon excitation as a possible imaging tool for laser scanning microscopy.
(Gryczynski et al., Photochem, Photobiol. 62 (1995) 804-808; S
See zmacinski et al, Biophys, J. 70 (1996) 547-555).

Additional studies using multiphoton excitation have revealed physical and chemical properties of agents using complex multiphoton excitation methods, and one or more transient or spectroscopic tailored lasers. Efforts have been made to provide quantum control on the excitation-state reaction path using pulses. However, these reported multiphoton methods generally require stepwise and continuous applications of light energy for periods well above 10 fs to allow reorganization to occur inside the molecule. FIG. 1c shows a generalized representation of such multiphoton excitation.

In FIG. 2, the 3 + 2 photon excitation (14) is provided by the interaction of the three photons P ₁ ″, P ₂ ″ and P ₃ ″ that the photoactive agent initially co-occurs with the agent. Due to the absorption of the specific additional energy E ₁ generated, it is excited to the first high quantum mechanical permissive state Sn (this interaction is tuned by two virtual states V ₁ ″ and V ₂ ″). Is caused by the absorption of a particular additional energy E ₂ provided by the interaction of the agent with two additional photons P ₄ ″ and P ₅ ″ so as to promote to the second higher state Sp. In fact, there is a short temporary delay between these two stages E ₁ and E ₂ .

Surprisingly, multiphoton spectroscopic use and the widely used ultrashort pulse source (
That is, despite considerable theoretical and experimental work, having a laser resource capable of producing pulses of light having a pulse width, generally less than about 10 picoseconds, the inventors of the present invention have Ultrafast, a multiphoton method for the practical diagnostic imaging of human or animal tissue involving selective activation of endogenous (naturally occurring) or exogenous (externally supplied) molecular agents I have not seen any earlier reports than the present invention for pulse applications.

Denk et al. (W. Denk, JP Strickler and WWWebb, “Two-Photon Laser Microscopy”, US Pat. No. 5,034,613) have shown that in the laboratory various cells or sub-cellular scales are used. Exogenous agents have been disclosed for special epi-illumination confocal laser scanning microscope structures and uses that use non-linear laser excitation to achieve essentially higher three-dimensional control in photoactivation. The emitted fluorescent light is collected by an excitation objective lens that uses an epi-illumination structure.

This light is then used to generate images based on luminescence in cells and subcells for specimens arranged in slides or other shapes at the sample stage. In subsequent work, Denk et al. (W. Denk, DW Piston and WW Webb, "Two-photon molecular excitation with laser scanning microscopy", Biological Confocal Microscopy Handbook, Second Edition, JB Pawley, ed. , Plenum Press, New York, 19
95, pp. 445-458) discusses external global area detection methods useful for the collection of microscopic imaging data generated from so-called two-photon excited fluorescent tags. This method, which the authors describe as "untested", eliminates the requirement to collect backscattered fluorescent light using so-called epi-illumination. Denk said that such an approach would be useful if the microscope objective did not transfer the emitted fluorescence wavelength, but it was
It is vulnerable to contamination from ambient room light. ”In these two studies, the obvious methods used or foreseen for background interference removal from ambient light or scattered excitation light are Absent.

Indeed, the low efficiency of non-linear excitation can be varied with a very high proportion of excitation light scattered as diagnostically useful radiation. The use of various modulation methods for the removal of interference from scattered excitation light, as well as from interference from ambient light and other environmental and mechanical background sources, has been attempted by numerous studies. It was In the field of two-photon excited fluorescence, Lytle and coworkers (RG Freeman, DLGililand and FELytle,
"Second harmonic detection of sinusoidally modulated two-photon excited fluorescence", Analytical Chemistry, 62 (
1990) 2216-2219) and "Second harmonic detection of spatially filtered two-photon excited fluorescence", Analytical Chemistry, 65 (1993) 631-635), using the second harmonic detection method to scatter. We discuss a complicated method for the elimination of the laser excitation light that has been generated: the sinusoidal modulation of the excitation light is performed at one frequency and the detection of the excitation fluorescence of two photons is The interference from the scattered excitation light has been so-called virtually eliminated when performed twice (in harmonics of 2). And the elimination of mechanical and environmental interference is very important by proper selection of the modulation frequency so as to eliminate other noise frequencies, electronically.

Thus, nonlinear excitation can be used under laboratory conditions to excite a variety of luminescent molecular agents that use light at longer wavelengths, and the excitation achieved thereby is 3D spatial control over the position of the cell, reducing interference from absorption and scattering of excitation light in an optically dense medium, and excitation for live cell samples to be tested using spectroscopy. Secondary damage associated with the route can be reduced.

[0016] Thus, prior to our work, many studies of the use of ultrashort pulsed sources and multiphoton excitation to validate a variety of photoactivated materials in a laboratory setting have been made. While explaining interesting features, this study aims to achieve selective photoactivation of one or more agents with high spatial control and efficiency to meet the diverse needs of the medical field. Failed. For example, previous work illustrated by these cited examples illustrates many interesting features of various nonlinear excitation methods applicable to spectroscopy and imaging applications, but medical imaging and diagnostics It does not disclose a general method for achieving selective photoactivation of one or more agents with a high degree of spatial control to meet the diverse needs of the industry. Explicitly, no practical use or method emerges to enable such control to target agents or materials and physical scales that are significant for medical and other imaging and diagnostic applications. Not not. Therefore, it is an object of the present invention to overcome the deficiencies of previous studies.

SUMMARY OF THE INVENTION The present invention relates to methods and apparatus for photoactivation of molecular agents, particularly for improving the volume of material used for multiphoton excitation and the detection of the resulting diagnostic signal. The invention relates to a method and a device for photoactivation for producing.

More specifically, the present invention provides one or more intrinsic or extrinsic diagnostics to achieve improved spatial control over photoactivation of such agents through multiphoton excitation processes. Alternatively, use the unique physical features of the nonlinear optics excitation of the imaging agent. Such multiphoton excitation results from the interaction of one or more photons with one or more intrinsic or extrinsic agents, which are essentially co-occurring, but which have a duration of approximately 10 ps or less. Provided by a single ultrashort laser pulse having. In the preferred embodiment of the present invention, two or more photon energies and wavelengths are the same, and such an excitation process is called degeneracy. However, the invention is not limited to processors in which more than one photon energy and wavelength are the same.

The multiphoton excitation of the present invention allows for a large number of detectable endpoints, including those that result from one or more of the following events: one or more at high quantum mechanical permissive states. Electronic excitation of agents; Vibrational excitation of one or more agents at high quantum mechanically permissive state; Electronic vibrational excitation (combined vibrational and electronic excitation) of one or more agents at high quantum mechanically permissive state And photoionization of one or more agents. From such an excited state endpoint one or more photoactivated agents are engineered to enhance the desired diagnostic effect, such as luminescence or loss of luminescence.

In a preferred embodiment of the invention, the photoactivating step comprises modulating the light from the light source with a special form of modulation, thereby producing modulated light and on the target object, or Processing the target object with the resulting modulated light to enhance multiphoton excitation of one or more intrinsic and extrinsic diagnostics or imaging agents present therein. More preferably, the invention includes demodulating the resulting detected signal with a special form of modulation and producing a demodulated energy signal that is characteristic of the particular target object.

More preferably, the step of demodulating the energy signal detected by the special form of modulation demodulates the detected energy signal at a frequency that is more than twice the frequency of the special form of modulation, and thereby the special form of modulation. Detecting the harmonics of the modulation of. It is also desirable that the demodulated energy signal, which is characteristic of the special object, causes a change in the lifetime of at least one photoactivated agent present in the special object. However, the method of the present invention can be performed without such selective steps of modulation or demodulation.

The multiphoton excitation method presented here offers special advantages over previous methods, but is active including the reduction of interference from absorption and scattering processes due to the surrounding environment surrounding the excited agent. There is improved depth of activation, improved efficiency, and better control of location and specificity for excited agents.

(Detailed Description of Preferred Embodiments) Hereinafter, preferred embodiments will be described with reference to the accompanying drawings. The invention described herein is unique to the nonlinear optical excitation of one or more intrinsic or extrinsic imaging or diagnostic agents so as to achieve improved control over photoactivation of such agents through a multiphoton excitation process. Take advantage of different physical features. "Nonlinear optical excitation" is defined for the purposes of this patent specification as an excitation process that involves the interaction of one or more agents and two or more photons that are essentially co-incident. For the purposes of this patent specification, "essentially coincidental interaction" refers to one or more agents and photons provided by a single ultrashort laser pulse having a duration of about 10 ps or less. It is defined as the excitation process that occurs as a result of the interaction of.

Thus, “multi-photon photoactivation” is one of essentially co-occurring for the purposes of this patent specification to produce one or more photoactivated imaging or diagnostic agents. It is defined as the nonlinear optical excitation resulting from the interaction of the imaging or diagnostic agent above with two or more photons generated from a single ultrashort laser pulse. By this definition, the physical process in which multiphoton photoactivation occurs is multiphoton excitation of one or more agents. Finally, an "endogenous agent", for the purposes of this specification, is melanin, hemoglobin, carotene, NA.
With photoactivators pre-existing on the patient or other target, such as examples of various natural chromophoric agents such as DH and NADPH, and other photoactivators such as tattoo dyes. Define. In contrast, an "exogenous agent", like a variety of diagnostic dyes or other photoactivating agents administered to increase the efficiency of conversion of light energy in diagnostic or therapeutic signals, may It is defined as a photoactivator that does not already exist in another target.

The endpoints of multiphoton photoactivation according to the present invention may include one or more of the following detectable events: electronic excitation of one or more agents with high quantum mechanical acceptance; Vibrational excitation of one or more agents in high quantum mechanically permissive state; Electronic vibrational excitation (combined vibrational and electronic excitation) of one or more agents in high quantum mechanically permissive state; and one or more agent Photoionization. From such excited state endpoints, one or more photoactivated agents are engineered to enhance the desired diagnostic effect, such as luminescence or loss of such luminescence.

The multiphoton excitation according to the present invention has a wavelength of about 2 compared to the wavelength used for conventional single photon excitation.
More than double the wavelength. By focusing the beam of this optical excitation at or within the sample under test, one or more diagnostic agents can cause a new vertical resolution according to the optical path, while providing a focal area provided by the focused beam. Can be photoactivated in substantially restricted positions.

Since the photoactivation according to the invention takes place at long wavelengths, which are associated with an equivalent linear excitation process, the scattering and absorption of excitation energy is greatly reduced. This means that for thick sections of optically dense samples such as human tissue, multiphoton excitation is possible with greater depth than when using conventional linear excitation methods. Furthermore, the spatial information associated with the source of the emitted light is incoded and related to the focal point of the activating beam so that it can be directly detected or imaged from the diagnostic agent or agents without scatter. Unnecessary for emitted light.

Therefore, the resulting emitted energy signal is caused by the movement of the position of this focal point relative to the specimen, by the movement of one or both of the light source or its target, or otherwise by the alteration of the optical path. Can develop 2 or 3 dimensional images. Also, the elimination of scattered excitation light and other interference can be significantly improved by the use of a suitable demodulation method for the activating light modulating and detecting device.

The non-linear optical excitation of the present invention includes reduced exposure of excitation and damage associated with the excitation path, reduced exposure to harmful optical wavelengths and enhanced specificity from excitation of the agent for imaging or diagnostic and other applications. The agent has the additional advantage of being light activated. The approach of nonlinear optical excitation used in the present invention provides an excellent tool for the detection and imaging of many diseases.

The present invention was primarily intended for in vivo detection and imaging of tissue diseases and other features such as cancer in the human breast. However, the disclosed method and apparatus has numerous additional applications, and these methods and apparatus are applicable to and used in other fields such as laser scanning microscopy. It will be fully apparent that the present invention has been disclosed to achieve substantial improvements in carrying out the features of the disclosed method and apparatus.

The basic configuration of the multiphoton photoactivation method and apparatus for achieving such imaging or diagnostic results is assigned to the assignee of the present invention and is a US patent application with the present application and co-inventors. Nos. 08 / 741,370 and 09 / 096,832. U.S. Patent Application No. 08 / filed October 30, 1996
741,370, and 09 / 096,83, filed June 12, 1998
Issue 2 is hereby incorporated by reference in its entirety.

In summary, in an embodiment of the invention, the light source and details are described below, but the resulting light is directed to image in a tissue or matrix. The modulator and method are also included in the description. The imaged area can be moved in relation to the light beam or resource, or alternatively the light beam can be moved. A detector is provided and can be equipped with a suitable demodulator if modulation is used. A detailed description of the light used for photoactivation is mentioned in the discussion of the scientific principles used in the present invention.

Energy Level Model for Multiphoton Photoactivation The embodiment of the invention disclosed herein is to selectively and efficiently photoactivate one or more imaging or diagnostic agents with a high degree of spatial control. It is in the use of multiphoton processes. Such selective photoactivation is accomplished by means of utilizing the special feature of nonlinear optical excitation to enhance the agent from one molecular energy state to another. To fully understand the striking features of this process, a conceptual model of multiphoton excitation was initiated in this patent specification. This is easily accomplished through the use of energy level diagrams for the exemplary case. Figure 2 (a)-(d)
Shows a generally modified Yavwoniski energy level diagram for several linear and nonlinear optical excitation processes.

FIG. 2A shows that the agent has an initial quantum mechanical permissible state Si (generally a ground state).
Shows the single-photon excitation (20) when excited to the final quantum mechanically permissible state Sf by absorption of some energy E ₁ provided by the interaction between the agent and the single-photon P. Many other allowed final states are possible. Each permissible state can be further subdivided into an ensemble of discrete substates, such as various oscillatory states that overlap many over a particular electronic state. Therefore, each allowable state S _i and
S _f can constitute a complex band of permissive states that reflects the basic characteristics of the agent and its local environment. In the case of luminescent imaging agents, the increase in agent from S _i to S _f can include, for example, fluorescent or phosphorescent emission of light, which can initiate multiple radiative emission of light from the excited state. As an example, the dye molecule Coumarin-480 absorbs a single photon of light at 400 nm and emits a photon at 465 nm through fluorescent re-emission (which can also be described as a relaxation process). In single photon excitation (20), the excitation probability has a linear relationship with the radiant emissivity of the incident light, so single photon excitation (20) is referred to as the linear excitation process.

In FIG. 2 (b), co-occurring two-photon excitation (22) is the mutual activation of photo-activated agent from the initial quantum mechanical permissive state S _i with co-occurring two-photon P ₁ and P ₂ . Occurs when excited to the final quantum mechanically permissible state S _f by absorption of some energy E ₁ provided by the action. In all examples considered below, it is assumed that the energies and wavelengths of the two (or more) photons involved in the excitation are the same, and such an excitation process is called "degeneracy".
However, the present invention is not limited to the degeneration process, and is applicable even when the energy and wavelength of two (or more) photons are not the same. When the agent increases to the final quantum mechanically acceptable state S _f , its photochemical properties are identical to those resulting from single photon excitation (20).

It is often explained that the co-occurring two-photon interaction shown in FIG. 2 (b) has a lifetime of the unit of 10 fs or less and is regulated by the temporary virtual state V ₁ . If two photons do not interact with the agent during the lifetime of this virtual state, no excitation occurs and the agent returns to the permissible state Si. Due to the very short lifetime of the virtual state V ₁ , the instantaneous irradiation, or W m ^{-2 of the} incident excitation light, causes the second photon P ₁ to pass before the virtual energy state V ₁ undergoes reaction relaxation to S _i . _It must be high enough to calculate a significant efficiency in absorption of ₂ . Therefore, pulsed excitation resources with very high pick power are generally used to efficiently stimulate this process; such resources are excited during the short lifetime of virtual state V _1. It is desirable because it can provide a large number of photons to the agent.

An example of co-occurring two-photon excitation (22) is the increase in fluorescent emission of light from coumarin-480 to 465 nm by absorption of co-occurring two-photons at 800 nm. In this example, the excitation probability is related to the product of the instantaneous irradiation of the first photon P ₁ and the second photon P ₂ . It can be conceptualized in the form of photochemical reactions.

Agent _{ground state} +2 hν _{800 nm} → agent _{excited state} (1) Here, the agents in the ground state each have two photons hν ₈ simultaneously generated at 800 nm.
Promotion to excited state following absorption at _{00 nm} . The ratio R that describes the generation of excited states is R
= k [agent _{ground state} ] [hν _800nm ] ² , where k is the intrinsic constant of the agent, such as absorption cross section, and [agent _{ground state} ] and [hν _800nm ”are the ground state agent molecule and It is a symbolic representation of the concentration of excited photons, where the quadratic equation, which depends on the ratio representation for instantaneous illumination,
The simultaneous two-photon excitation (22) is called a nonlinear excitation process.

In FIG. 2 (c), the simultaneous three-photon excitation (24) indicates that the photoactivating agent has three photons P ₁ ′, P ₂ ′, P ₂ ′, P ₂ ′, which are simultaneously generated with the agent from the initial quantum mechanical permissive state Si. It occurs when excited to the final quantum mechanically permissive state S _f by absorption of some energy E ₁ provided by the interaction of P ₃ ′. The number of simultaneous three-photon interactions is 10 each.
It has a lifetime of fs and below and is regulated by the temporary virtual states V ₁ 'and V ₂ '. If all three photons do not interact with the agent during the lifetime of this virtual state, no excitation occurs and the agent returns to S _i . Virtual state
Due to the very short lifetimes of V ₁ 'and V ₂ ', the instantaneous irradiation, or the incident excitation light, causes a second photon to reach the second photon before the agent performs reaction relaxation to S _i . P ₂ '
And must be high enough to calculate a significant efficiency in the absorption of the third photon P ₃ '.

An example of co-occurring three-photon excitation (24) may include an increase in fluorescent emission of light at coumarin-480 to 465 nm due to absorption of co-occurring three photons at 1200 nm. In this example, the excitation probability is the first photon P ₁ ′, the second photon P ₂ ′, and the third photon P ₂ ′.
Related to the product of P ₃ 'momentary irradiation. It can be conceptualized in the form of photochemical reactions.

[0041]     Agent_{Ground state}+ 3hν_{800 nm}→ Agent_{Excited state}  (2)   Here, the agents in the ground state are three photons hν co-generated at 1200 nm each.
_{1200 nm}To the excited state following the absorption of. The ratio R is R = k [agent_base
Status] [hν_{1200 nm}]^ThreeGiven by, where [hν_{1200 nm}"Is the excitation light
It symbolizes the density of the child. Where the cubic equation that depends on the instantaneous irradiation
Due to this, the simultaneous two-photon excitation (24) is called a non-linear excitation process.

In FIG. 2 (d), simultaneous multiphoton excitation (26) is provided by the photoactivated agent from the interaction of two or more simultaneous photons with the agent from the initial quantum mechanical permissive state Si. Occurs when the final quantum mechanically permissive state S _f is excited by the absorption of a certain energy E ₁ . As the agent increases to the final quantum mechanically acceptable state S _f , its photochemical properties are identical to those resulting from single photon excitation (20). The interaction of two or more photons occurring at the same time is described as having unit lifetimes of 10 fs and less, respectively, coordinated by one or more multiple virtual states of equality. If the desired photon does not interact with the agent during these lifetimes, no excitation to S _f occurs and the agent returns to S _i . Due to the very short lifetime of one or more virtual states, the instantaneous irradiation of incident excitation light yields a significant efficiency in absorbing the desired photons before the agent performs reaction relaxation to S _i . Must be high enough to do.

An example of simultaneous multiphoton excitation (26) is a simultaneous n photon excitation at a wavelength (λ) of (400 · n) nm.
The increase in fluorescent emission of light from coumarin-480 to 465 nm due to absorption of photons can be mentioned (for example, λ = 800 nm when n = 2; λ = 1200 nm when n = 3; n ≧
2). In this example, the excitation probability is related to the product of the instantaneous irradiation of n photons. This can be conceptualized in the form of general photochemical reactions.

Agent _{ground state} + nhν → agent _{excited state} (3) Here, the agent in the ground state is promoted to the excited state following the simultaneous absorption of n photons. The ratio R is given by R = k [agent _{ground state} ] [hν] n, where [hν ”symbolizes the concentration of n excited photons, where it depends on the instantaneous irradiation. Due to this non-linearity, the simultaneous multi-photon excitation (26) is called a non-linear excitation process.

As mentioned above, a more general definition of multiphoton excitation is substantially the same as, for example, any interaction that occurs during a single ultrashort laser pulse having a duration of about 10 ps or more. It only requires that two or more photons that occur simultaneously interact with one or more agents. Under such restrictions, the interaction of light with one or more agents substantially limits the direct photoactivation effect on internal molecular processes such as electronic excitation or photoionization of the agent (and You have to get up fast (while localizing). Substantial molecular reorganization or activity or a vast time course should not occur during such excitations, and some transitional frames that are normally observable and affect one or more agents are: , Essentially single, should occur at the joint stage. Such a more general definition is utilized in the following discussion of multiphoton excitation and resulting multiphoton photoactivation of imaging or diagnostic agents.

In addition to the specific examples of photochemical processes and energy diagrams shown in connection with FIG. 2, many other possible transition and energy level conditions are related to their temporal and spatial correlations. This is possible due to a number of factors, including molecular systems, their environment and properties including specific energies of absorbed and excreted forms.

Nonlinear Relationships in Multiphoton Photoactivation For the previous example, the agent evolved into a final quantum mechanically permissive state S _f following absorption of a special amount of energy E ₁ , which is diagnostically useful. Its photochemical properties, including the luminescent emission of light, should be the same regardless of the scheme used for such enhancement. This property is determined by the intrinsic properties of the excited agent and its local environment. The special final energy state S _{f obtained} is determined mainly by the magnitude of the total energy E ₁ transferred to the molecule, and therefore the arrival at S _f will ultimately determine the properties of the agent. _{It is one} size. When the ultrashort pulsed excitation method is used, the mechanism that induces the agent to progress to the excited state is such that the excitation process itself does not directly impact the resulting properties of the excited agent or its environment. It is clear that it has no significant influence on the outcome.

The multiphoton excitability for a given agent is a function of the pump power (
It can be easily evaluated through examination of agent response (such as luminescence), which correlates directly with momentary exposure. By measurement of such agents respond in some excitation power, successful multiphoton excitation, it can be confirmed by illustrating the log _{1 0 (response)} against log _{10 (Power).} This must produce a straight line with slope n, where n represents the number of excited photons absorbed by the agent under special test conditions. This is easily demonstrated for the fluorescent probe agent Indo-1, but with strong irradiance with light between 300 and 400 nm, strong single photon excitation (
It emits fluorescence at 500 nm). This agent was illuminated using focused light at 810 nm and when the log ₁₀ (fluorescent signal) is plotted against log ₁₀ (average power), the two-photon excitation response (30) is shown in FIG. Is expressed as follows (slope m = 1.9
6, correlation coefficient R ² = 0.99994). For example, the focused light is a beam composed of a 76 MHz repetition rate train of approximately 200 fs pulses generated by a mode-locked titanium: sapphire laser. When similar tests are performed at 910 nm, Indo-1 is expressed as a function of the laser power of ^{(m = 2.97, R 2 =} 0.991) 3 -photon excitation response (32).

By comparison, the fluorescent agent Coumarin-540A, which exhibits only a two-photon response at this wavelength, exhibits slopes of 2.01 and 2.00 at 810 nm and 910 nm, respectively.
At these wavelengths, it was confirmed that if the laser is not pulsed, multiphoton excitation leads to the observed response (an unpulsed laser beam is sufficient to sustain the multiphoton process with the agent at this wavelength. (Because it does not provide instantaneous irradiation),
The multiphoton signal disappears completely.

Also, the excitation cross section for a multiphoton process generally decreases as the number of essential photons for a given transition increases. For example, the three-photon cross section for a particular transition in a given agent will typically be smaller than each two-photon cross section for the same transition. This is due, at least in part, to the reduced probability that all photons required for a particular multiphoton process will interact with the agent in a substantially simultaneous fashion. This is Indo-1 (Fig. 3) at a given average pump power.
It becomes clear by comparing the magnitudes of the two-photon ruling KI response (30) and the three-photon excitation response (32). However, since the slope of the three-photon process is steeper than that of the two-photon process, such a difference in the relative cross-sections is substantially due to the instantaneous irradiation of the excitation light, for example, for a given average power. Reduced pulse width or increased pulse energy for the beam (e.g., regenerated and amplified titanium: sapphire laser or chirp (c
hirp) -by using an amplified light source such as a pulse-amplified Nd: YAG laser).

Spatial Properties of Multiphoton Photoactivation Since multiphoton excitation is non-linear with instantaneous illumination, such processes are important and dramatic differences in spatial excitation properties associated with linear excitation processes. Represents For example, as shown in FIG. 4, when the laser beam is focused on the material, the beam intensity profile (40), as predicted by classical Gaussian optical theory,
It should be produced to vary as a function of distance through the sample reaching the maximum level in the center of the focus. For single photon processes, the linear relationship between beam intensity (or instantaneous irradiation) and excitation efficiency results in a single photon excitation efficiency profile (42) that follows the beam intensity profile (40). On the other hand, for multiphoton processes, beam intensity (or instantaneous irradiation) and excitation efficiency (for each two or three photon process I ² or
A non-linear relationship between (such as proportional to I ³ ) results in a much more sensitive excitation efficiency profile (44) than the beam intensity profile (40).

Therefore, such spatial localization of the excitation may be limited to a focal region where the extent of the excitation is substantially small when using multiphoton excitation. On the other hand, when using a linear excitation, the excitation occurs along the entire optical path, creating a local space that is substantially very small. Such spatial localization can be observed both in transparent media such as the eye and in optically dense media such as skin tissue. In this way, the spatially localized multiphoton excitation can be performed by using an agent existing under the surface of the tissue (50) or the tissue (52) as shown in FIGS. 5 (a) and 5 (b), respectively. Can be used to activate locally.

Localized and remotely photoactivated response simulations in an optically dense medium are shown in FIGS. 6 (a) and 6 (b). FIG. 6 (a) shows a photograph of a solid block of agarose gelatin (60) in which the dye molecule coumarin-480 is evenly distributed. Figure 6 (b) shows
It is a figure which shows the photograph of Fig.6 (a). Agarose gelatin (60) creates an optically dense medium to strongly scatter visible light. Coumarin-480 is 350-4450nm
It emits blue fluorescent light when excited by the single photon method at 1, or when excited by the two photon method at 700-900 nm.

In this example, the luminous flux (62) is focused on the agarose gelatin (60) from one side so that a focal point (64) of about 2 cm is formed in the gelatin. Especially visible light
A beam (62) of NIR light (at 400 nm) or (at 730 nm from a mode-locked titanium: sapphire laser) expands and produces a collimated beam EL of about 50 mm diameter using a beam expansion telescope. The laser produces a continuous train of light below 200 fs at a pulse repetition frequency of 76 MHz. Then the expansion beam
Focus on agaros gelatin (60) using a 0 mm focal length (fl), 50 mm diameter double-sided convex single body glass lens. Next, agaros gelatin (60)
The focus (64) of the 0-mm fl lens is positioned so that it is at a position 2 cm within the agarose gelatin (60). From this, from a perspective view looking down directly on the agaros gelatin (60) from the optical axis provided by the luminous flux (62), FIGS. 6 (a) and 6 (b) clearly show Coumarin-480. Represents that the fluorescence from E. coli is stimulated only at the focus of the NIR beam (66), while the fluorescence is emitted along the entire flight line for the beam (68). Furthermore, the majority of the NIR beam (70) continues through the entire thickness of the agaros gelatin (60) with only some attenuation, while the visible beam (72) is one of the thickness of the agaros gelatin (60). It completely disappears before passing / 3.

The features observed in FIGS. 6 (a) and 6 (b) are associated with shorter wavelength light (such as agarose gelatin (60) or human or animal tissue) in an optically dense medium, Dramatically represents the unique spatial localization achievable with multiphoton excitation by generally improved NIR light transmission. Due to the non-linear relationship between multiphoton excitation and instantaneous irradiation efficiency, agent simulations may be neglected at locations along the beam path before and after focus.

Therefore, outside the focal region, photoactivation is very slight or absent. Also, the NIR excitation light is only weakly absorbed or scattered by the gelatin, so that a sharp focus is maintained at a deep penetration depth within the block (actually, by the movement of the gelatin along the optical axis, A sharp focus is achievable through the entire 8-cm thickness of gelatin). Since the sharpness of the focus observed in FIGS. 6 (a) and 6 (b) is determined by the Gaussian optical properties, the length and area of the focus area can be determined by, for example, beam expansion and subsequent refocusing (and thereby refocusing). It is easily adjusted by changing the optical variables used to adjust the beam (such as adjusting the numerical range of the resulting focused beam).

As the number of photons used for excitation increases, it is shown in FIG. 4 and FIG.
) And 6 (b), the multiphoton excitation efficiency profile (44) is more dense (tight
ly) should be. Therefore, three-photon excitation provides a tighter spatial localization than is possible with two-photon excitation and the like. This means that the multi-photon order (the number of photons used for a particular excitation) can be optimized so that the spatial properties of the resulting focal region match those of the target. . For example, the thickness of the focal region is shown in FIG.
As shown in (a), the use of three-photon excitation may be denser in order to optimize the selective excitation of agents at the surface of the tissue (50).

Spectral Response Characteristics of Multiphoton Photoactivation Along with dramatic differences in spatial excitation characteristics, the selection rules governing excitation efficiency as a function of wavelength can differ significantly for single photon and multiphoton excitation. .. However, the properties of the particular excited state accessed will be the same regardless of the excitation mechanism used to promote the agent to that particular excited state.

FIG. 7 shows the relative absorption cross section as a function of excitation wavelength from ethanol to the weakly luminescent molecule Hp-IX when using conventional single photon excitation (74) and simultaneous two-photon excitation (76). Examples are provided to represent different comparisons.

Furthermore, the data for simultaneous two-photon excitation (76) shows that simultaneous two-photon excitation is twice the energy (or 1/2 of the wavelength) of each two-photon and equal to the energy for absorption of a single photon. It is shown using a wavelength scale divided into two (78) so as to reflect that A comparison of the relative end views for the single photon excitation (74) and the simultaneous two-photon excitation shown on the 1/2 wavelength scale (78) shows a significant difference as a function of wavelength. In particular, the sorret (Sorrett), which is clearly influential in single photon excitation (74)
The) band (80) is absent for simultaneous two-photon excitation (76) or (78) because it is quantum mechanically unacceptable.

Such differences are due to differences in the selection rules for particular molecular transitions that depend on the mechanism used for excitation, which are multi-photon selection rules and specific multi-photon properties. It is useful to optimize the excitation efficiency or selectivity on the basis of difference in designing the agent. In general, for centrosymmetric agents (including symmetric centers), even-photon-based excitations (eg, two-photon excitations) must promote the agent from an initial state to an excited electronic state with the same parity. . This is exactly the opposite selection rule for the excitation process using odd photons (single photon excitation, etc.), and the difference in the response to Hp-IX observed in FIG. explain.

On the other hand, agents with little or no symmetry generally have the same selection rule regardless of the number of photons used in their excitation. Thus, for centrosymmetric agents, it is generally beneficial to carefully determine the multiphoton spectral response as a function of wavelength to determine the optimal excitation wavelength, while for non-centrosymmetric agents, In general, single photon excitation spectra can be used to estimate the response of multiphoton spectra, generally as a function of wavelength.

Meaning of absorption and scattering properties of tissue in multiphoton photoactivation Although the cross-section for multiphoton excitation can be much lower than that observed for single photon excitation, the longer wavelength optical emission Due to the lower matrix absorption and optical scattering, the use of the multiphoton method under many conditions may also be compatible with conventional excitation methods. For example, FIG. 8 represents typical absorption and scattering characteristics for tissues covering the UV and IR spectral regions. Some conclusions become clear from FIG.

First, the use of longer wavelength light generally reduces the relative effects of scattering,
This improves the depth of optical penetration. Therefore, replacing the single photon method with the multiphoton method according to the embodiment of the present invention should reduce the scattering effect on the transmission of the activating light to the agent existing in the tissue. For example,
Adjusting the excitation using 2 photons at 700 nm or 3 photons at 1050 nm to activate the same UV-activated fluorophores as Indo-1 (normally activated using 350 nm light) It should result in a scattering reduction of about 100 to 2000 times for each of the 700 nm and 1050 nm light.

Second, interference from tissue absorption, which can reduce penetration of activating light, resulting in light-induced tissue damage, is generally associated with longer wavelength light, eg, about 70%.
It is reduced by the use of so-called tissue transmission window NIR light, which extends from 0 nm to 1300 nm. A generally negligible effect is observed in tissues irradiated with low energy NIR light even when the optical power of NIR light is several times greater than the optical power of UV or visible radiation. Therefore, replacing the multiphoton method of the present invention with a conventional single photon method will generally reduce the effect of tissue absorption on the transmission of activating light to agents residing in the tissue.

For example, a UV-activated PD such as Indo-1 which was normally activated using 350 nm light.
Two-photon excitation or 10 at 700 nm to activate T-fluor
Use with three-photon excitation at 50 nm is equivalent to 700 nm and 1 in normal human tissue.
It would result in an approximately 260 to 3500 fold reduction in absorption for each of the 050 nm lights. Therefore, the use of agents characterized as single photon excitation wavelengths that overlap with the spatial region of high tissue absorption overlap multiple times, although the use of multiphoton excitation methods makes them suitable for deep tissue applications. Since such a method allows delivery of activating light to deep tissue sites without interference from tissue absorption.

The tissue-reduced scattering and absorption allows the spatial local excitation to be efficiently realized by the multiphoton method, as shown in FIGS. 5 and 6, because the matrix interference is substantially reduced. . Furthermore, the reduced scattering and absorption by the tissue offers the advantage of additional safety against multiphoton excitation. For example, when UV light reaches human tissue, most of the optical energy is immediately absorbed and scattered in the outermost layers, such as the epidermis and skin. Such absorption can occur due to the excitation of certain molecules within the cells of this tissue, as well as the organization of genetic material within the cell nucleus. Absorption of high-energy light by cellular components can lead to a variety of additional photochemical changes within these cells, including the induction of genetic damage and cancer thereby unaltered. On the other hand, the NIR light used in the two-photon or three-photon method is not appreciably absorbed and scattered by the tissue, and the possibility of additional damage to cells should be substantially less.

The additional effects of absorption and scattering are considered in FIG. High energy UV depending on organization
Alternatively, the inherent high absorption and scattering of visible photons 90 should occur at a very low tissue penetration depth, while generally lower energy NIR photons 92 have a greater penetration depth. In addition, the scattered higher energy photons 94 can guide radiation from the diagnostic agent along the scattering path, causing the higher energy photons to penetrate the tissue so that the higher energy photons spread out radially from the excitation path. Is easier to generate.

However, because of the non-linearity that depends on multiphoton excitation, irradiation with lower energy photons can cause scattering of lower energy photons (generally such scattered photons are spatial and transient). The radiation cannot be guided due to the loss of significant coherence
) Should produce a more sharply provided excitation pattern without being significantly blurred by its presence. Therefore, irradiation and subsequent detection of subsurface features is difficult or impossible when using high energy photons 90 such as in the UV or visible spectral range, while irradiation and subsequent detection of subsurface features is NIR
Or it is easier when using lower energy photons 92, such as in the IR spectrum region.

It should also be noted that the light emitted from the diagnostic agent may be more highly absorbed and scattered by the tissue or other optically dense medium under test. However, in order to satisfactorily detect the emitted light, such a small portion of the light need only make its path to the detector. This large area in which the emitted light can be scattered requires that elaborate methods are required to differentiate the scattered light and the emitted light produced by agents excited from other optical or mechanical noise sources. Means Such important differences in absorption and penetration depth characteristics for higher and lower energy light are graphically represented in FIG.
When visible light 90, such as UV or light at 400 nm, for example, affects human tissue 96, most of the optical energy is immediately absorbed (98) and scattered (98) in the outermost layers 100, such as the epidermis and skin. (94).

Therefore, the depth of optical penetration is low and the potential for the induction of collateral light-induced damage is similar to that used for linear excitation of conventional diagnostic agents, UV.
Or higher than excitation with visible light 90. On the other hand, for example, NIR light at 800 nm 9
2 will result in empirically lower absorption and scattering by tissue 96. The overall penetration depth should be even greater and the extent of collateral damage to the cells will be substantially lower. Therefore, if long wavelength excitation light is used in a multiphoton excitation process to replace higher energy single photon excitation, it uses a relatively non-damaged wavelength with a large penetration depth. It will be possible to photoactivate specific diagnostic agents present in deep tissues.

Selection of Excitation Wavelengths for Multiphoton Photoactivation Previous discussions on the function of tissue properties that are useful in terms of the overall efficiency of multiphoton excitation suggests an equally important point: the order of multiphoton processes. (Ie the number of photons used for excitation) can be selected to simultaneously optimize the excitation wavelength of one or more desired photoactivating agents and the transmission properties of the tissue. In particular, in the photoactivation of the desired exogenous agent or agents, allowing excitation using light within the transmission region to the matrix (such as tissue) to activate one or more exogenous photoactivating agents. It is generally desirable to choose an efficient, special multiphoton process.

Similarly, in order to activate one or more endogenous photoactivating agents, it is common to select a particular multiphoton process that is efficient in photoactivating the desired endogenous agent or agents. Desirable for. When one or more endogenous agents consist of multiple components of the tissue to be tested, a suitable choice for multiphoton processes is
In general even sub-surface locations (since interference from direct and linear absorption of photoactivation can be minimized) should allow spatial local photoactivation of such agents. This is clearly illustrated in Figures 6 (a) and 6 (b). In this figure, the selective and efficient activation of the agent 66 is illustrated by the depth within the sample where the agent is evenly distributed. On the other hand, conventional activation methods produce activation of the agent (68) that exhibits insufficient efficiency at such depths by absorbing the activation light by the agent according to a spatial non-local optical path.

Thus, an example of selection of multiphoton process sequences for optimizing concomitant photoactivation efficiency and tissue transmission properties can include activation of Indo-1 in normal human tissue. In this example, the large two-photon cross-section of Indo-1 (shown in FIG. 3), which is related to the corresponding three-photon cross-section, shows a 260-fold increase in tissue absorption at 350 nm for single-photon excitation (shown in FIG. 8). Indo-1, which produces a reduction and uses two-photon excitation at 700 nm
Enables efficient photoactivation of. As in other examples, activation of Indo-1 in human tissues with melanosis can be additionally illustrated. In this example, the large absorption of light at 700 nm by melanin indicates that the use of longer wavelengths is more desirable to avoid matrix interference, and that such excitation of the Indo-1 three-photon at 1000 nm is 1000 nm. The absorption of light in tissue is about 15 for 700 nm.
And about 3500 for 350 nm (in absorption of tissue light at 1000 nm)
It means that the result is doubled to be optimal.

It should be noted that the generally most desirable wavelengths for multiphoton excitation are between 500 nm and 4000 nm as a result of matched tissue transmission properties within this band. In addition, in addition to spectroscopic tissue (such as tissue and agent absorption properties), the order of the particular multiphoton process is tailored to suit optimal spatial localization performance for the particular specimen or target to be tested. You can choose. For example, generally higher order multiphoton processes (≧ 3 photons) provide greater spatial localization than that observed with two-photon excitation. Therefore, for some specimens it may be desirable to use such a higher order excitation process for photoactivation of the agent, even though it may result in reduced detection sensitivity. .

Excitation Sources for Multiphoton Photoactivation A cross section for a particular multiphoton excitation process is generally for an equivalent single photon excitation process that produces the same activation state, such as a multiphoton process. Several times smaller than the ones. This is 2
Or due to the relatively low probability that three photons interact with the agent in a substantially simultaneous fashion. However, the present invention is even more desirable when used to utilize optical excitation sources capable of providing high instantaneous illumination, such as mode-locking lasers (including titanium: sapphire lasers and Nd: YAG lasers) and amplifications. -Type mode-locking lasers (including regeneratively amplified titanium: sapphire lasers and chirp-pulse-amplified Nd: YAG lasers) increase the incident instantaneous illumination and substantially improve such low-efficiency shocks. Yes, this can greatly increase the effective efficiency of multiphoton efficiency.

Such a typical laser has a suitable average power (ranging from 1 mW to 10 W).
) With high pulse repetition rate (range from 1KHz to 100MHz) pulsed output of ultra-short wave (
With a pulse width range from 10 fs to 10 ps). The attainable high instantaneous irradiation can stimulate multi-photon processes while short pulse widths and appropriate average powers surround the medium to minimize undesired photoactivation, so such output characteristics Facilitates selective and efficient multiphoton excitation. For example,
When using continuous wavelength excitation, the efficiency of three-photon excitation for a specified agent can be a factor less than 10 ⁷ compared to what can be achieved with single-photon excitation. However, if the same average optical power is emitted in the form of an ultrashort pulse train, the movement can change at a rate that is close to unity in instantaneous generation and average irradiation. The special properties of ultrashort pulsed excitation enable such a dramatic improvement without increasing by-products due to additional damage.

Excitation sources capable of emitting relatively low energy pulses (eg, having typical pulse energies in the range of 1-10 nJ, and mode-locking titanium: sapphire lasers, etc.) under focused illumination conditions It would be most suitable for diagnostic photoactivation or imaging agents using two or three photons, while
Resources capable of emitting relatively high energy pulses (eg, regeneratively amplified titanium: sapphire lasers with typical pulse energies in the 1-10 nJ range) are, for example, large areas of tissue or large volumes. It would be best suited for photoactivation of such agents using 2 or 3 photons under non-focused illumination conditions to activate the agent therein. Both of the two are intended to be included within the scope of the present invention. This description describes possible optical excitation sources and limits the present invention to using only the optical excitation sources described above so that other optical excitation sources may be used within the scope of the present invention. is not.

Detection of Multiphoton Excited Radiation from Diagnostic and Imaging Agents Spatial information about the origin of emitted light from multiphoton excited diagnostic imaging agents can be incoded by the excitation focus and associated with the excitation focus. .. This includes those based on the movement of photons and is in complete contrast to the single photon excitation imaging method, where the diagnostic imaging signal is from radiation generated by scattered excitation light and along the entire excitation path. Carefully deconvolved from the emitted synchrotron radiation. Therefore, it is unnecessary to directly detect or image the emitted light from a multi-photon excited diagnostic agent without scattering. Effectively, the collection and detection process is collected so as not to distort the correlation between the detected signal and the originating emission point, and requires only a portion of the detected emitted light.

More specifically, multiphoton imaging requires less light for detection than that required for excitation. Therefore, the loss of light from scattering in the radiation is acceptable. Moreover, only the emitted light selectively emits more easily, while emitting at precisely the location provided (excitation area). Preferably, the present invention uses agents that emit relatively long wavelengths. However, the invention is not limited to such agents.

To understand the importance of the relationship between signal detection and the origin of multiphoton stimulated emission, it is useful to consider that the excited light occurs immediately with the emission instant. During imaging on an optically dense specimen such as biological tissue, light from a multiphoton excited diagnostic imaging agent emits in an essentially isotropic manner. Some of this emitted light travels directly to the detector, which is located remotely from the point of emission, while other parts of the detector are at the detector as a result of one or more scattering events occurring between emission and detection. Travel through indirect routes. Unscattered or ballistic emitted photons if attempts are made to image at a depth of 10 cm on a biological specimen.
The transition time for (ie, the total transition time from the emission instant emanating from the surface of the specimen) is about 0.3 ns.

Therefore, in order to obtain maximum efficiency with this sample, it is desirable to integrate all emitted light for a sufficient period of time to capture the ballistic and heavily scattered photons. This is about 10 ns for imaging at a depth of 10 cm or less.
It means that the integration cycle of is appropriate.

When the image is generated by moving or scanning the position of the excitation focus relative to the specimen, previous analysis means that the excitation point must move more than once every 10 ns. In practice, practical limits for scanning processes and mechanisms combined with signal-to-noise discussions regarding the use of minimal dwell times and additional possible modulation methods are generally above 1 μm. Request scanning done using the number of pauses.

Therefore, for intensity of the imaging substrate with pause times exceeding the possible modulation frequencies of, for example, 1 μm and 100 MHz or less, the detector was placed so that it could focus a significant part of the ballistic and radiant scattering. If you are located as long as anything,
Try not to make much difference. The position of the detector relative to the origin of the point of emission depends on the ability to correlate with the signal detected as the origin, while the length of time is introduced by the optical delay, while the short transition times for other measurement variables. On the other hand, it has little or no effect. It is therefore clear that the detector can be located in such a manner that it can be in an epi-illumination arrangement with the excitation light or it can be located outside the excitation beam.

Epi-illumination configuration (or other possible co-linear excitation and detection configuration)
Minimizes potential jet lag for detection of surfaces or nearby surface objects, but such an arrangement is more sensitive to interference from elastically scattered and reflected excitation light. It should be noted that Staggered loss can be measured from the sample by using individually optimized multiplex detection assemblies for focusing radiation from other regions within the sample, or by minimizing staggered loss. By positioning the detection system far enough away, the matching registry (reg
It can be minimized for external detection arrangements by actively located detection systems, such as maintaining the istry). Therefore, if the fluorescent agent has no control over where it travels, as shown in FIG. 12, imaging must rely on knowing where the illuminated area is.

The discussion on the detection of light emitted from a multi-photon excitation diagnostic imaging agent focuses on points relating to intensity-based methods, where the image is the emission detected at the location of the excitation relative to the multiple excitation points by the sample. It can be configured by a correlation with intensity. However, strength-based methods are not always optimized because they are sensitive to many complex factors, which are:

Sample-as the abnormal optical density region located between the excitation source and the intended excitation point may change with unexpected differences within the effective excitation level at the intended excitation point Deviations in excitation light scattering and absorption due to heterogeneity. The artifacts caused by such a phenomenon can result in several excitation pathways that adversely affect other regions due to the heterogeneity following the subsequent deconvolution of the resulting multiple dataset. It can be improved by obtaining data along, but this is difficult or impossible for some samples.

Emission due to heterogeneity in the specimen-heterogeneity, such that the region of abnormal optical density located between the emission point and the detection system can be changed by an unexpected difference in the focusing efficiency for the emitted light from the excitation point Deviations in diffuse and absorption. The products caused by such phenomena acquire data along several focusing pathways that adversely affect other regions due to subsequent heterogeneity due to deconvolution associated with the resulting multiple datasets. Can be improved, but this is difficult or impossible for some specimens.

• Changes in the concentration or local environment of diagnostic imaging agents that are not directly related to shape or function-changes in the radiation level due to the specimen are intensity-based that may be associated with the structural or physiological tissue of the specimen. Assume imaging. However, if the imaging agent is not properly distributed over the sample, or if it adversely affects the radiation of the imaging agent such that other factors such as heterogeneity in the local environment within the sample may be associated with morphology or function, Obtaining meaningful data from a sample becomes even more difficult. The consequences of such phenomena can be improved by using or designing imaging agents that are not sensitive to such factors, which is difficult or impossible for some specimens.

The detection approach, which is less sensitive to the optical heterogeneity of the specimen, can serve as a basis for measuring changes in the excited state lifetime rather than to the radiant intensity. Excited state lifetime is an intrinsic property of the excited state of the molecular agent and its immediate environment (and, in particular, the mechanism or method used for photoactivation). Coincidentally, accurate measurements of lifetime are almost unaffected by the largest changes in excitation level and focusing efficiency. A convenient way to measure the excited state lifetime may be to use a phase excitation photometric method that correlates the phase shift between the emission signal with the modulated excitation source and the resulting lifetime. In particular, the above discussion on photon transition times means that the phase photometric method is applicable for imaging in optically dense media, especially for agents with lifetimes in excess of 1-10 ns.

Therefore, using a diagnostic imaging agent as having a radiative lifetime associated with a morphology or function within the sample allows for the implementation of change-based imaging rather than radiant intensity over lifetime. Such lifetime-based methods are equally applicable to laser scanning spectroscopy and remote imaging of magnified objects such as tumors in human patients.

Suitable focusing devices for intensity conversion or phase-based emission data include photomultiplier tubes, micro-channel plate devices, photodiodes, avalanche photodiodes), charge-coupled devices and arrays of charge-coupled devices, charge injection. Including but not limited to devices and charge injection device arrays and photographic films.

Noise Reduction for Recovery of Multiphoton Excited Radiation from Diagnostic Imaging Agents-
Modulation and Harmonic Detection: The inherent low efficiency of the multiphoton excitation process is scattered at a very high rate in multiphoton excitation radiation, and non-absorbing excitation light can be converted to multiphoton excitation radiation. In addition, the importance of other possible linear interferences due to the use of high pump power levels, along with the requirement to eliminate interference from ambient light and other optical or electronic noise sources, test, Raman scattering, and Modulation excitation methods involving agents or other types of single-photon excitation fluorescence present in the sample under other phenomena, coupled with appropriate demodulation of all detector signals, have improved interference with analytical signals. It represents that the best decision for recovery must be provided.

In effect, background interference reported as a disadvantage in US Pat. No. 5,034,613 to Denk et al. , Most can be evaded. Substantially, the use of such a method is useful in signal-to-noise ratio (S
(NR) performance can be dramatically improved. In general, modulation can improve detection performance for some hypothetical measurement in one or more of the following ways:

(1) Persistent background or noise source removal. For example, when using a microscope such as Denk's two-photon laser scanning microscope, the modulation of the excitation resource by demodulation with the detector signal while using a device such as a lock-in amplifier (LIA) or heterodyne demodulator It will limit the response of the detection system in a band of frequencies closely related to. By adjusting the phase sensitivity of the demodulation in this way, additional discrimination can be achieved for signals that are not linked to the modulation pattern or are closely matched to the modulation pattern. Therefore, by proper selection of the modulation frequency and demodulation phase, interference from room light or noise sources such as electronic noise at a particular frequency, for example near the electric motor, can be strongly eliminated. Such access is equally effective for remote imaging of magnified objects such as tumors in human patients.

(2) Removal of broadband or "pink noise" sources. This measurement environment, along with the electronics and other equipment used for a given measurement, may provide a wide band noise, sometimes referred to as pink noise, to a given measurement. The impact of such intrinsic noise can be greatly reduced by using a wide band limited detection method. In particular, for a given optical measurement, the observed signal voltage, V _{SI GNAL} , emanating from the detected optical emission of the _{photoactivation assay} is the detector input current i generated by photons interacting with the detector. _INPUT
The input impedance Z _INPUT is multiplied by the gain G of the detection system by the following equation: V _SIGNAL = i _INPUT · Z _INPUT · G (4)

On the other hand, the observed noise voltage V _NOISE can be approximated by the noise current i _NOISE , the input impedance, the square root of the electronic and optical bandwidth B of the detection system and the square of the gain by the following equation: V _NOISE = I _NOISE , Z _INPUT , B ^1/2 , G (5)

[0098] Thus, the signal-to-noise ratio (SNR) can be estimated from the ratio of the two voltages _{(V SIGNAL / V NOIS E)} . When a conventional optical detector such as a photomultiplier tube (PMT) is used for the detection of unmodulated fluorescent signal, this detector should produce a certain signal level with noise current. For example, it has an optical input at the level of 10 pW
A standard PMT such as the Hamamatsu R928 (7.4x105A / W radiation anode sensitivity) is 7.4.
Generate μAi _SIGNAL . If this signal current is converted to a voltage by a low noise amplifier with a gain of 100, an input impedance of 50Ω, an input noise level of 5nV / √Hz and a bandwidth of 1MHz, the following signal is generated: V _SIGNAL = 7.4μA ・ 50Ω ・ 100 = 37mV; (5a) V _NOISE = 5nV / √Hz ・ (10 ⁶ Hz) ^1/2・ 100 = 0.5mV (5b)

It should be noted that Ohm's law, or V = I · R, is replaced by the noise current and impedance shown in equation 5. Thus, for this wideband embodiment, SNR = 74. If this excitation energy is modulated to a sine wave at 1 MHz, for example with 100% depth modulation, the value V _SIGNAL = approx. Of about 55% loss (assumed to be introduced by other loss-based modulation methods). However, if the detection system uses a limited demodulation bandwidth at 1 MHz with a bandwidth of 1 kHz, the pink noise will decrease much faster than the signal; V _NOISE = 5 nV / √Hz · (10 ³ Hz) ^1/2・ 100 = 16μV (5c)

Then, the overall SNR increases to about 1200. Therefore, even though it resulted in some absolute signal level loss when using many forms of modulation,
Overall, compensating for this loss increases SNR. Furthermore, if there is any linear interference in the detector response from the ambient light leakage to the detector, for example, the wideband detection scheme will detect this with an additional noise source, while limiting as shown. The resulting modulation bandwidth will eliminate such interference. Assume that the peripheral leakage produces a 1 μA background signal for the PMT. For this non-modulated case, the optical imaging noise from this background B is identical to the square root of the total photons detected, SNR = S / (S + B) ^1/2 ; this is an estimated SNR of about 5.7. Produce. Such analyzes include laser scanning speculums and tumors in human patients,
The same applies to remote imaging of magnified objects.

(3) Removal of linear interference at the modulation frequency. The inherent low efficiency results of multiphoton excitation, scattering ratio, and nonabsorption excitation for multiphoton excitation radiation are generally very high. This includes linear interference at the modulation frequency that comes from elastic and inelastic scattering as well as from single photon excited radiation. Optical filtering is often used in an effort to spectrally distinguish multiphoton excitation from optical background phenomena. Unfortunately, such interference is very difficult or impossible to eliminate using spectral means alone. As an alternative to ignoring such residual interference sources, one approach for the recovery of pure multiphoton signals utilizes the recovery of the signal detected at several pump power levels relative to the pump power level, As a result, the nonlinear multiphoton excitation component can be mathematically extracted from the linear interference. It uses a predetermined total fluorescent response If the following _{model: I f = αI L + βI} N L (6) where, IL moment excitation intensity, alpha is proportional constants for various linear effects, beta is Proportional constant for multiphoton excited radiation, n is the number of photons used for photoactivation.

On the other hand, such a reversion-based method is suitable for experiments in which a small number of measurements are required per unit time, but in the case of multipoint scan optical imaging that can be used for cancer or tumor detection. As described above, it takes a lot of time each time the acquisition time of the total data is minimized, which is complicated and impractical. Much faster results can be obtained by using a temporary removal method such as second harmonic detection, which eliminates the need to make multiple measurements at some power levels. Freeman et al. (RGFr
eeman, DL Gilliland and FELytle, “Detection of a sinusoidally modulated, two-photon excited fluorescent second harmonic,” Analytical Chemistry, 62 (1990) 2216-2219).
A second harmonic detection method useful for the analysis and characterization of chemical samples in test tubes is discussed, where the sinusoidal modulation of the excitation resource produces a signal at twice the modulation frequency solely related to the two-photon excitation fluorescence. Used to do.

However, the point that Freeman used imaging does not appear. The lock-in amplifier mentioned for the modulation frequency is used to recover a pure two-photon signal at the second harmonic of the modulation frequency. On the other hand, the second harmonic fluorescence signal is only about 12% of the total two-photon fluorescence generated, and the improved rejection of linear interference results in an increase in overall SNR, resulting in a loss in absolute signal level. Will be more than compensated. Therefore, the second harmonic detection method is ideal for laser scanning spectroscopy and remote imaging of magnified objects such as tumors in human patients as a result of the elimination of scatter and the inherent efficiency at high data bandwidth potentials. Is applicable. Thus, the advantage is that in an imaging system using second harmonic detection, the minimum dewll time at each point and the use of maximum pump power for each measurement at each point This means that a two-photon excitation radiation signal can be easily obtained.

For the more general case of multiphoton excitation, a similar harmonic analysis can be used with the nth harmonic, where n is defined in Equation 6. A signal containing only the excitation response of n photons should appear at the nth harmonic. Such an approach for demodulation provides a general and powerful means to avoid linear interference for a variety of nonlinear excitation processes.

The advantages mentioned above for the use of the modulation method in multiphoton excitation diagnostic imaging apply equally as to whether the data are obtained based on the measurement of the emission intensity or the excited state lifetime. In fact, lifetime measurements are mostly easy and sensitive to measurements using phase photometric methods based on the determination of the phase shift between the modulating waveform and the detected signal. Therefore, it is clear that the modulation method has important utility in the efficient detection of multiphoton excitation phenomena, as it is not only from the scattering and other phenomena that occur in the specimen under test, but also from surrounding and instrumental. Provides removal of interference from noise sources. For optically dense media such as human tissue, the extremely high ratio of non-absorbed pump diffuse to multiphoton pump radiation makes the use of such methods important. Therefore, the use of modulation methods as described herein is beneficial for clinical imaging applications or for multiphoton laser scanning spectroscopy.

Contrast Agents-Intrinsic and Extrinsic Agents in Multiphoton Excitation Imaging: In the preceding discussion, multiphoton excitation was specific and penetration depth for optically reactive molecular agents present in optically dense media. , And shows that detection performance can be improved through the use of incoding and decoding methods for each excitation and detection process. The exceptional spatial localization of the excitation possible when using the multiphoton method can be used to significantly improve the contrast at the excitation point. When such local excitation is performed, the radiometric analysis light caused thereby can be detected using various detection means. Such an excitation point may move relative to the sample under test, for example by scanning the focal position relative to the sample or by scanning the sample position relative to the focus. Then, a two- or three-dimensional image of the sample can be produced by forming a correlation between the position of the excitation point and the emitted light produced thereby.

However, the useful contrast in such images also depends on the presence of differences in the molecular agent or concentration of agents or local environment causing the radiation. This agent is either intrinsic or extrinsic to the specimen, and imaging is ultimately based on contrast in local emission properties that can be correlated with structural or functional heterogeneity within the specimen. Therefore, the role of such contrast agents in non-linear diagnosis or imaging is also important.

A wide variety of endogenous plastid agents are particularly useful for the diagnosis or imaging of diseased tissue. Storage of aromatic amino acids, proteins, nucleic acids, cellular energy conversion due to structural and physiological differences between diseased and healthy tissue, or other areas of healthy or moderately healthy tissue. The concentration or local environment of natural plastid agents (such as adenosine triphosphate), enzymes, hormones, or other agents turns into a useful method for examining structural and functional heterogeneity. obtain. Therefore, such heterogeneous endogenous indicators can be interrogated non-intrusively using multiphoton excitation.

Unfortunately, in many cases the possible specificity of such agents is inadequate to achieve meaningful results and exogenous agents must be added to the sample. Traditional exogenous agents are semi-selectively divided into specific tissues, institutions or other structural units of the specimen following administration. The route of administration of this agent is generally by topical application or symmetrical administration. Under ideal conditions, such agents may be split, otherwise focused, or preferentially removed from the tissue of interest. Such concentration is probably the result of local isolation directly on top of the superficial structure or by direct differences in the physical or chemical properties of the structure leading to the partitioning of the agent into that structure. The contrast between high and low density areas can be used as a basis for investigating structural or physiological heterogeneity. Alternatively, the exogenous agent can penetrate into the specimen uniformly; if radiation properties such as plastid shift, quenching, or lifetime are sensitive to physiological specificity, such variables in contrast agents can Available as a basis for contrast in.

Since the radiative properties of the molecular agent are determined by the fundamental properties of the excited state and its environment, the mechanism leading to the promotion to the excited state of the agent has no significant impact on the radiated properties of the excited state. Therefore, molecular diagnostics or contrast agents that perform well under single photon excitation conditions can be expected to exhibit similar behavior under multiphoton excitation conditions. In general, certain contrast agents useful for single photon excitation can be used for multiphoton excitation and provide increased control over the excitation site for improved image resolution. Suitable contrast agents include many molecular agents used not only for photodynamic therapy (PDT) but also as biological dyes or stains. Standard PDT agents generally have tissue specificity based on the combined chemical-physical properties of agents and tissues such as cancerous diseases. Such agents are efficient absorbers of optical energy and are often luminescent. Examples of such agents include, but are not limited to:

Diverse Psoralen Derivatives; Diverse Porphyrin and Hematoporphyrin Derivatives; Diverse Chlorine Derivatives; Diverse Phthalocyanine Derivatives; Diverse Rhodamine Derivatives; Diverse Coumarin Derivatives; Diverse Benzophenoxazine Derivatives; Chlorpromazine and Its Derivatives; Chlorophyll and bacteriochlorophyll derivatives; Pheophorbide [pheo a]; Merocyanine 540 [MC540]; Vitamin D; 5-Aminolevallinic acid [ALA]; Photosan; Pheophorbide-
a [Ph-a]; Phenoxazine Nile blue-derivatives (including a wide variety of phenoxazine dyes); A wide variety of charge transfer and radiation transfer agents;

Such agents are generally semi-selectively accumulated at or near the point of application or within a particular tissue, due to differences in the tissue's physical or chemical properties that lead the tissue to compartmentalize with the PDT agent. It Such a storage agent is
Sensitive to multiphoton excitation, its emissive or other emissive properties can be used for diagnostic or image data acquisition. One that can absorb light and transmit energy continuously to one or more other agents, either alone or by accepting the energy thus transmitted and transformed into radiant radiation. It can be used in connection with the above response agents.

Biological Contrast Agents for Multiphoton Excitation Imaging Under ideal conditions, standard contrast agents gain target specificity based on their chemical or physical affinity for specific tissues. Thus, the contrast agent should be split or concentrated on or in the tissue of interest. Unfortunately, such target specificity is not complete. Consequently, improved methods of increasing specificity are desirable in targeting agent applications. In particular, one embodiment of the invention that achieves such improvement is based on the use of a particular biological symbol of structure, function, or disease. Binding of antisense oligonucleotide agents to one or more light-activating moieties, such as FITC, allows new bioimaging agents to contain specific genetic cells such as cancer cells containing complementary genetic incoding. Are created to be selectively displayable.

Furthermore, the basic approach is easily extended to a number of genetically-based diseases, as well as other diseases, by altering the oligomeric code used for bioprobes. The use of multiphoton excitation can be applied by using such strong access to the bio-specificity of bioprobing coupled with high spatial localization inherent to the multiphoton photoactivation process.
Thus, very high contrast, very high resolution imaging is possible at the genetic level using a specific targeted agent for a particular organ, tissue or injury.

The optimal design for biopsy uses one or more photoactive moieties with radiative properties that vary with the complexation between the bioagent and the location of the target. In particular,
Changes in emission wavelength or lifetime due to complexation increase the sensitivity of general methods because such changes help to increase the contrast between regions containing complex agents and regions containing non-complex agents. Can be used for An example is a bioagent based on a photoactivatable moiety that is suppressed until complexation occurs by making the radiation generation uncontrollable. Another example is an agent that is based on an intervening photoactivating moiety, such as psoralen bound to an antisense genetic sequence; by complexing between the antisense sequence and its target sequence. , It becomes possible to derive a shift of dyeability in the radiation characteristics of the interlayer photoactivated portion of the photoactivated portion.

Other organism-specific modal targeting methods, such as those immunological rather than genetic only, are apparent from previous discussions that are within the scope of the present invention. More specifically, the specificity of antigen-antibody method-based agents offers powerful new modalities for the diagnosis of diseases and infections, while antibody studies are linked to photoactivation groups. Additional ways to achieve biospecificity with agent targets are:
Including aldehydes, ketones, alcohols, esters, amides, amines, nitriles, azides, or those containing other hydrophilic or hydrophobic moieties, including DNA, RNA, amino acids, proteins, antibodies, ligands, haptens, carbohydrate receptors Or including, but not limited to, complex compounds, protein receptors or complex compounds, lipid receptors or complex compounds, chelates, encapsulated solutions, nanoparticles, short- or long-chain fatty or aromatic hydrocarbons. Not a thing.

Detailed Description of the Preferred Embodiments One of the preferred embodiments of the present invention is intended to induce multiphoton photoactivation of one or more endogenous or exogenous photoactivating agents. , A mode-locking titanium-sapphire laser or a regeneratively amplified titanium-sapphire laser, utilizing a high instantaneous illumination power that is an ultrashort pulse source.
Including this, another preferred embodiment is shown in FIGS. 10-12.

First Embodiment of the Invention: One particular preferred embodiment of the present invention uses light at a wavelength about twice or more than that required for conventional single photon photoactivation. The output of the NIR source is used to induce intrinsic or extrinsic diagnostics present in the specimen, or to induce multiphoton photoactivation of the imaging agent. Such a preferred embodiment is shown in FIG. The NIR source 108 is a rapid series of high pick powers of NIR radiation.
Produces a NIR radiation beam 110 composed of pulses, for example with a duration <20
It can consist of a commercially available standard mode-locking titanium: sapphire laser capable of outputting pulse energies up to about 20 nJ at 0 fs mode-locking pulses and pulse repetition frequencies in excess of about 10 MHz. Such sources are relatively low, but quasi-continuous beams of light of average power with a continuously tunable high pick power (for orders of 100 kW) over the 690-1080 nm to NIR wavelength band. (Up to a few Watts). The pulse train emitted by the NIR source 108 consists of a beam of NIR radiation 110 that is easily focused using standard optical means such as reflective or refractive optics 112.

The focused NIR beam 114 can be sent for imaging onto the specimen 116. Multi-photon photoactivation (eg, two-photon, three-photon) of a diagnostic or imaging agent will be substantially confined to the focal area 118 of the focused beam 114 due to the high instantaneous levels present only at the focal point. Let's do it. The excitation light 120 scattered by the specimen 116 may not have an instantaneous illumination level sufficient for significant photoactivation of certain diagnostic or imaging agents that may be within the outer region of the focal region 118. The light 122 emitted by the diagnostic or imaging agent molecules present in the focal area 118 is in a substantially isotropic manner.
8 (light isotropically emitted during fluorescence or phosphorescence).

A portion of the emitted light 124 is captured by a detection device 126, such as a photomultiplier tube, mounted inside or outside the specimen 116. Modulated radiation 1
A portion of 46 is captured by a detection device 126, such as a photomultiplier tube mounted inside or outside the specimen 116. This detection device 126 is mounted on a wavelength selection device 128, such as an optical bandpass filter, which provides for processing the trapped portion of the emitted light 124, which selection device 128 is primarily emitted as radiation from the diagnostic agent. Removes a major portion of the elastically scattered light while passing a major portion of the light at one or more wavelengths corresponding to having the characteristics of

Therefore, the signal 130 generated by the above-mentioned detection device 126 is transmitted to the processor 1
Captured by 32, the first purpose is to record the radiation response from the diagnostic or imaging agent as a function of the location of the focal area 118. Since the location of the focal area 118 is scanned over the volume of the specimen 116, a complete image of the specimen 116 can be obtained by examining the contents of the processor 132 as a function of the location of the focal area 118. This image can be used to identify regions of interest 134, such as subcutaneous tumors or other diseased regions.

Second Embodiment of the Invention: As an alternative to the preferred embodiment described above, a modulator can be combined with the general embodiment shown in FIG. Such modulators improve the removal of environmental or instrumental noise sources, allow the recovery of pure multiphoton excitation radiation, or facilitate the measurement of emitted light using phase photometric proximity. In addition, it can be used to improve the overall performance of the image system.

In particular, FIG. 11 shows modulator driver 1 providing drive signal 140 to modulator 136.
NI that can be used to incode with modulation patterns registered at 38 outputs
Shown is a modulator 126, such as an electro-optical or acousto-optical modulator, chopper, or other device, positioned to interact with the NIR beam 110 emitted by the R source 108. The modulated beam 142 of NIR radiation produced thereby is directed onto the sample 116, as described above in connection with FIG. The multiphoton excitation radiation 144 produced thereby exits the focal region 118 in an essentially isotropic manner.

However, in contrast to the similar emitted light 122 described above in connection with FIG. 10, this emitted light 144 is sequentially synchronized with the drive signal 140 generated by the modulator driver 138 while modulating beam 142 of NIR radiation. Shows modulation that is essentially synchronous with the modulation of.
The detection device 126 is installed in a wavelength selection device 128, such as an optical bandpass filter that provides for processing the trapped portion of the modulated emission light 146, which selection device 128 emits primarily from diagnostic agents. Removes a major portion of the elastically scattered light while the major portion of the light passes through at one or more wavelengths corresponding to having

Therefore, the modulated signal 148 resulting from the detection device 126 described above is captured by the processor device 150. The processor 150 serves two main purposes, the first is to demodulate the modulated signal 148 generated by the detector 126 using the demodulation reference output 152 generated by the modulator driver 138, and the second is Recording the radiated response demodulated from the diagnostic or imaging agent as a function of the location of the focal area 118. Therefore, focal area 11
8 positions are scanned over the volume of the specimen 116, so that the specimen 116
A complete image of can be obtained by examining the contents of processor 150 as a function of the location of focal area 118. This image can be used to identify regions of interest 134, such as subcutaneous tumors or other diseased regions.

Also, the pulse frequency of the NIR source 108 is such that the NIR radiation 14
It can be used as the modulation source itself while producing two modulated beams. In this alternative embodiment, source 108 provides the roles of modulator 136 and modulator driver 138 to generate the source of modulation reference output 152 for processor 150. As a result, no separate modulator or driver is needed.

Third Embodiment of the Invention As a second alternative to the preferred embodiment, a non-focused NIR radiation beam is used to illuminate the apparent specimen features so as to provide a direct image detection mode. be able to. This is shown in FIG. In particular, the output of an NIR source, such as a mode-locking titanium: sapphire laser, uses light at about twice or more the wavelength required for conventional single-photon photoactivation, on or near the surface of the specimen. It can be used to induce multi-photon photoactivation of an endogenous or extrinsic diagnostic or imaging agent.

The NIR source 108 produces a beam of NIR radiation 110 consisting of a series of high pick power pulses of fast NIR radiation. This beam is modulated using a modulator 136 positioned to interact with the NIR radiation beam 110 emitted by the NIR source 108. Modulator 136 incodes NIR radiation beam 110 with a modulation pattern registered at the output ole of modulator driver 138 which provides drive signal 140 to modulator 136. The modulated beam 142 of NIR radiation produced thereby is divergent to the divergent excitation beam 1 to form an image on the specimen 116.
Focused using standard scientific equipment, such as reflective or refractive optics 154, to produce 56. The diagnostic or image agent multiphoton photoactivation present on or near the specimen 116 is substantially synchronized with the modulation of the modulated beam 142 of NIR radiation, in turn synchronized with the driver signal 140 generated by the modulator driver 138. A modulated multiphoton excitation radiation 144 having a modulation that

A portion of the modulated radiation beam 146 is captured by an image detection device 158, such as a charge coupled device array located outside the specimen 116. The image detection device 158 is installed on a wavelength selection device 128, such as an optical bandpass filter that provides to process the captured portion of the modulated radiation 146, which selection device 128 is primarily used by diagnostic agents. Eliminates a major portion of the elastically scattered light while passing a major portion of the light at one or more wavelengths, which corresponds to having a characteristic as radiation. Therefore, the modulated signal 160 generated by the image detection device 158 described above is captured by the processor 162. Processor 162
Provides two primary purposes, the first is to demodulate the modulated signal 160 generated by the image detector 158 using the demodulation reference output 152 generated by the modulator driver 138, and the second is Recording the radiation response demodulated from a diagnostic or imaging agent as a function of the location of the radiation. Thus, this alternative embodiment allows direct videographic imaging of surface features 164, such as skin cancer lesions, to be based on spatial differences in multiphoton excited radiation across the illuminated surface of specimen 116. It can be so.

Also, the pulse frequency of the NIR source 108 is such that the NIR radiation 14
It can be used as the modulation source itself while producing two modulated beams. In another embodiment, source 108 provides the role of modulator 136 and modulator driver 138 to generate the source of modulation reference output 152 for processor 150. As a result, no separate modulator or driver is needed. Whereas the previous disclosure is focused primarily on the application of an embodiment using multi-photon excitation of an agent with ultrashort pulsed NIR optical radiation produced by a mode-locking titanium: sapphire laser, the present invention is It is clear, that it is not limited to such a narrowly defined optical source and such excitation. In effect, an aspect of the invention is that when optical excitation is achieved using linear or other non-linear methods,
Applicable. For example, continuous wave and pulse lamps, diode light sources, semiconductor lasers; argon ion lasers; krypton ion lasers; helium-neon lasers; helium-cadmium lasers; ruby lasers; Nd: YAG, Nd: Y
LF, Nd: YAP, Nd: YV04, Nd: glass, and Nd: CrGsGG laser; Cr: LiSF laser; Er: YAG laser: F-center laser; Ho: YAF and Ho: YLF laser; Copper vapor laser; Nitrogen laser Optical variable oscillators, amplifiers and generators; regeneratively amplified lasers; chirp-pulse width lasers; and other forms of gas, dye and solid state continuous, pulsed, or mode-locking lasers including; A variety of other optical sources can be applied, either alone or in combination.

Such a source may be continuous or pulsed, for example with a pulse repetition frequency in the range of about 1 kz or less to 10 GHz or more, and energy in the range of about 10 picojoules or less to about 50 millijoules. A focused light beam can be formed and is thus particularly suitable for use in the present invention.

Also, while the previous disclosures have focused on diagnostic and imaging applications for in vivo detection or disease characterization in plant or animal tissues, the present invention provides response target agent agents. It is clear that it has additional utility whenever selective activation is desired. Applications of certain aspects of the invention, especially in other areas such as biological microscopy and other analytical methods and devices, are within the scope of the invention. As an example, the use of modulation and demodulation methods as described herein has direct application in various forms of speculum such as single photon laser scanning speculum, two photon laser scanning speculum and multiphoton laser scanning speculum. Has applicability. It should be understood that each, or more than one, of the aforementioned elements may also find structures or useful applications that differ from the forms referred to above.

While the present invention has been illustrated and described as being modified in a general way for improved selectivity in photoactivation of molecular diagnostic or imaging agents, the present invention has been illustrated. Various omissions, modifications, substitutions and changes in the method forms and detailed descriptions and operations thereof can be naturally manufactured by those skilled in the art without departing from the spirit of the present invention, and thus are limited to only those described in the detailed description. And that was not what I intended. For example, in the third embodiment, the details of the modulation and demodulation may be omitted to produce a simpler imaging device, although the modulation of this embodiment results in an overall reduction in the performance of the imaging. it can.

This detailed description is provided for purposes of illustration only and is not intended to limit the invention of application as defined in the following claims. Without further analysis, the above examples fully illustrate the point of the invention, others apply the current knowledge, and in view of the prior art, reveal the unique and specific essential features of the invention. It is possible to easily apply it to various applications without omitting properly configured features. What is new and desired to be protected by a patent is set forth in the following claims.

[Brief description of drawings]

1 is an exemplary diagram of energy levels for conventional linear and nonlinear optical excitation processes.

FIG. 2 is a diagram of generally modified Yavwoniski energy levels for several linear and nonlinear optical excitation processes.

FIG. 3 is a comparison of the two-photon and three-photon excitation responses for Indo-1 as a function of pump power.

[Figure 4]   FIG. 5 is a comparison diagram of spatial excitation properties for linear and nonlinear excitation processes.

FIG. 5 is an illustration of spatially localized multiphoton excitation utilized for localized activation of an agent residing at or below the surface of a tissue.

FIG. 6 is a comparison diagram of single-photon and two-photon excitation fluorescence of dye molecule coumarin-480 uniformly distributed in blocks of agarose gelatin.

FIG. 7 compares absorption cross sections as a function of excitation wavelength for Hp-IX when using single photon excitation and simultaneous two-photon excitation.

FIG. 8 is an exemplary diagram of absorption and scattering spectra for human tissue that covers ultraviolet rays in the infrared spectrum region.

FIG. 9 shows general trends in the optical absorption and scattering properties of tissue for incident short and long wavelength light.

FIG. 10 is a diagrammatic view of a preferred embodiment of the present invention for imaging an intrinsic or extrinsic diagnostic imaging agent.

FIG. 11 is a diagrammatic view of another preferred embodiment of the present invention for imaging intrinsic or extrinsic diagnostic imaging agents, where modulation is used to improve imaging performance.

FIG. 12 is a diagrammatic view of a second preferred embodiment of the present invention for superficial feature videographic imaging.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C N, CR, CU, CZ, DE, DK, DM, EE, ES , FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, K R, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG , UZ, VN, YU, ZA, ZW (72) Inventor H Craig Dees             37923, Tennessee, United States             Cusbile Windham Way 1006             Apartment 1517 (72) Inventor Eric Wakuta             37830 Oh, United States             Cleridge Bay Pass Drive 138 F-term (reference) 2G043 AA03 BA16 CA05 EA01 FA01                       FA06 GA02 GA08 GB01 GB21                       HA01 JA03 KA01 KA02 KA03                       NA04 NA06

Claims (78)

[Claims] 1. A method for diagnosing or imaging a specific volume of a substance comprising at least one photoactive molecular agent, comprising: (a) enhancing multiphoton excitation of a photoactive molecular agent contained in the specific volume of said substance. Treating the specific volume of the substance with sufficient light to cause (b) at least one photoactive molecule by photoactivating the at least one photoactive molecule agent within the specific volume of the substance. Generating an agent, the at least one photoactivated molecular agent emitting energy, (c) detecting the energy emitted by the at least one photoactivated molecular agent, and (d) the substance. Generating a sensed energy signal characterizing a particular volume of the. 2. The method of claim 1, wherein the light sufficient to enhance multiphoton excitation of the at least one photoactive agent is laser light. 3. The method of claim 2, wherein the laser light comprises one or more ultrashort pulse trains. 4. The method of claim 3, wherein each of the one or more pulses has a duration of at most about 10 ps. 5. The method of claim 1, wherein the light sufficient to enhance multiphoton excitation of the photoactivated molecular agent is a focused beam. 6. The method according to claim 5, wherein the focused light flux is a focused laser light. 7. The method of claim 1, wherein the at least one photoactive molecular agent is an endogenous agent. 8. The method further comprises a first step of treating a substance having at least one photoactive molecular agent, wherein the particular volume of the substance comprises at least a portion of the at least one photoactive molecular agent. The method of claim 1, wherein the method is retraining. 9. The at least one photoactive molecular agent comprises psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 4, 5 '.
, 8-trimethylpsoralen (TMP), 4'-aminomethyl-4,5 ', 8-trimethylpsoralen (AMT), 5-chloromethyl-8-methoxypsoralen (HMT), anzellysin (isopsoralen), 5-methylan Zerricin (5-MIP), 3-carboxypsoralen, porphyrin, hematoporphyrin derivative (HPD), potophrin II, benzoporphyrin derivative (BPD), protoporphyrin IX (PIX), dye hematoporphyrin ether (DHE), polyhemato Porphyrin ester (PHE), Protoporphyrin (PH1008) 13,17-N, N, N-dimethylethylethanolamine
Ester, tetra (3-hydroxyphenyl) -porphyrin (3-THPP), tetraphenylporphyrin monosulfonate (TPPSI), tetraphenylporphyrin disulfonate (TPPS2a), dihematoporphyrin ether, mesotetraphenylporphyrin, mesotetra ( 4N-methylpyridyl) porphyrin (T4MpyP),
Octa - (4-tert-butylphenyl) tetra pyrazinoporphyrazine (OPTP), phthalocyanine, tetra - (4-tert-butyl) phthalocyanine (t ₄ -PcH _2), tetra - (4-tert-butyl) phthalate Russia Natomagnesium (t ₄ -PcMg), Chloroaluminum sulfonated phthalocyanine (CASPc), Chloroaluminum phthalocyanine tetrasulfate (AIPcTS), Mono-sulfonated aluminum phthalocyanine (AISPc), Disulfonated aluminum phthalocyanine ( AIS2Pc), tri-sulfonated aluminum phthalocyanine (AIS3Pc), tetra-sulfonated aluminum phthalocyanine (AIS4Pc), silicon phthalocyanine (SiPcIV), zinc (
II) Phthalocyanine (ZnPc), bis (di-isobutyl octadecylsiloxy) silicon 2,3-naphthalocyanine (isoBOSINC), germanium (IV) octabutoxy
Phthalocyanine (GePc), Rhodamine 101 (Rh-101), Rhodamine 110 (Rh
-110), Rhodamine 123 (Rh-123), Rhodamine 19 (Rh-19), Rhodamine 560 (Rh-560), Rhodamine 575 (Rh-575), Rhodamine 590
(Rh-590), Rhodamine 610 (Rh-610), Rhodamine 640 (Rh-640)
, Rhodamine 6G (Rh-6G), Rhodamine 700 (Rh-700), Rhodamine 800
(Rh-800), Rhodamine B (Rh-B), sulforhodamine 101, sulforhodamine 640, sulforhodamine B, coumarin 1, coumarin 2, coumarin 4, coumarin 6, coumarin 6H, coumarin 7, coumarin 30, coumarin 47, Coumarin 10
2, coumarin 106, coumarin 120, coumarin 151, coumarin 152, coumarin 152A, coumarin 153, coumarin 311, coumarin 307, coumarin 3
14, coumarin 334, coumarin 337, coumarin 343, coumarin 440, coumarin 450, coumarin 456, coumarin 460, coumarin 461, coumarin 4
66, coumarin 478, coumarin 480, coumarin 481, coumarin 485, coumarin 490, coumarin 500, coumarin 503, coumarin 504, coumarin 5
10, Coumarin 515, Coumarin 519, Coumarin 521, Coumarin 522, Coumarin 523, Coumarin 535, Coumarin 540, Coumarin 540A, Coumarin 548, 5-Ethylamino-9-diethylaminobenzo [a] phenoxazinium (EtNBA), 5- Ethylamino-9-diethylaminobenzo [a] phenothiazinium (EtNBS), 5-ethylamino-9-diethylaminobenzo [a] phenoselenazinium (EtNBSe), chlorpromazine, chlorpromazine derivatives, chlorophyll derivatives, bacteriochlorophyll (green) Bacteria) derivative, metal ligand complex salt,
Tris (2,2'-bipyridine) ruthenium (II) dichloride (RuBPY), Tris (2
2'-bipyridine) rhodium (II) dichloride (RhBPY), tris (2, 2'-bipyridine) prattum (II) dichloride (PtBPY), pheophorbide a, merocyanine 540, vitamin D, 5-amino-laevulinic acid, photo Sun (photosa
n), chlorin e6, chlorin e6 ethylenediamine, mono-L-aspartyl chlorin e6, phenoxazine nile blue derivative, stilbene, stilbene derivative, 4- (N- (2-hydroxylethyl) -N-methyl-aminophenyl) -4'-
9. The method of claim 8 selected from the group consisting of (6-hydroxylhexylsulfonyl) stilbene (APSS), and standard biological dyes and pigments. 10. The multiphoton excitation photoactivation comprises n photons, where n is 2
The method of claim 1, wherein the photons are above and the agent can be varied to optimize the volume of photoactivated tissue. 11. Treating the specified volume of said substance with sufficient light to enhance multiphoton excitation of said at least one photoactive molecular agent contained in said specified volume of said substance, comprising: (a1) a light source. Modulating the light from and producing modulated light; (a2) with sufficient modulated light to enhance multiphoton excitation of the at least one photoactivated molecular agent contained in a specific volume of the substance. Demodulating a detected energy signal having a specific form of modulation, and (f) characterizing the specific volume of the material, the demodulated energy signal comprising: Generating the method of claim 1. 12. The method of claim 11, wherein the modulated light is modulated as a specific form of modulation and the detected energy signal comprises the specific form of modulation. 13. The method of claim 11, wherein a microscope is used in the step of treating the specific volume of tissue with modulated light. 14. The method of demodulating a detected energy signal having a specific form of modulation comprises demodulating the detected energy signal at a frequency more than twice that of the specific form of modulation to obtain a specific form of modulation harmonics. Claim 1 including detecting
The method described in 2. 15. A demodulated energy signal characterizing a specific volume of the material is
12. The method of claim 11, which represents a change during the lifetime of at least one photoactivatable molecular agent present in a particular volume of the substance. 16. The method of claim 11, wherein the modulated light is generated by the operation of a laser that produces a pulsed output having a pulse repetition frequency of 10 megahertz or greater and a sub-nanosecond pulsed downnation. . 17. The processing step focuses the light beam over a range of focal lengths such that the focal plane of the light beam extends to a position between the surface of the material and a point substantially beyond the surface of the material. The method of claim 1, wherein the processing step can be extended to penetrate deep into the material. 18. The method of claim 1, wherein the detected energy signal is used to form an image of a specific volume of the material. 19. The step of treating with light is performed over a range of focal lengths such that the focal plane of the light flux extends to a position between the surface of the material and a point substantially beyond the surface of the material. Focusing, so that the processing step can be extended to penetrate deeply into the material, and further the steps of photoactivating, detecting, and generating a detected energy signal include: 19. The image is three-dimensional, including changing the position of the focal length of the light bundle within the material, as caused by a change in position between a site located substantially beyond the surface. the method of. 20. The method of claim 2, wherein the laser light is produced by the operation of a laser that produces a pulsed output having a pulse repetition frequency of 10 megahertz or greater and a duration of sub-nanosecond pulses. 21. The method of claim 20, comprising actuating a laser that produces near infrared light. 22. The method of claim 21, wherein the laser produces pulse energy of up to about 20 nanojoules. 23. The method of claim 1, wherein the detecting step comprises detecting emitted light that does not follow the optical path of incident light from the laser. 24. The processing step further comprises using modulated laser light, and in the detecting step, a wavelength selective device is used to filter energy emitted by the photoactivating agent. The method of claim 1. 25. The method of claim 20, wherein the processing and photoactivating step produces synchrotron radiation emitted from a molecular agent within the substance, the synchrotron radiation generation being substantially synchronized with the modulation of the laser light. Method. 26. The method according to claim 1, wherein the substance is a plant or animal tissue. 27. The at least one photoactivated molecular agent is an endogenous agent selected from the group consisting of aromatic amino acids, nucleic acids, proteins, cellular energy exchange stores such as adenosine triphosphate, enzymes and hormones. The method of claim 1, comprising: 28. The at least one photoactivated molecular agent is bound to a target agent before the step of treating the substance with the at least one photoactivated molecular agent, wherein the target substance is a nucleic acid. , Amino acids, proteins,
Protein receptor, protein complex compound, carbohydrate, carbohydrate receptor, carbohydrate complex compound, lipid, lipid receptor, lipid complex compound antibody, ligand, hapten, complex compound, chelate, encapsulated solution, liposome, fullerene, crown 9. The method of claim 8 selected from the group consisting of ethers and cyclodextrins. 29. A device for diagnosing or imaging a specific volume of a substance containing at least one photoactivated molecular agent, wherein the substance has a frequency effective to penetrate substantially into the substance and is contained in the substance. A light source adapted to enhance multi-photon excitation of a molecular agent, and a focusing device for focusing light from a surface of the material over a range of focal lengths extending to a depth substantially beyond said surface. A focusing device for working with the light source to enhance multi-photon excitation of the molecular agent, the focus or focal plane being adjustable with respect to the substance, and the detector being emitted by the molecular agent. A device positioned to detect the light, the detector being arranged to generate a detection signal characterizing a particular volume of focus of the light source. 30. The apparatus of claim 29, wherein the light source produces a pulsed output having a pulse repetition frequency ranging from about 1 kilohertz to about 10 gigahertz. 31. The apparatus of claim 30, wherein the pulse repetition frequency is about 10 megahertz or higher. 32. The apparatus of claim 30, wherein the light source has a sub-nanosecond pulse duration. 33. The apparatus of claim 29, wherein the light source produces near infrared light. 34. The apparatus of claim 30, wherein the light source operates within a pulse energy range of about 10 picojoules to about 50 millijoules. 35. The apparatus of claim 34, wherein the light source produces pulse energy of about 20 nanojoules or less. 36. The apparatus of claim 33, wherein the light source comprises a laser. 37. The apparatus of claim 36, wherein the light from the laser comprises one or more microwave pulse trains. 38. The method of claim 37, wherein each of the one or more pulses has a duration of at most about 10 ps. 39. The apparatus of claim 29, further comprising a processor coupled to the detector. 40. The apparatus of claim 39, further comprising a modulation system associated with the light source, the processor being coupled to the modulation system and arranged to demodulate the detection signal. 41. The multiphoton photoactivation comprises n photons, where n is two or more photons, which the agent can vary to optimize the volume of photoactivated material. 30. The device of claim 29, which is 42. The device of claim 29, wherein the at least one photoactivated molecular agent is an endogenous agent. 43. The device of claim 29, wherein the at least one photoactivated molecular agent is an exogenous agent. 44. The device of claim 40, wherein the light focusing device is a microscope. 45. The device of claim 29, wherein the substance is plant or animal tissue. 46. In a medical diagnostic imaging method, the step of introducing a photoactive molecular agent into a tissue, wherein the agent is selected for specificity of the tissue of interest and is sensitive to multiphoton excitation. Allowing the agent to accumulate in the tissue of interest, and directing light to a region of interest within the tissue, including substantially below the surface of the tissue, wherein the light directs the tissue Frequency and energy are selected to penetrate and substantially enhance multiphoton excitation in the focal region alone, controlling the position of the focal region over a range of depths in the tissue, and using multiphoton excitation. Generating a photoactivated agent in a focal region by photoactivating the agent within the tissue over a range of depths, wherein the photoactivation The sexual molecular agent is a medical diagnostic imaging method comprising the steps of emitting energy, detecting radiant energy, and generating a detected energy signal that characterizes tissue in the focal region. 47. The step of delivering light generates near-infrared light using a pulsed laser operating at short pulse width and long pulse repetition rate to focus the light on the tissue. 47. The method of claim 46, including. 48. The position control step of claim 46, comprising changing the position of the focal region for a particular tissue under test or changing the position of the tissue under test for a fixed focal region. Method. 49. The method of claim 46, wherein the method further comprises modulating the light and demodulating the detected energy signal prior to being incident on the tissue. 50. The method of claim 46, wherein the detected energy signal is used to image the focal area. 51. A substantially imaged region in which the frequency and energy are selected to produce light directed onto or into the tissue for imaging and to penetrate into or under the surface of the tissue. A light source that enhances multiphoton excitation only, and a focusing device that can change the position of light within the depth of the region of tissue to image, and the agent is excited using multiphoton excitation And a detector arranged to receive and detect the radiation emitted by the photoactivated molecular agent within the substance. 52. The apparatus of claim 51, wherein the light source produces near infrared light. 53. The device of claim 52, wherein the light source is a laser. 54. The apparatus of claim 51, wherein the apparatus further comprises a modulator cooperating with the light source to modulate the light, the light being modulated in the form of a modulation to produce modulated light. Equipment. 55. The device of claim 54, wherein the device further comprises a demodulator coupled to the detector to generate a demodulated energy signal characterizing a particular photoactivated molecular agent. 56. The apparatus of claim 51, wherein the light source produces a pulsed output having a pulse repetition frequency of 10 megahertz or greater and a sub-nanosecond pulse duration. 57. The apparatus of claim 51, wherein the light source produces pulse energy of about 20 nanojoules or less. 58. The multiphoton photoactivation comprises n photons, where n is 2
52. The device of claim 51, wherein the photons are those that are variable such that the agent optimizes tissue volume to be photoactivated. 59. A method of characterizing a substance comprising at least one photoactivating molecular agent, the method comprising incoding light from a light source with a modulation pattern to produce modulated light, and said at least one excitation. The molecular agent is photoactivated in the substance,
And treating the substance with the modulated light to enhance multiphoton excitation of the at least one photoactivated molecular agent to emit modulated energy, and detecting a portion of the modulated radiant energy. And producing the detected modulated energy signal characterizing a substance. 60. The method of claim 59, wherein the method further comprises demodulating the detected modulated energy signal. 61. The method of claim 59, wherein the characterization method is used for imaging the material. 62. The method comprises demodulating the detected modulated energy signal and as a function of position of the at least one photoactivated molecular agent.
60. The method of claim 59, further comprising recording the demodulated energy signal. 63. The method of claim 59, wherein the substance is selected from the group consisting of plant tissue and animal tissue. 64. The method of claim 63, wherein the animal tissue is located within the body of an animal. 65. The method of claim 59, wherein the light source is a laser. 66. The method comprises at least one step prior to treating the material with light.
60. The method of claim 59, further comprising treating the substance with one photoactivated molecular agent. 67. The method of claim 59, wherein a microscope is used in the step of treating the substance with modulated light. 68. The multiphoton photoactivation comprises n photons, where n is two or more photons, which the agent can vary to optimize the volume of photoactivated material. 60. The device of claim 59, which is 69. A method of characterizing a specific volume of tissue comprising at least one photoactive molecular agent, the steps of: incoding light from a light source with a modulation pattern to produce modulated light; Directing light to specific regions of interest in the tissue, including subsurface regions;
Controlling the position of the focal region over a range of depths in the tissue, the light being selected to enhance multiphoton excitation substantially only at a position within the focal region, the light penetrating the tissue; Photoactivating the agent over a range of depths in the tissue using photon excitation to photoactivate the at least one excited molecular agent substantially only in the focal region; The activated molecular agent emits modulated energy, the method comprising detecting a portion of said radiated modulated energy and producing a detected modulated energy signal characterizing tissue. 70. The method of claim 69, wherein the method further comprises demodulating the detected modulated energy signal. 71. The method of claim 69, wherein the characterization method is used to image the substance. 72. The method of claim 70, wherein the method further comprises recording the demodulated energy signal as a function of the position of the focal area. 73. The method of claim 69, wherein the tissue is located within the body of an animal. 74. The method of claim 69, wherein the light source is a laser. 75. The method comprises at least one step prior to treating the tissue with light.
70. The method of claim 69, further comprising treating the tissue with one photoactive molecular agent. 76. A method of diagnosing a property of a tissue having a surface and having a predetermined property and being relatively transparent to light, comprising introducing a photoactive molecular agent into the tissue, wherein: The agent is sensitive to multiphoton excitation, allowing the agent to accumulate at a particular feature, if present in the tissue, and a laser to obtain a light flux having the predetermined feature. And directing the laser beam to a particular region of interest within the tissue, including a region substantially below the surface of the tissue, causing the beam to penetrate the tissue and only at a position within the focal region. Substantially enhancing multi-photon excitation of the agent and moving the position of the focal region over a cross-sectional region located at a depth range within the tissue to define a diagnostic volume. And a multi-photon excitation is used to photoactivate the agent that has accumulated the feature of interest in the diagnostic volume that passes through the focus region, thereby causing the focus region to detect the feature of interest. Generating a photoactivating agent at each of the features of interest when intersecting, wherein the photoactivating agent emits energy and detects radiant energy; and Generating a detected energy signal. 77. The method of claim 76, wherein the characterization method is used to image the tissue. 78. The method comprises: modulating the laser beam; the photoactivating agent being caused by modulation energy, the detecting step comprising detecting modulation energy, and the generating step the detected modulation. 77. Generating an energy signal; using the detected modulated energy signal to form an image of any of the features of interest in the tissue.