EP1187555A1 - Procedes et appareil ameliores pour la photo-excitation multiphotonique et la detection d'agents moleculaires - Google Patents

Procedes et appareil ameliores pour la photo-excitation multiphotonique et la detection d'agents moleculaires

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Publication number
EP1187555A1
EP1187555A1 EP00937691A EP00937691A EP1187555A1 EP 1187555 A1 EP1187555 A1 EP 1187555A1 EP 00937691 A EP00937691 A EP 00937691A EP 00937691 A EP00937691 A EP 00937691A EP 1187555 A1 EP1187555 A1 EP 1187555A1
Authority
EP
European Patent Office
Prior art keywords
light
photo
agent
tissue
coumarin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00937691A
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German (de)
English (en)
Other versions
EP1187555A4 (fr
Inventor
Walter Fisher
John Smolik
H Craig Dees
Eric Wachter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Photogen Inc
Original Assignee
Photogen Inc
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Filing date
Publication date
Application filed by Photogen Inc filed Critical Photogen Inc
Publication of EP1187555A1 publication Critical patent/EP1187555A1/fr
Publication of EP1187555A4 publication Critical patent/EP1187555A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A61B5/444—Evaluating skin marks, e.g. mole, nevi, tumour, scar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0082—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes
    • A61B5/0091—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence adapted for particular medical purposes for mammography

Definitions

  • the present invention relates generally to methods and apparatus for achieving selective photo-activation of one or more molecular agents with a high degree of spatial control and for improving the detection of the diagnostic signals thereby produced.
  • the methods taught herein for the present invention for achieving selective photo-activation utilize the special properties of non-linear optical excitation for promotion of an agent from one molecular energy state to another with a high degree of spatial and molecular specificity.
  • the special features of the methods of the present invention are applicable for activation of various endogenous and exogenous diagnostic or imaging agents, and in particular afford distinct advantages in the diagnosis of diseases in humans and animals.
  • the non-linear, multi-photon excitation methods of the present invention facilitate controlled activation of diagnostic or imaging agents in deep tissue or in other specimens using near infrared to infrared radiation, which is absorbed and scattered to a lesser extent than during the methods and radiations currently used.
  • sensitivity in the detection of diagnostic signals is greatly increased.
  • certain aspects of the present invention have direct applicability in microscopy and other related fields.
  • the desired improvements in activation include ( 1 ) enhancements in spatial or temporal control over the location and depth of activation, (2) reduction in undesirable activation of other co-located or proximal molecular agents or structures, (3) increased preference in the activation of desirable molecular agents over that of undesirable molecular agents, and (4) improved sensitivity in detection of the resultant diagnostic signals thereby produced.
  • Various linear and nonlinear optical methods have been developed to provide some of these improvements for some agents under very specialized conditions.
  • FIGURE 1 A generalized Jablonski diagram for such activation is shown in FIGURE 1 (a), wherein single-photon excitation (10) occurs when a photo-active agent is excited from a lower quantum-mechanically allowed state S m to a higher quantum-mechanically allowed state S n upon absorption of a certain energy E, which is provided by interaction of a single photon P, with the agent.
  • Relaxation R may then occur from this transient excited state S n . Additionally, similar relaxation processes R 2 may occur from a secondary long-lived activated state T m the latter occurring following intersystem crossing IX.
  • the performance of such excitation methods has not been as successful as desired.
  • the high energy optical radiation P typically used can often produce disease or other undesirable side effects and may have a less than desirable penetration depth because of optical scatter and the absorbance of the UV or visible activating optical radiation.
  • simultaneous two-photon excitation has been used as a means for stimulating fluorescence emission from molecules present in optically dense media.
  • FIGURE 1 (b) A generalized mechanism for such activation is shown in FIGURE 1 (b), wherein simultaneous two-photon excitation (12) occurs when a photo-active agent is excited upon absorption of a certain energy E, that is provided by the simultaneous, combined interaction of two photons P,' and P 2 ' with the agent.
  • FIGURE 1(c) shows a generalized representation of such multi-photon excitation, wherein 3 +2 -photon excitation (14) occurs when a photo-active agent is initially excited to a first higher quantum- mechanically allowed state S n upon absorption of a certain energy E, that is provided by the simultaneous, combined interaction of three photons P,", P 2 " and P 3 " with the agent
  • ultrashort pulsed sources i.e., laser sources capable of producing pulses of light having pulse widths generally less than approximately 10 picoseconds
  • the inventors of the present application are aware of no reports earlier than the present invention of the application of ultrashort pulsed, multi -photon methods for practical diagnostic imaging of human or animal tissue involving selective activation of endogenous (naturally present) or exogenous (externally supplied) molecular agents.
  • Denk et al. (W. Denk, J.P. Strickler and W.W. Webb, "Two-Photon Laser Microscopy," U.S. Patent No. 5,034,613) disclose the construction and use of a special epi-illumination confocal laser scanning microscope utilizing non-linear laser excitation to achieve intrinsically high three-dimensional control in the photo-activation of various exogenous agents in the laboratory on a cellular or sub-cellular scale. Emitted fluorescent light is collected by the excitation objective using an epi-illumination configuration. This light is then used for generation of luminescence-based images at the cellular and sub- cellular level for specimens mounted on a slide or other form of sample stage. In later work, Denk et al. (W.
  • Lytle "Second Harmonic Detection of Spatially Filtered Two-Photon Excited Fluorescence," Analytical Chemistry, 65 (1993) 631-635) discuss complex methods for rejection of scattered laser excitation light by making use of second-harmonic detection methods: i.e. when sinusoidal modulation of the excitation light is performed at one frequency, and detection of the two-photon excited fluorescence is performed at twice that frequency (which is the second harmonic of the excitation modulation frequency), interferences from scattered excitation light are allegedly virtually eliminated. And by proper selection of the modulation frequency to avoid electronic and other noise frequencies, rejection of instrumental and environmental interferences is extremely high.
  • non-linear excitation can be used under laboratory conditions to excite various luminescent molecular agents using light at longer wavelengths than that used for linear single-photon excitation, and that the excitation thereby effected can improve three- dimensional spatial control over the location of excitation, can reduce interference from absorption and scatter of the excitation light in optically dense media, and can reduce collateral damage along the excitation path to living cell samples undergoing microscopic examination.
  • the present invention is directed to a method and apparatus for photo-activating a molecular agent in a particular volume of material using multi-photon excitation and for improving the detection of the resultant diagnostic signals thereby produced.
  • the present invention utilizes the unique physical properties of nonlinear optical excitation of one or more endogenous or exogenous diagnostic or imaging agents to effect improved spatial control over the photo-activation of such agents via multi-photon excitation processes.
  • multi-photon excitation results from an essentially simultaneous interaction of two or more photons with one or more endogenous or exogenous agents, wherein said photons are provided by a single ultrashort laser pulse having a duration of approximately 10 ps or less.
  • the energy and wavelength of the two or more photons are identical, and as such the excitation processes are termed degenerate.
  • the present invention is not limited to a process wherein the energy and wavelength of the two or more photons are identical.
  • the multi-photon excitation of the present invention allows for a number of detectable end points, including those resulting from one or more of the following events: electronic excitation of the one or more agents to a higher quantum-mechanically allowed state; vibrational excitation of the one or more agents to a higher quantum-mechanically allowed state; vibronic excitation (combined vibrational and electronic excitation) of the one or more agents to a higher quantum-mechanically allowed state; and photoionization of the one or more agents. From such excited state end points, the one or more photo-activated agents are made to precipitate desired diagnostic effects, such as luminescence or loss of such luminescence.
  • the step of photo-activation includes the step of modulating light from a light source with a particular type of modulation, thereby producing a modulated light, and treating a target subject with the resultant modulated light so as to promote multi-photon excitation of one or more endogenous or exogenous diagnostic or imaging agents present on or within the target subject. It is further preferred that the present invention include the steps of demodulating a resultant detected energy signal with the particular type of modulation, and producing a demodulated energy signal which is characteristic of the particular target subject.
  • the step of demodulating the detected energy signal with the particular type of modulation includes demodulating the detected energy signal at a frequency twice or more of that of the particular type of modulation, thereby detecting the harmonic of the particular type of modulation. It is also more preferred that the demodulated energy signal which is characteristic of the particular subject represents a change in lifetime of at least one photo-activated agent present in the particular subject. The method of the present invention, however, can be performed without these optional steps of modulation or demodulation.
  • the multi-photon excitation methods taught herein offer specific advantages relative to prior methods, including reduction of interference from absorption and scattering processes originating from the environment surrounding the excited agent, improved activation depths, improved efficiency, and enhanced control over location and specificity for the excited agent.
  • FIGURES 1 (a)-(c) illustrate example energy level diagrams for typical linear and nonlinear optical excitation processes
  • FIGURES 2(a)-(d) illustrate typical modified Jablonski energy level diagrams for several linear and non-linear optical excitation processes
  • FIGURE 3 shows a comparison of two-photon excitation response and three-photon excitation response for Indo-1 as a function of excitation power
  • FIGURE 4 shows a comparison of spatial excitation properties for linear and nonlinear excitation processes
  • FIGURES 5(a)-(b) illustrate examples of spatially localized multi-photon excitation used to locally activate agents present at the surface of tissue or below the surface of tissue;
  • FIGURES 6(a)-(b) show a comparison of single-photon and two-photon excited fluorescence of the dye molecule Coumarin-480 distributed evenly throughout a block of agarose gelatin;
  • FIGURE 7 compares absorption cross-sections as a function of excitation wavelength for Hp-IX when using single-photon excitation and simultaneous two-photon excitation;
  • FIGURE 8 shows example absorption and scatter spectra for human tissue covering the ultraviolet to infrared spectral region;
  • FIGURE 9 shows the general trends in optical absorption and scattering properties of tissue for incident short wavelength and long wavelength light
  • FIGURE 10 shows a diagram of a specific preferred embodiment of the present invention for imaging endogenous or exogenous diagnostic imaging agents
  • FIGURE 1 1 shows a diagram of an alternate preferred embodiment of the present invention for imaging endogenous or exogenous diagnostic imaging agents, wherein modulation is used to improve imaging performance;
  • FIGURE 12 shows a diagram of a second alternate preferred embodiment of the present invention for videographic imaging of superficial features.
  • Non-linear optical excitation is defined for the purposes of this patent document as those excitation processes involving the essentially simultaneous interaction of two or more photons with the one or more agents.
  • Essentially simultaneous interaction is defined for the purposes of this patent document as those excitation processes occurring as a result of interaction of the one or more agents with photons provided by a single ultrashort laser pulse having a duration of approximately 10 ps or less.
  • Multi-photon photo-activation is thus defined for the purposes of this patent document as non-linear optical excitation occurring as a result of the essentially simultaneous interaction of two or more photons originating from a single ultrashort laser pulse with one or more imaging or diagnostic agents to produce one or more photo-activated imaging or diagnostic agents. According to this definition, the physical process resulting in multi-photon photo-activation is multi-photon excitation of the one or more agents.
  • endogenous agents are defined for the purposes of this patent document as photo-active materials which are pre-existent in a patient or other target, such as for example various natural chromophoric agents, such as melanin, hemoglobin, carotenes, NADH and NADPH, and other photo-active materials, such as tattoo dyes.
  • exogenous agents are defined as photo-active materials which are not pre-existent in a patient or other target, such as for example various diagnostic dyes or other photo-active agents administered for the purpose of increasing efficiency of conversion of optical energy into a diagnostic or therapetic signal.
  • the end point of such multi-photon photo-activation according to the present invention may include one or more of the following detectable events: electronic excitation of the one or more agents to a higher quantum-mechanically allowed state; vibrational excitation of the one or more agents to a higher quantum-mechanically allowed state; vibronic excitation (combined vibrational and electronic excitation) of the one or more agents to a higher quantum-mechanically allowed state; and photoionization of the one or more agents. From such excited state end points, the one or more photo-activated agents are made to precipitate desired diagnostic effects, such as luminescence or loss of such luminescence.
  • Multi-photon excitation is performed at a wavelength approximately twice or more of that used for conventional single-photon excitation.
  • one or more diagnostic agents may be photo-activated at a location substantially limited to a focal region defined by the focused beam, resulting in unprecedented resolution along and perpendicular to the optical path.
  • the non-linear optical excitation of the present invention has additional advantages during photo-activation of imaging or diagnostic and other agents, including reduction of collateral excitation and damage along the excitation path, reduction in exposure to harmful optical wavelengths, and enhanced specificity in the excitation of the agent.
  • the non-linear optical excitation approach employed in the present invention provides a superior means for the detection and imaging of many diseases.
  • the present invention is intended primarily for in vivo detection and imaging of disease and other characteristics of tissues, such as cancer in the human breast.
  • the methods and apparatus taught have numerous additional applications, and that these methods and apparatus can be applied to other fields, such as laser scanning microscopy, in order to achieve substantive improvements in the performance characteristics of methods and apparatus used in such fields.
  • the basic configurations of the multi-photon photo-activation method and apparatus for achieving such imaging or diagnostic outcomes are described in U.S. Patent Application Serial nos. 08/741,370 and 09/096,832, which are assigned to the assignee of the present invention and which have inventors in common with the present application.
  • U.S.S.N. 08/741,370 filed October 30, 1996 and U.S.S.N. 09/096,832 filed on June 12, 1998, are incorporated herein by reference in their entirety.
  • embodiments of the present invention include a light source, details of which are described in detail below, where the resulting light is directed at a tissue or substance to be imaged.
  • a modulation device or method can be included, as also described.
  • the region to be imaged can be moved relative to the light beam or source or alternatively, the light beam can be moved.
  • a detector is provided, and may include a suitable demodulator if modulation has been used. Details of the light used for photoactivation will now be set forth through a discussion of the scientific principles used in this invention.
  • One aspect of the present invention taught in this disclosure lies in the use of multi- photon processes to selectively and efficiently photo-activate one or more imaging or diagnostic agents with a high degree of spatial control.
  • This selective photo-activation is achieved by means of harnessing the special properties of non-linear optical excitation to promote an agent from one molecular energy state to another.
  • a conceptual model of multi-photon excitation is developed herein. This is conveniently achieved through use of energy level diagrams for representative cases.
  • FIGURES 2(a)-(d) illustrate typical modified Jablonski energy level diagrams for several linear and non-linear optical excitation processes.
  • FIGURE 2(a) illustrates single-photon excitation (20) which occurs when an agent is excited from an initial quantum-mechanically allowed state S, (which is typically the ground state) to a final quantum-mechanically allowed state S f upon absorption of a certain energy E, that is provided by interaction of a single photon P with the agent.
  • S initial quantum-mechanically allowed state
  • S f final quantum-mechanically allowed state
  • E certain energy
  • promotion of the agent from S, to S f can initiate various radiative emissions of light from the excited state, including for example fluorescent or phosphorescent emission of light.
  • the dye molecule coumarin-480 may, upon absorption of a single photon of light at 400 nm, emit a photon via fluorescence re-emission (which may also be described as a relaxation process) at 465 nm.
  • fluorescence re-emission which may also be described as a relaxation process
  • single-photon excitation (20) the probability of excitation is linearly related to the irradiance of the incident optical radiation, so single- photon excitation (20) is referred to as a linear excitation process.
  • simultaneous two-photon excitation (22) occurs when a photoactive agent is excited from an initial quantum-mechanically allowed state S. to a final quantum-mechanically allowed state S f upon absorption of a certain energy E, that is provided by simultaneous interaction of two photons P, and P 2 with the agent.
  • An example of simultaneous two-photon excitation (22) is the promotion of fluorescence emission of light at 465 nm from coumarin-480 upon simultaneous absorption of two photons at 800 nm.
  • the probability of excitation is related to the product of the instantaneous irradiance of the first photon P, and the second photon P 2 . This can be conceptualized in the form of a photochemical reaction,
  • simultaneous three-photon excitation occurs when a photo- active agent is excited from an initial quantum-mechanically allowed state S, to a final quantum-mechanically allowed state S f upon absorption of a certain energy E, that is provided by simultaneous interaction of three photons P,', P 2 ' and P 3 ' with the agent.
  • the simultaneous interaction of the three photons is described as being mediated by two transient virtual states V,' and V 2 ' each having a lifetime on the order of 10 fs or less. If all three photons do not interact with the agent during these life times, excitation does not occur and the agent returns to S,.
  • the instantaneous irradiance of the incident excitation light must be sufficiently high to yield significant efficiency in absorption of the second photon P 2 ' and the third photon P,' before the agent undergoes relaxation back to S,.
  • An example of simultaneous three-photon excitation (24) is the promotion of fluorescence emission of light at 465 nm from coumarin-480 upon simultaneous absorption of three photons at 1200 nm.
  • the probability of excitation is related to the product of the instantaneous irradiance of the first photon P,', the second photon P 2 ' and the third photon P 3 '. This can be conceptualized in the form of a photochemical reaction,
  • simultaneous multi-photon excitation occurs when a photoactive agent is excited from an initial quantum-mechanically allowed state S, to a final quantum-mechanically allowed state S f upon absorption of a certain energy E, that is provided by simultaneous interaction of two or more photons with the agent.
  • a certain energy E that is provided by simultaneous interaction of two or more photons with the agent.
  • the agent has been promoted to the final quantum-mechanically allowed state S f its photochemical properties will be identical to those resulting from single-photon excitation (20).
  • the simultaneous interaction of the two or more photons is often described as being mediated by a corresponding multiplicity of one or more virtual states, each having a lifetime on the order of 10 fs or less.
  • the probability of excitation is related to the product of the instantaneous irradiance of the n photons. This can be conceptualized in the form of a general photochemical reaction,
  • a more general definition of multi-photon excitation than that given above requires only that the two or more photons interact with the one or more agents in a substantially simultaneous manner, for example any interaction occurring during a single ultrashort laser pulse having a duration of approximately 10 ps or less. Under such constraints, the interaction of light with the one or more agents must occur in a fast regime, substantially limiting (and localizing) the direct photo-activation effect to intra-molecular processes, such as electronic excitation or photoionization of the agent. No significant elapse of time nor substantial molecular reorganization nor motion will occur during such excitation, and on conventionally observable frames of reference any transitions thereby effected in the one or more agents will occur as an essentially single, concerted step. This more general definition shall be used in subsequent descriptions of the properties of multi-photon excitation and of the resultant multi-photon photo-activation of imaging or diagnostic agents.
  • the agent has been promoted to the final quantum- mechanically allowed state S f following absorption of a particular quantity of energy E, its photochemical properties, including diagnostically useful luminescent emission of light, will be identical regardless of the way used for such promotion. These properties will be determined by the intrinsic properties of the excited agent and its local environment.
  • the particular final energy state S f attained will be determined primarily by the magnitude of the total energy E, delivered to the molecule, and it is thus the magnitude of E, that will ultimately determine the properties of the agent upon arrival at S f .
  • the mechanism responsible for promoting the agent to the excited state has no significant impact on this fate since the excitation process itself does not directly impact the subsequent properties of the excited agent or its environment.
  • agent response such as luminescence
  • excitation power which correlates directly with instantaneous irradiance
  • Indo- 1 which exhibits strong single-photon excitation (emitting fluorescence at 500 nm) upon illumination with light between 300 and 400 nm.
  • the focused light is, for example, a beam composed of a 76 MHz repetition rate train of approximately 200 fs pulses produced by a mode-locked titanium: sapphire laser.
  • the fluorescent agent Coumarin-540A which exhibits only a two-photon response at these wavelengths, exhibits slopes of 2.01 and 2.00 at 810 nm and 910 nm, respectively.
  • these multi-photon signals completely disappear if the laser is not pulsed, confirming that multi-photon excitation is responsible for the observed response (since the non-pulsed laser beam does not provide sufficient instantaneous irradiance to support a multi-photon process with these agents at these wavelengths).
  • the excitation cross-sections for multi-photon processes generally decrease as the number of photons required for a given transition increases.
  • the three-photon cross-section for a particular transition in a given agent will generally be lower than the respective two-photon cross-section for the same transition. This is at least in part due to the reduced probability that all photons necessary for a particular multi- photon process will interact with the agent in a substantially simultaneous manner. This is evident when comparing the magnitude of the two-photon excitation response (30) and the three-photon excitation response (32) for Indo-1 (FIGURE 3) at a given average excitation power.
  • this difference in relative cross-section can be substantially ameliorated by increasing the instantaneous irradiance of the excitation light, for example by decreasing pulse width for a beam of a given average power, or by increasing pulse energy (for example by using an amplified light source, such as a regeneratively amplified titanium: sapphire laser or a chirp-pulse amplified NdNAG laser).
  • an amplified light source such as a regeneratively amplified titanium: sapphire laser or a chirp-pulse amplified NdNAG laser.
  • spatially localized multi-photon excitation may be used to locally activate agents present at the surface of tissue (50) or below the surface of tissue (52), as shown for example in FIGURES 5(a) and 5(b), respectively.
  • FIGURE 6(a) shows a photograph of a solid block of agarose gelatin (60) throughout which the dye molecule Coumarin-480 has been evenly distributed.
  • FIGURE 6(b) is a drawing of the photograph in FIGURE 6(a).
  • the agarose gelatin (60) constitutes an optically dense medium since it strongly scatters visible light.
  • Coumarin-480 emits blue fluorescent light when excited via single- photon methods at 350-450 nm or when excited via two-photon methods at 700-900 nm.
  • a beam of light (62) was focused into the agarose gelatin (60) from one side such that it formed a focus (64) approximately 2 cm into the gelatin.
  • a beam (62) of visible light (at 400 nm) or of NIR light (at 730 nm from a mode- locked titanium: sapphire laser) was expanded to produce a collimated beam approximately 50 mm in diameter using a beam expanding telescope.
  • the laser produced a continuous train of ⁇ 200 fs pulses of light at a 76 MHz pulse repetition frequency.
  • the expanded beam was then focused into the agarose gelatin (60) using a 250 mm focal length (f.l.),
  • FIGURES 6(a) and 6(b) clearly show that fluorescence from the Coumarin-480 is stimulated only at the focus of the NIR beam (66), while fluorescence is emitted along the entire line of flight for the visible beam (68).
  • FIGURES 6(a) and 6(b) dramatically illustrate the unique spatial localization achievable with multi-photon excitation, along with the generally improved penetration of NIR light relative to shorter wavelength light in optically dense media (such as an agarose gelatin (60) or human or animal tissue). Because of the non- linear relationship between efficiency of multi-photon excitation and instantaneous irradiance, agent stimulation at positions along the beam path prior to and after the focus is negligible. Hence, little or no photo-activation occurs outside the focal zone.
  • the NIR excitation light is only weakly absorbed or scattered by the gelatin, sharp focus is maintained at deep penetration depths into the block (in fact, by moving the gelatin along the optical axis, sharp focus was achievable through the entire 8-cm thickness of gelatin). Since the sharpness of the focus observed in FIGURES 6(a) and 6(b) is determined by Gaussian optical properties, the length and area of the focal zone is easily adjusted by changing the optical parameters used for beam manipulation, such as beam expansion and subsequent refocusing (thereby controlling numeric aperture of the resultant focused beam).
  • the multi-photon excitation efficiency profile (44) shown in FIGURE 4 and evidenced in FIGURES 6(a) and 6(b) will become increasingly tighter.
  • three-photon excitation will provide tighter spatial localization than that possible with two-photon excitation, and so on.
  • the multi-photon order number of photons utilized for a particular excitation
  • the thickness of the focal zone might be tightened through the use of three -photon excitation in order to optimize selective activation of agent at the surface of tissue (50), as shown in FIGURE 5(a).
  • FIGURE 7 provides an example showing a relative comparison of absorption cross- sections as a function of excitation wavelength for the weakly luminescent molecule Hp- IX in ethanol when using conventional single-photon excitation (74) and simultaneous two-photon excitation (76).
  • simultaneous two-photon excitation (76) data is also plotted using a wavelength scale that has been divided by two (78) to reflect that simultaneous two-photon excitation is equivalent in energy to absorption of a single photon at twice the energy (or one half the wavelength) of the each of two photons.
  • Comparison of the relative cross-sections for single-photon excitation (74) and for simultaneous two-photon excitation plotted at the one-half wavelength scale (78) shows significant differences as a function of wavelength. Specifically, the prominent Sorrett band (80) evident with single-photon excitation (74) is absent for simultaneous two- photon excitation (76) or (78), since it is quantum-mechanically disallowed.
  • agents with little or no symmetry will in general have identical selection rules regardless of the number of photons employed in their excitation.
  • centrosymmetric agents it will generally be advantageous to carefully determine multi-photon spectral response as a function of wavelength in order to determine optimal excitation wavelengths, while for non-centrosymmetric agents, single-photon excitation spectra may generally be used to estimate multi-photon spectral response as a function of wavelength.
  • FIGURE 8 shows typical absorption and scattering properties for tissue covering the UV to IR spectral region.
  • the use of longer wavelength light generally reduces the relative effects of scatter, and thereby improves optical penetration depth.
  • substitution of a multi- photon method according to embodiments of the present invention for a conventional, single-photon method will reduce the effects of scatter on the delivery of activating light to an agent present within tissue.
  • a UV-active fluorophor such as Indo-1 (which is normally activated using 350 nm light)
  • Indo-1 which is normally activated using 350 nm light
  • tissue absorbance which can reduce the penetration of activating light and which might lead to light-induced tissue damage, is generally reduced through the use of longer wavelength light, for example, NIR light in the so-called tissue transmission window extending from approximately 700 nm to 1300 nm.
  • Negligible effects are generally observed in tissues irradiated with lower-energy, NIR light, even when the optical power of the NIR light is many-fold higher than that of the UV or visible radiation.
  • substitution of a multi-photon method in accordance with the present invention for a conventional, single-photon method will usually reduce the effects of tissue absorbance on the delivery of activating light to an agent present within tissue.
  • Reduced scatter and absorption by tissue allows spatially-localized excitation to be efficiently realized with multi-photon methods, as illustrated in FIGURES 5 and 6, since matrix interference is substantially reduced. Furthermore, reduced scatter and absorption by tissue results in additional safety advantages for multi-photon excitation. For example, when UV light impinges on human tissue, the majority of the optical energy is immediately absorbed and scattered in the outermost layers, such as the epidermis and dermis. This absorption may occur due to excitation of certain molecules in the cells of this tissue, such as those composing the genetic material in the cellular nucleus. This absorption of high- energy light by cellular constituents can thereby initiate a variety of collateral photochemical changes in these cells, including irreversible genetic damage and induction of cancer. In contrast, NIR light used for two-photon or three-photon methods will not be appreciably absorbed or scattered by tissue, and as such the possibility for collateral damage to cells will be substantially lower.
  • FIGURE 9 Additional effects of absorption and scatter are considered in FIGURE 9.
  • the inherently high absorption and scatter of higher-energy U V or visible photons 90 by tissue can result in very shallow tissue penetration depths, while lower-energy NIR photons 92 generally have much greater penetration depths.
  • scattered higher-energy photons 94 can induce emission from diagnostic agents along their scatter path, higher- energy photons that manage to penetrate tissue will tend to produce a diffuse emission zone that extends radially from the excitation path.
  • FIGURE 9 These important differences in absorption and penetration depth properties for higher- energy and lower-energy light are shown schematically in FIGURE 9.
  • UV or visible light 90 for example light at 400 nm
  • the majority of the optical energy is immediately absorbed 98 and scattered 94 in the outermost layers 100, such as the epidermis and dermis.
  • optical penetration depth is low, and potential for induction of collateral photo-induced damage is high for excitation with UV or visible light 90, such as that conventionally used for linear excitation of diagnostic agents.
  • NIR light 92 for example at 800 nm, will experience much lower absorption and scatter by tissue 96.
  • the overall depth of penetration will be much greater and the extent of collateral damage to cells will be substantially lower.
  • the order of a multi-photon process i.e. the number of photons utilized for excitation
  • the order of a multi-photon process can be chosen to simultaneously optimize the excitation wavelength of the one or more desired photoactive agents and the transmissive properties of tissue.
  • an example of the selection of multi-photon process order for simultaneous optimization of photo-activation efficiency and tissue transmissive properties is the activation of Indo- 1 in normal human tissue.
  • the large two-photon cross- section of Indo- 1 relative to the corresponding three-photon cross-section would allow efficient photo-activation of Indo- 1 using two-photon excitation at 700 nm with an approximate 260-fold reduction in tissue absorbance relative to single- photon excitation at 350 nm (as illustrated in Fig. 8).
  • activation of Indo- 1 in melanotic human tissue affords further illustration.
  • the large absorbance of light at 700 nm by melanin indicates that use of longer wavelengths would be preferable to avoid matrix interferences and as such three-photon excitation of Indo- 1 at 1000 nm would be optimal as a consequence of the approximate 15 -fold reduction in tissue absorbance of light at 1000 nm relative to 700 nm and the approximate 3500-fold reduction in tissue absorbance of light at 1000 nm relative to 350 nm.
  • the order of a particular multi -photon process can be further chosen to match spatial localization performance to that which is optimal for the particular specimen or target to be examined. For example, in general, higher- order multi-photon processes (>3 photons) will provide greater spatial localization than that observed with two-photon excitation. Thus for certain specimens, it may be desirable to use such higher order excitation processes for agent photo-activation, even if this may result in reduced detection sensitivity.
  • the cross-section for a particular multi-photon excitation process is typically many- fold smaller than that for an equivalent single-photon excitation process yielding the same activated state as the multi-photon process. This is due to the relatively low probability that two or more photons will interact with an agent in a substantially simultaneous manner.
  • optical excitation sources capable of providing high instantaneous irradiance such as for example mode-locked lasers (including titanium: sapphire lasers and Nd: YAG lasers) and amplified mode-locked lasers (including the regeneratively amplified titanium: sapphire laser and chirp-pulse amplified Nd: YAG lasers), can substantially ameliorate the impact of this low efficiency by increasing the incident instantaneous irradiance and thereby dramatically increase the effective efficiency of multi-photon excitation.
  • mode-locked lasers including titanium: sapphire lasers and Nd: YAG lasers
  • amplified mode-locked lasers including the regeneratively amplified titanium: sapphire laser and chirp-pulse amplified Nd: YAG lasers
  • Such lasers typically offer ultrashort pulsed output (having pulse widths ranging from 10 fs to 10 ps) at high pulse repetition rates (ranging from 1 kHz to 100 MHz) with modest average powers (ranging from 1 mW to 10 W).
  • Such output properties facilitate selective, efficient multi-photon excitation, since the high instantaneous irradiance obtainable is capable of stimulating multi-photon processes while the short pulse widths and modest average powers minimize undesirable photo-activation of surrounding media.
  • the efficiency of three-photon excitation for a particular agent may be a factor of 10 7 or more smaller than that achievable with single-photon excitation.
  • sources capable of emitting relatively low energy pulses are optimally suited to photo-activation of diagnostic or imaging agents using two- or more photons under focused illumination conditions
  • sources capable of emitting relatively high energy pulses are optimally suited to photo-activation of such agents using two- or more photons under non-focused illumination conditions, for example to activate agents over a large area or within a large volume of tissue.
  • sources capable of emitting relatively high energy pulses are optimally suited to photo-activation of such agents using two- or more photons under non-focused illumination conditions, for example to activate agents over a large area or within a large volume of tissue.
  • Spatial information concerning the origin of the emitted light from a multi-photon excited diagnostic imaging agent is encoded by and may be correlated to the excitation focus. This is in stark contrast with single-photon excited imaging methods, including those based on photon migration, where the diagnostic imaging signal must be carefully deconvolved from emission light generated along the entire excitation path and from emission produced by scattered excitation light. Hence, it is not necessary for the light emitted from the multi-photon excited diagnostic agent to be detected or imaged directly without scatter. In fact, it is only necessary that a fraction of this emitted light be collected and detected in such a way that the collection and detection process does not distort the correlation between detected signal and emission point of origin.
  • multi-photon imaging requires far less light for detection than is needed for excitation. Hence, a loss of light from scattering during emission is acceptable. Further, the emitted light only comes from a well defined location (the region of excitation), making it easier to selectively detect.
  • the present invention uses agents that emit at relatively long wavelengths. The present invention, however, is not limited to such agents.
  • the transit time for an unscattered, or ballistic, emitted photon (that is, the total transit time from instant of emission to exit from a surface of the specimen) will be approximately 0.3 ns.
  • this transit time could be as high as 3-10 ns.
  • the detector may be located in such a way that it comprises an epi-illumination configuration with the excitation beam, or that it may be located externally to the excitation beam. It is notable that the epi-illumination configuration (or other possible co- linear excitation and detection configurations) minimizes potential parallax losses for detection of surface or near surface objects, but that such configurations are more susceptible to interference from elastically scattered or reflected excitation light.
  • Parallax losses may be minimized for external detection configurations by actively orienting the detection system such that it maintains consistent registry with the point of excitation, by using multiple detection assemblies that are individually optimized for collection of emitted light from different zones within the specimen, or by locating the detection system sufficiently far from the specimen such that parallax losses are minimal.
  • imaging depends on knowing where the illuminated region is, unless the fluorescent agent is controlled as to where it goes, as illustrated in Fig. 12.
  • intensity based methods wherein an image may be constructed by correlating detected intensity of emission with location of excitation for multiple excitation points throughout a specimen.
  • intensity based methods are not always optimal, since they are susceptible to a number of complicating factors, including:
  • a detection approach that is less susceptible to optical heterogeneity of the specimen could be based on measurement of change in excited state lifetime rather than on intensity of emission.
  • Excited state lifetimes are an intrinsic property of the excited state of a molecular agent and its immediate environment (and, notably, are unrelated to the mechanism or method used for photo-activation).
  • the accurate measurement of lifetimes are immune to all but the grossest variations in excitation level and collection efficiency.
  • a convenient way for measuring excited state lifetimes uses phase photometric methods to correlate phase shift between a modulated excitation source and the resultant emission signal to lifetime. Specifically, the preceding discussion on photon transit times implies that phase photometric methods are applicable for imaging in optically dense media, especially for agents with lifetimes in excess of 1-10 ns.
  • diagnostic imaging agents that have emission lifetimes that correlate with form or function within the specimen, such as quenching of fluorescence of an imaging agent in the presence of oxygen or concentration of an imaging agent within a structure, then imaging based on change in lifetime rather than on emission intensity becomes practical.
  • lifetime based methods would have equal applicability to laser scanning microscopy and to remote imaging of extended objects, such as a tumor in a human subject.
  • Appropriate collection devices for transduction of intensity or phase based emission data include, but are not limited to, photomultiplier tubes, microchannel plate devices, photodiodes, avalanche photodiodes, charge coupled devices and charge coupled device arrays, charge injection devices and charge injection device arrays, and photographic film.
  • Noise reduction methods for recovery of multi-photon excited emission from diagnostic imaging agents - modulation and harmonic detection The inherently low efficiency of the multi -photon excitation process can translate into a very high ratio of scattered, unabsorbed excitation light to multi-photon excited emission. Furthermore, the importance of other possible linear interferences attributable to the use of high excitation power levels, including single-photon excited fluorescence of the agent or other species present in the specimen under examination, Raman scatter, and other phenomena, along with the need to eliminate interferences from ambient light and other optical or electronic noise sources, all indicate that a modulated excitation method coupled with appropriate demodulation of the detector signal should provide optimal discrimination against interferences and enhanced recovery of the analytical signal. In fact, background interferences, reported to be disadvantageous in Denk et al.
  • U.S. Patent No. 5,034,613 could be largely circumvented if suitable modulation and demodulation methods were used, including demodulation at the pulse repetition frequency of the laser. In fact, use of such methods would dramatically improve signal-to- noise (SNR) performance in a microscope, such as for example, the microscope disclosed in Denk. In general, modulation can improve detection performance for virtually any measurement in one or more of the following ways:
  • ⁇ SIGNAL ' resulting from the detected optical emission of a photo-activated analyte, is related to a detector input current, ⁇ NPl j T , produced by photons interacting with a detector, multiplied by the input impedance, Z INPUT , and the gain of the detection system, G, according to the following:
  • NOISE noise current
  • B the square root of the electronic or optical bandwidth
  • the signal-to-noise ration may be estimated from the ratio of these two voltages, (K SIGNAL / F NOISE ).
  • K SIGNAL / F NOISE the ratio of these two voltages
  • a standard PMT such as the Hamamatsu R928 (7.4x10 5 A/W radiant anode sensitivity)
  • an optical input at a level of 10 pW produces 7.4 ⁇ A / SIGNAL - I this signal current is converted to voltage in a low noise amplifier having a gain of 100, an input impedance of 50 ⁇ , an input noise level of 5 nV/JHz, and a bandwidth of 1 MHz, the following signals are produced:
  • the overall SNR increases to approximately 1200.
  • the broadband detection scheme will detect this as an additional noise source, while the modulated, bandwidth limited scheme will reject this interference.
  • ambient leakage produces a background signal of 1 ⁇ A on the PMT, which translates to 5 mV of background signal.
  • optical shot noise from this background, B is equal to the square root of the total photons detected, and SNR ⁇ SI(S + B) v2 ; this yields an estimated SNR of approximately 5.7.
  • the SNR for the modulated case is essentially unchanged. This analysis is equally applicable to laser scanning microscopy and to remote imaging of extended objects, such as a tumor in a human subject.
  • one approach for recovery of a pure multi- photon signal utilizes regression of the detected signal at several excitation power levels against the excitation power level, so that the non-linear multi -photon excited component can be extracted mathematically from linear interferences.
  • I L is the instantaneous excitation intensity
  • a is a proportionality constant for various linear effects
  • /? is a proportionality constant for multi-photon excited emission
  • n is the number of photons used for photo-activation.
  • Lytle "Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence," Analytical Chemistry.62 (1990) 2216-2219) discuss second harmonic detection methods useful for the analysis and characterization of chemical samples in test tubes, wherein sinusoidal modulation of the excitation source is used to generate a signal at twice the modulation frequency that is related only to two- photon excited fluorescence. Freeman, however, does not appear to be used for imaging. A lock-in amplifier referenced to the modulation frequency is used to recover the pure two-photon signal at the second harmonic of the modulation frequency.
  • the second harmonic fluorescence signal is only approximately 12% of the total two-photon fluorescence produced, the improved rejection of linear interferences more than compensates for the loss in absolute signal level, resulting in an increase in the overall SNR.
  • the second harmonic detection method is ideally applicable to laser scanning microscopy and to remote imaging of extended objects, such as a tumor in a human subject, as a consequence of its intrinsic efficiency in rejection of scatter and its high data bandwidth potential.
  • n is as defined in Eq. 6.
  • the signal that contains only the n-photon excited response will be at the nth harmonic.
  • modulation methods have important utility in the efficient detection of multi-photon excited phenomena, where they serve to eliminate interferences from ambient and instrumental noise sources as well as from scattering and other phenomena occurring within the specimen undergoing examination.
  • modulation methods For optically dense media, such as human tissue, the extremely high ratio of scattered, unabsorbed excitation light to multi-photon excited emission makes use of such methods vital.
  • Contrast agents in multi-photon excited imaging - endogenous and exogenous agents The foregoing discussion has shown that multi-photon excitation can be used to effect important improvements in the specificity and depth of penetration for optically excitable molecular agents present in optically dense media, and that detection performance can be improved by use of encoding and decoding methods on the respective excitation and detection processes.
  • the exceptional spatial localization of excitation possible when using multi-photon methods can be harnessed to significantly improve contrast in the point of excitation. Once this localized excitation is effected, the analytic light thereby emitted may be detected using a variety of detection means.
  • this excitation point is moved relative to the specimen under examination, for example by scanning the position of the focus relative to the specimen or by scanning the position of the specimen relative to the focus, then a two- or three-dimensional image of the specimen can be generated by making a correlation between the location of the excitation point and the emitted light thereby produced.
  • Useful contrast in this image also depends on the existence of differences in the concentration or local environment of the molecular agent or agents responsible for emission. These agents may be endogenous or exogenous to the specimen, and imaging is ultimately based on contrasts in their localized emission properties that can be correlated to heterogeneity in structure or function within the specimen. Hence, the role of these contrast agents in non-linear diagnostics or imaging is also important.
  • chromophoric agents may be useful for diagnostics or imaging, particularly of diseased tissue. Because of structural or physiological differences between diseased and non-diseased tissues, between various internal substructures and organs in higher animals, or between different ranges of healthy or sub-healthy tissues, the concentration or local environment of natural chromophoric agents, such as aromatic amino acids, proteins, nucleic acids, cellular energy exchange stores (such as adenosine triphosphate), enzymes, hormones, or other agents, can vary in ways that are useful for probing structural or functional heterogeneity. Thus, these endogenous indicators of heterogeneity can be probed non-invasively using multi-photon excitation.
  • exogenous agents semi-selectively partition into specific tissues, organs, or other structural units of a specimen following administration.
  • the route for administration of these agents is typically topical application or via systemic administration.
  • these agents will partition into or otherwise become concentrated on or in the structures of interest, or may be excluded preferentially from these structures. This concentration is possibly a consequence of isolated topical application directly onto a superficial structure, or through intrinsic differences in the physical or chemical properties of the structure which lead to partitioning of the agent into the structure. Contrasts between areas of high concentration and low concentration can thereby be used as a basis for probing structural or physiological heterogeneity.
  • exogenous agents may permeate throughout a specimen; if their emission properties, such as chromatic shift, quenching, or lifetime, are sensitive to physiological heterogeneity, then these parameters of the contrast agent can be used as the basis for contrast in imaging.
  • a molecular diagnostic or contrast agent that works well under single-photon excitation conditions may be expected to exhibit similar behavior under multi-photon excitation conditions.
  • any contrast agent that is useful for single-photon excitation can be used with multi-photon excitation, where the enhanced control over the site of excitation will serve to improve resolution of the image.
  • Appropriate contrast agents include many molecular agents used as biological dyes or stains, as well as those used for photodynamic therapy (PDT).
  • Standard PDT agents have tissue specificities that in general are based on the combined chemical and physical properties of the agent and the tissue, such as a cancerous lesion. These agents are efficient absorbers of optical energy, and in many cases are luminescent. Examples of these agents include, but are not limited to: various psoralen derivatives; various porphyrin and hematoporphyrin derivatives;
  • agents will in general become accumulated either at or near a point of application or semi-selectively within a specific tissue due to differences in the physical or chemical properties of the tissue which lead to partitioning of the PDT agent into the tissue. Once accumulated, such agents will be susceptible to multi-photon excitation, and their luminescent or other emission properties can then be used for acquisition of diagnostic or imagery data.
  • Other photo-active agents that absorb light and are capable of subsequent energy transfer to one or more other agents may also be used, either alone or in conjunction with one or more responsive agents that are capable of accepting this transferred energy and transforming it into a radiative emission.
  • ⁇ contrast agents derive target specificity based on chemical or physical affinity for specific tissues. In this way, contrast agents partition into or otherwise become concentrated on or in tissues of interest. Unfortunately, this target specificity is usually not perfect. As a result, an improved method for increasing specificity in the targeting of agent destination is desired.
  • One embodiment of the present invention to achieve such improvement in specificity is based on utilization of specific biological signatures of structure, function, or disease. For example, by coupling anti- sense oligonucleotide agents to one or more photo-active moieties, such as FITC, new biogenic contrast agents are created that are capable of selectively tagging only specific cells, such as cancerous cells, that contain complementary genetic encoding.
  • the basic approach is easily extended to numerous genetic-based diseases or other disorders by changing the oligomeric code used for the biogenic probe.
  • Employment of multi-photon excitation enables this powerful approach to be applied using the combined bio-specificity of the biogenic probe and the high spatial localization inherent to the multi- photon photo-activation process.
  • very high contrast very high resolution imaging becomes possible at the genetic level using agents that are specifically targeted for a particular organ, tissue, or lesion.
  • An optimal design for biogenic probes utilizes one or more photo-active moieties that have emission properties that change upon complexation between the biogenic agent and the target site. Specifically, changes in emission wavelength or lifetime upon complexation can be used to increase sensitivity of the general method, since such changes will help to increase contrast between areas containing complexed agent and those containing uncomplexed agent.
  • An example is a biogenic agent based on a photo-active moiety that is quenched until complexation occurs, upon which occurrence emission becomes unquenched.
  • Another example is an agent based on an intercalating photo-active moiety, such as psoralen, that is tethered to an anti-sense genetic sequence; upon complexation between the anti-sense sequence and its target sequence, intercalation of the photo-active moiety is enabled that leads to a chromatic shift in emission properties of the photo-active moiety.
  • an intercalating photo-active moiety such as psoralen
  • agent specificity based on antigen-antibody methods where an antibody probe is coupled to a photo-active group, provides a powerful new way for diagnosis of disease and infection.
  • Additional ways for achieving biospecificity in agent targeting include, but are not limited to, use of DNA, RNA, amino acids, proteins, antibodies, ligands, haptens, carbohydrate receptors or complexing agents, protein receptors or complexing agents, lipid receptors or complexing agents, chelators, encapsulating vehicles, nanoparticles, short-or long-chain aliphatic or aromatic hydrocarbons, including those containing aldehydes, ketones, alcohols, esters, amides, amines, nitriles, azides, or other hydrophilic or hydrophobic moieties.
  • One of the preferred embodiments of the present invention is to employ the output of a high instantaneous irradiance, ultrashort pulsed source, such as for example a mode- locked titanium-sapphire laser or a regeneratively amplified titanium: sapphire laser, to induce multi-photon photo-activation of one or more endogenous or exogenous photoactive agents.
  • a high instantaneous irradiance, ultrashort pulsed source such as for example a mode- locked titanium-sapphire laser or a regeneratively amplified titanium: sapphire laser
  • One specific preferred embodiment of the subject invention is to employ the output of a NIR source to induce multi-photon photo-activation of endogenous or exogenous diagnostic or imaging agents present in a specimen using light at a wavelength approximately twice, or more, than necessary for conventional single-photon photo- activation.
  • This preferred embodiment is shown in FIGURE 10.
  • the NIR source 108 produces a beam of NIR radiation 1 10 consisting of a rapid series of high peak power pulses of NIR radiation, and may consist, for example, of a standard commercially available mode-locked titanium: sapphire laser capable of outputting mode-locked pulses with durations ⁇ 200 fs and pulse energies of up to about 20 nJ at pulse repetition frequencies in excess of about 10 MHz.
  • Such a source produces a quasi-continuous beam of light having a relatively low average power (up to several watts) but high peak power (on the order of 100 kW) that is continuously tunable over a NIR wavelength band from approximately 690-1080 nm.
  • the pulse train emitted by the NIR source 108 constitutes a beam of NIR radiation 1 10 that is easily focused using standard optical means, such as reflective or refractive optics 1 12.
  • the focused NIR beam 1 14 can then be directed onto a specimen 1 16 to be imaged.
  • Multi-photon photo-activation e.g. two photo, three photon
  • the diagnostic or imaging agent will be substantially limited to the focal zone 1 18 of the focused beam 1 14 due to the high instantaneous level that is only present at the focus.
  • Excitation light that is scattered 120 by the specimen 1 16 will not have a sufficient instantaneous irradiance level for significant photo-activation of any diagnostic or imaging agent that may be present in areas outside of the focal zone 1 18.
  • Light 122 emitted by diagnostic or imaging agent molecules present in the focal zone 1 18 will exit (light is emitted isotropically during fluorescence or phosphorescence) the focal zone 1 18 in a substantially isotropic manner.
  • a portion of the emitted light 124 is captured by a detection apparatus 126, such as a photomultiplier tube, that is mounted at a position inside or outside of the specimen 116.
  • This detection apparatus 126 is fitted with a wavelength selection apparatus 128, such as an optical bandpass filter, that serves to pre- process the captured portion of the emitted light 124 in such a way that the selection apparatus 128 rejects a major fraction of the elastically scattered light while passing a major fraction of light at the wavelength or wavelengths corresponding to that which is principally characteristic of emission from the diagnostic agent.
  • the signal thus issued 130 from the detection apparatus 126 is captured by a processor 132, the primary purpose of which is to record emission response from diagnostic or imaging agent as a function of location of the focal zone 118.
  • a complete image of the specimen 116 may be obtained by examining the contents of the processor 132 as a function of location of the focal zone 118. This image may be used to identify zones of interest 134, such as subcutaneous tumors or other diseased area.
  • modulation apparatus may be incorporated into the general embodiment shown in FIGURE 10.
  • Such modulation apparatus may be used to improve overall performance of the imaging system, such as to improve rejection of environmental or instrumental noise sources, to enable recovery of pure multi-photon excited emission, or to facilitate detection of emitted light using phase photometric approaches.
  • FIGURE 1 1 shows a modulator 126, such as an electro-optic or acousto-optic modulator, a chopper, or other apparatus, located so as to interact with the beam of NIR radiation 110 emitted by the NIR source 108 that can be used to encode the beam of NIR radiation 1 10 with a modulation pattern that is registered to the output of a modulator driver 138 that provides a drive signal 140 to the modulator 136.
  • the modulated beam of NIR radiation 142 thereby produced is then directed onto the specimen 1 16 as described previously for FIGURE 10.
  • the multi-photon excited emitted light 144 thereby produced will exit the focal zone 1 18 in an essentially isotropic manner.
  • this emitted light 144 will exhibit a modulation that is essentially synchronous with the modulation of the modulated beam of NIR radiation 142, which in turn is synchronous with the drive signal 140 issued by the modulator driver 138.
  • a portion of the modulated emitted light 146 is captured by a detection apparatus 126, such as a photomultiplier tube, that is mounted at a position inside or outside of the specimen 1 16.
  • This detection apparatus 126 is fitted with a wavelength selection apparatus 128, such as an optical bandpass filter, that serves to process the captured portion of the modulated emitted light 146 in such a way that the selection apparatus 128 rejects a major fraction of the elastically scattered light while passing a major fraction of light at the wavelength or wavelengths corresponding to that which is principally characteristic of emission from the diagnostic agent.
  • the modulated signal thus issued 148 from the detection apparatus 126 is captured by a processor apparatus 150.
  • the processor 150 serves two primary purposes, first to demodulate the modulated signal thus issued 148 from the detection apparatus 126 using a demodulation reference output 152 issued by the modulator driver 138, and second to record the demodulated emission response from the diagnostic or imaging agent as a function of location of the focal zone 118.
  • a complete image of the specimen 1 16 may be obtained by examining the contents of the processor 150 as a function of location of the focal zone 1 18. This image may be used to identify zones of interest 134, such as subcutaneous tumors or other diseased areas.
  • the pulse frequency of the NIR source 108 can be used as a modulation source itself, producing a modulated beam of NIR radiation 142 at such pulse frequency.
  • the source 108 serves the role of modulator 136 and modulator driver 138, and provides a source of the demodulation reference output 152 for the processor 150. As a result, no separate modulator or driver is needed.
  • an unfocused beam of NIR radiation may be used to illuminate superficial features of a specimen to provide a direct imaging mode of detection.
  • a NIR source such as for example a mode-locked titanium: sapphire laser
  • the NIR Source 108 produces a beam of NIR radiation 1 10 consisting of a rapid series of high peak power pulses of NIR radiation.
  • This beam is modulated using a modulator 136 located so as to interact with the beam of NIR radiation 1 10 emitted by the NIR source 108.
  • This modulator 136 encodes the beam of NIR radiation 1 10 with a modulation pattern that is registered to the output ole of a modulator driver 138 that provides a drive signal 140 to the modulator 136.
  • the modulated beam of NIR radiation 142 thereby produced is then defocused using standard optical apparatus, such as reflective or refractive optics 154, to produce a divergent excitation beam 156 that is directed onto a specimen 116 to be imaged.
  • Multi-photon photo-activation of diagnostic or imaging agent present on or near the surface of the specimen 116 produces modulated multi-photon excited emitted light 144 having a modulation that is essentially synchronous with the modulation of the modulated beam of NIR radiation 142, which in turn is synchronous with the drive signal 140 issued by the modulator driver 138.
  • a portion of the modulated emitted light 146 is captured by an imaging detection apparatus 158, such as a charge coupled device array, that is mounted at a position outside of the specimen 116.
  • This imaging detection apparatus 158 is fitted with a wavelength selection apparatus 128, such as an optical bandpass filter, that serves to process the captured portion of the modulated emitted light 146 in such a way that the selection apparatus 128 rejects a major fraction of the elastically scattered light while passing a major fraction of light at the wavelength or wavelengths corresponding to that which is principally characteristic of emission from the diagnostic agent.
  • the modulated signal thus issued 160 from the imaging detection apparatus 158 is captured by a processor 162.
  • the processor 162 serves two primary purposes, first, to demodulate the modulated signal thus issued 160 from the imaging detection apparatus 158 using a demodulation reference output 152 issued by the modulator driver 138, and second, to record the demodulated emission response from the diagnostic or imaging agent as a function of the location of emission.
  • this alternate embodiment enables direct videographic imaging of surface features
  • the pulse frequency of the NIR source 108 as a modulation source itself, producing a modulated beam of NIR radiation 142 at such pulse frequency.
  • the source 108 serves the role of modulator 136 and modulator driver 138, and provides a source of the demodulation reference output 152 for the processor 150. As a result, no separate modulator or driver is needed.
  • optical sources are applicable, alone or in combination, such as continuous wave and pulsed lamps, diode light sources, semiconductor lasers; other types of gas, dye, and solid-state continuous, pulsed, or mode-locked lasers, including: argon ion lasers; krypton ion lasers; helium-neon lasers; helium-cadmium lasers; ruby lasers; Nd:YAG, Nd:YLF, Nd: YAP, Nd: YV04, Nd:Glass, and Nd:CrGsGG lasers; Cr:LiSF lasers; Er:YAG lasers: F-center lasers; Ho:YAF and Ho:YLF lasers; copper vapor lasers; nitrogen lasers; optical parametric oscillators, amplifiers and generators; regeneratively amplified lasers; chirped-pulse amplified lasers; and sunlight.
  • Such sources are capable of producing continuous or pulsed beams of light, for example with pulse repetition frequencies ranging from less

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Abstract

L'invention porte sur un procédé et un appareil utilisé dans le diagnostic ou l'imagerie d'un volume déterminé de matière, ce procédé consistant à traiter ce volume déterminé de matière avec une lumière suffisante de façon à faciliter l'excitation multiphotonique d'au moins un agent photosensible contenu sur ou dans un volume déterminé de matière. Au moins un agent photosensible est excité dans le volume déterminé de matière, ce qui permet la génération d'un signal optique qui est capturé sous forme électronique pour le diagnostic et l'imagerie du volume déterminé de matière.
EP00937691A 1999-05-26 2000-05-23 Procedes et appareil ameliores pour la photo-excitation multiphotonique et la detection d'agents moleculaires Withdrawn EP1187555A4 (fr)

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TW487565B (en) 2002-05-21
JP2003500094A (ja) 2003-01-07
WO2000071028A1 (fr) 2000-11-30

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