Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/0613—Apparatus adapted for a specific treatment
  • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0066—Psoralene-activated UV-A photochemotherapy (PUVA-therapy), e.g. for treatment of psoriasis or eczema, extracorporeal photopheresis with psoralens or fucocoumarins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/008—Two-Photon or Multi-Photon PDT, e.g. with upconverting dyes or photosensitisers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0039—Coumarin dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
  • A61K49/0043—Fluorescein, used in vivo
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods
  • A61B2017/00017—Electrical control of surgical instruments
  • A61B2017/00022—Sensing or detecting at the treatment site
  • A61B2017/00057—Light
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors

Definitions

• the present invention relates generally to methods and apparatus for remotely effecting spatially-selective photo-activation of one or more molecular agents and for improving the detection of the diagnostic signals thereby produced.
• the method taught for effecting photo- activation utilizes the special properties of non-linear optical excitation for promoting an agent from one molecular energy level to another with a high degree of spatial and molecular specificity.
• the special features of this method are applicable for activation of various endogenous and exogenous imaging agents, and in particular afford distinct advantages in the diagnosis of diseases in humans and animals.
• use of non-linear excitation methods facilitate controlled activation of diagnostic agents in deep tissue using near infrared to infrared radiation, which is absorbed and scattered to a lesser extent than methods and radiations currently used. Combination of these non-linear excitation methods with advanced signal encoding and processing methods greatly increases sensitivity in the detection of diagnostic signals.
• the desired improvements in activation include enhancements in spatial or temporal control over the location and depth of activation, reduction in undesirable activation of other co-located or proximal molecular agents or structures, and increased preference in the activation of desirable molecular agents over that of undesirable molecular agents.
• Various linear and non-linear optical methods have been developed to provide some such improvements for some such agents under very specialized conditions. However, in general the performance and applicability of these methods have been less than desired.
• improved photo-activation methods are needed that may be used to selectively photo-activate a variety of molecular diagnostic agents while providing improved performance in the control of application of this photo-activation.
• linear optical excitation methods have been used extensively as a means for achieving semi-selective activation of molecular diagnostic agents.
• Linear optical excitation occurs when a target agent, such as a molecular diagnostic agent, undergoes a specific photo-chemical or photo- physical process, such as fluorescent emission, upon absorption of energy provided by a single photon.
• a target agent such as a molecular diagnostic agent
• a specific photo-chemical or photo- physical process such as fluorescent emission
• Vo-Dinh "Spectroscopic Diagnosis of Esophageal Cancer: New Classification Model, Improved Measurement System," Gastrointestinal Endoscopy, 41 (1995) 577-581) teach of the use of similar linear optical probe methods for detection of diseased tissues in humans; however, this approach is also plagued by less than desirable penetration depth and is limited to detection of superficial lesions due to scatter and absorption of the incident probe radiation. Also, because this type of excitation is linearly related to excitation power, such methods provide no effective means for limiting the location of probe excitation along the optical path.
• non-linear optical excitation methods have been employed in an effort to achieve specific improvements in the selectivity of photo-activation for certain applications, and to address many of the limitations posed by linear excitation methods.
• the non-linear process consisting of simultaneous absorption of two photons of light by a molecule to effect excitation equivalent to that resulting from absorption of a single photon having twice the energy of these two photons is very well known, as are the specific advantages of this process in terms of reduced absorption and scatter of excitation photons by the matrix, enhanced spatial control over the region of excitation, and reduced potential for photo-chemical and photo- physical damage to the sample.
• Excitation sources ranging from single-mode, continuous wave (CW) lasers to pulsed Q-switched lasers having peak powers in excess of 1 GWhave been employed for numerous examples of two-photon excitation methods.
• CW continuous wave
• Excitation sources ranging from single-mode, continuous wave (CW) lasers to pulsed Q-switched lasers having peak powers in excess of 1 GWhave been employed for numerous examples of two-photon excitation methods.
• Wirth and Lytle M.J. Wirth and F.E. Lytle, "Two-Photon Excited Molecular Fluorescence in Optically Dense Media," Analytical Chemistry, 49
• Wirth teaches a method for achieving extremely high spatial selectivity in the excitation of target molecular agents using a microscopic imaging system.
• Fluorescent light then travels back along that same optical path, through an objective lens, a series of mirrors and ultimately a photomultiplier tube. Hence, light is merely focussed on the object plane of a slide and reflected back.
• the special properties of non-linear two-photon excitation are utilized to substantially limit excitation and subsequent detection of the fluorescent signal thus produced to a confocal region occurring at the focus of an objective lens, thereby enhancing contrast in three-dimensional imaging by sharply controlling the depth of focus.
• Emitted fluorescent light is collected by the excitation objective using an epi-illumination configuration. Control of photo-excitation for generation of luminescence -based images at the cellular and sub-cellular level is shown in target samples mounted on a stage.
• Denk teaches that reduction in photo-induced necrosis of cells located at the focal plane is a primary benefit of this microscopy approach, based on the replacement of ultraviolet excitation radiation with less damaging near infrared excitation radiation.
• Denk et al. W. Denk, D.W. Piston and W.W. Webb, "Two- Photon Molecular Excitation in Laser-Scanning Microscopy," in Handbook of Biological Confocal Microscopy, Second Edition, J.B. Pawley, ed., Plenum Press, New York, 1995, pp. 445-458) an external whole area detection method is taught for collection of microscopic imaging data produced from two-photon excited fluorescent tags.
• two-photon excitation of fluorescence can be used under laboratory conditions to excite molecular fluorophors using light at approximately twice the wavelength of 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.
• an object of the present invention to provide a general method for achieving selective photo-activation of one or more molecular agents with a high degree of spatial control.
• the present invention generally provides for a method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photoactive molecular agent.
• the method comprises the steps of treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent contained in the particular volume of the plant or animal tissue, photo-activating at least one of the at least one photo-active molecular agent in the particular volume of the plant or animal tissue, thereby producing at least one photo-activated molecular agent, wherein the at least one photo- activated molecular agent emits energy, detecting the energy emitted by the at least one photo-activated molecular agent, and producing a detected energy signal which is characteristic of the particular volume of plant or animal tissue.
• the present invention also provides a method for the imaging of a particular volume of material, wherein the material contains at least one photo-active molecular agent.
• the light sufficient to promote a simultaneous two-photon excitation of the at least one photo-active molecular agent is laser light. It is also preferred that the light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent is a focused beam of light, and more preferred that the focused beam of light is focused laser light.
• Another preferred embodiment of the present invention further includes a first step of treating the material, plant tissue or animal tissue with at least one photoactive molecular agent, wherein the particular volume of the material, plant tissue or animal tissue retains at least a portion of the at least one photo-active molecular agent.
• the at least one photo-active molecular agent is selected from the group consisting of 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), angelicin (isopsoralen), 5-methylangelicin (5-MIP), 3-carboxypsoralen, porphyrin, haematoporphyrin derivative (HPD), photofrin II, benzoporphyrin derivative (BPD), protoporphyrin IX (PpIX), dye haematoporphyrin ether (DHE), polyhaematoporphyrin esters (PHE), 13,17-N,N,N-dimethylethylethanolamine ester of protoporphyrin (PH100
• the at least one photo-active molecular agent is at least one biogenic photo-active molecular agent that is specific to a particular material or tissue within the particular volume of material, plant tissue or animal tissue, even more preferred that the at least one biogenic photo-active molecular agent includes a segment selected from the group consisting of DNA, RNA, amino acids, proteins, antibodies, ligands, haptens, carbohydrate receptors or complexing agents, lipid receptors or complexing agents, protein receptors or complexing agents, chelators, and encapsulating vehicles and yet further more preferred that the at least one biogenic photo-active molecular agent further includes a segment which is photo-activated when subject to light sufficient to promote a simultaneous two-photon excitation.
• the step of treating the particular volume of the material, plant tissue or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the at least one photo- active molecular agent contained in the particular volume of the material, plant tissue or animal tissue further includes the step of modulating light from a light source with a particular type of modulation , thereby producing a modulated light, and the step of treating the particular volume of the material, plant tissue or animal tissue with the modulated light sufficient to promote a simultaneous two-photon excitation of the at least one photo-active molecular agent contained in the particular volume of the material, plant tissue or animal tissue.
• the present invention further include the steps of demodulating the dectected energy signal with the particular type of modulation, and producing a demodulated energy signal which is characteristic of the particular volume of the material, plant tissue or animal tissue. It is more preferred that the step of demodulating the dectected energy signal with the particular type of modulation includes demodulating the detected energy signal at a frequency twice that of the particular type of modulation, thereby detecting the second harmonic of the particular type of modulation. It is also more preferred that the demodulated energy signal which is characteristic of the particular volume of the material, plant tissue or animal tissue represents a change in lifetime of at least one photo-activated molecular agent present in the particular volume of the material, plant tissue or animal tissue.
• FIGURE 1 shows example energy level diagrams for linear and non-linear optical excitation
• FIGURE 2 shows the relationships between incident power distribution and excitation efficiency for single-photon and two-photon excitation
• FIGURE 3 shows an example absorption spectrum for animal tissue covering the ultraviolet to near infrared spectral region
• FIGURE 4 shows a scattering spectrum for animal tissue covering the ultraviolet to near infrared spectral region
• FIGURE 5 shows the general trends in optical absorption and scattering properties of tissue for incident short wavelength and long wavelength light
• FIGURE 6 compares optically-induced excitation regions in tissue when single- photon and two-photon excitation methods are used
• FIGURE 7 shows typical properties of linear excitation of a diagnostic agent in solution
• FIGURE 8 shows typical properties of non-linear excitation of a diagnostic agent in solution
• FIGURE 9 shows a photograph of two-photon excited fluorescence of the dye molecule coumarin 480 distributed evenly throughout a tissue phantom;
• FIGURE 11 shows a diagram of a specific preferred embodiment of the subject invention for imaging endogenous or exogenous diagnostic imaging agents
• the confocal region, Z c is defined as the zone extending a distance of 2 ⁇ w 0 2 1 ⁇ , where w> 0 is the diameter of the minimum beam waist and ⁇ is the wavelength of the optical radiation.
• excitation occurs substantially along the entire optical path, making spatial localization of excitation considerably less defined.
• use of the two-photon excitation process greatly increases the resolution of excitation along the optical path.
• scatter and absorption of the excitation energy is greatly reduced. For thick, optically dense samples, such as human tissue, this means that two-photon excitation is possible at depths considerably greater than is possible using linear excitation methods.
• the light emitted from the diagnostic agent it is not necessary for the light emitted from the diagnostic agent to be detected or imaged directly without scatter, since spatial information concerning the origin of the emitted light is encoded by and may be correlated to the excitation focus. By moving the location of this focus relative to the specimen, a two- or three-dimensional image of the emitted light may be developed. Also, by modulating the excitation light and using an appropriate demodulation method on the detection apparatus, rejection of scattered excitation light and other interferences may be markedly improved.
• This internal conversion 16 is typically very fast, occurring on a time scale on the order of 10 "12 to 10 "15 sec.
• the excited molecule can undergo further relaxation, such as through collisional deactivation 20, to return to the initial, first energy level 6.
• Alternative relaxation processes include fluorescent emission of a photon 21, which occurs directly from S t to S 0 , and phosphorescence, which occur following intersystem crossing 22 from a singlet state to a triplet state 10.
• singlet to singlet electronic transitions such as those shown for S t ⁇ S 0 , constitute quantum mechanically allowed transitions according to the Pauli exclusion principle.
• transitions from a singlet to a triplet state 10 such as S t ⁇ T 1?
• An example of single-photon excitation to an allowed energy level 2 is promotion of the dye molecule coumarin from a ground electronic state to an excited electronic state through the absorption of a single photon 4 at 400 nm, followed by internal conversion 16 and subsequent fluorescent emission of a photon 21 at 480 nm.
• the probability of excitation is linearly related to the power of the incident optical radiation, thus single-photon excitation to an allowed energy level 2 is referred to as a linear excitation process.
• simultaneous two-photon excitation to an allowed energy level 34 occurs upon simultaneous absorption of a first of two photons 36 and a second of two photons 38.
• the combined energy of the first of two photons 36 and the second of two photons 38 is sufficient to promote the molecule from a first allowed energy level 6 to a second allowed energy level 8.
• the individual energies of neither the first of two photons 36 nor the second of two photons 38 is sufficient to directly promote this or any other allowed electronic transition.
• the first of two photons 36 promotes the molecule to a very short lived virtual energy level 30. This is the same virtual energy level as that shown in the second Jablonski diagram.
• the second of two photons 38 immediately promotes the molecule to a second allowed electronic energy level 8.
• the result is excitation that is equivalent to that achieved using linear single-photon excitation to an allowed energy level 2. Note that the first of two photons 36 and the second of two photons
• FIGURE 4 shows a scattering spectrum 66 for animal tissue, such as human dermis or liver, under similar conditions.
• FIGURE 3 demonstrates how higher-energy photons 60, such as those used for linear excitation of diagnostic agents, may experience considerably greater tissue absorption than lower-energy photons 62, such as those used for non-linear excitation of diagnostic agents.
• human skin strongly absorbs higher-energy photons 60 at 400 nm, but is relatively transparent to lower- energy photons 62 at 800 nm. This is a consequence of the relatively high natural absorbance of higher-energy photons 60, having ultraviolet or visible wavelengths, by pigments, proteins, and genetic materials, among other natural components, of skin.
• FIGURE 6 compares the penetration depth and spatial localization characteristics expected for single-photon excitation 86 and simultaneous two-photon NIR excitation 88 of imaging agents present in a subcutaneous tumor 90.
• single-photon excitation 86 produces an excitation zone 92 that extends substantially along the entire optical path and has no significant specificity. Note that the efficiency of single-photon excitation 86 will vary along the optical path due to absorption and scatter, being highest 94 near the point of introduction of optical radiation and dropping off rapidly 96 along the optical path. Note also that the potential for induction of collateral photodamage will follow this same trend. Hence, single-photon excitation produces an extended excitation zone 92 that cannot be effectively limited to a finite volume, especially in deep tissues. Also, significant collateral damage can occur throughout surrounding tissues 98, and especially in surface tissues 100. If the single-photon excitation 86 is focussed, the excitation zone 92 will be slightly enhanced at the focus 102.
• laser radiation at 730 nm was used to excite a dilute solution of the dye molecule coumarin 480 in methanol.
• the NIR output of a mode-locked titanium:sapphire laser which emitted a continuous train of 730 nm wavelength, ⁇ 200 fs pulses of light at a 78 MHz pulse repetition frequency in a beam approximately 1 mm in diameter, was focused through the same 20x microscope objective into a cuvette containing the dye solution.
• FIGURE 8 clearly shows that fluorescence response from the dye molecule is limited to the focus of the NIR beam. Because of the quadratic relationship between two-photon excitation and instantaneous laser power, stimulation at positions along the excitation path prior to and following the focus is negligible.
• NIR output of the mode-locked titanium:sapphire laser which emitted a continuous train of 730 nm wavelength, ⁇ 200 fs pulses of light at a 78 MHz pulse repetition frequency in a beam approximately 1 mm in diameter, was expanded to produce a collimated beam approximately 50 mm in diameter using a beam expanding telescope.
• This expanded beam was then focused into the gelatin block using a 100 mm focal length, 50 mm diameter biconvex singlet glass lens.
• the gelatin block was then positioned such that the focus of this 100-mm f.l. lens fell at a position 40 mm into the block.
• FIGURE 9 clearly shows that fluorescence response from the coumarin 480 is only stimulated at the focus of the NIR beam.
• FIGURE 10 Similar results are obtained if an equivalent excitation process is applied to a labeled tumor specimen, as shown in FIGURE 10.
• pulsed or mode-locked lasers have applicability, including: argon ion lasers; krypton ion lasers; helium-neon lasers; helium-cadmium lasers; ruby lasers; Nd:YAP, Nd:YV04, Nd:Glass, and Nd:CrGsGG lasers; regeneratively amplified lasers; Cr:LiSF lasers; Er:YAG lasers; F-center lasers; Ho:YAF and Ho:YLF lasers; and copper vapor lasers.
• Various continuous wave lasers may also be used, but with considerably lower efficiency than that achieved using pulsed lasers.
• 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; for a highly scattered emitted photon, this transit time could be as high as 3-10 ns.
• the epi-illumination configuration 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.
• 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:
• 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.
• the observed signal voltage, K SIGNAL is related to a detector input current, impm produced by photons interacting with a detector, multiplied by the input impedance, Z ⁇ ., and the gain of the detection system, G, according to the following:
• J NOISE * mav be approximated by the product of the noise current, I NOISE» tne input impedance, the square root of the electronic or optical bandwidth, B, of the detection system, and the gain, according to following:
• V slGSAL I J NOISE the ratio of these two voltages
• the overall SNR increases to approximately 1200.
• the overall increase in SNR more than compensates for this loss.
• 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
• Lytle "Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence," Analytical Chemistry, 62 (1990) 2216-2219) teach of second harmonic detection methods useful for the analysis of chemical samples, 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. 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. While 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.
• modulation methods including those based on second-harmonic detection, have important utility in the efficient detection of two-photon excited fluorescence, 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 including those based on second-harmonic detection, have important utility in the efficient detection of two-photon excited fluorescence, 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.
• optically dense media such as human tissue
• the extremely high ratio of scattered, unabsorbed excitation light to two-photon excited fluorescence emission makes use of such methods vital.
• employment of modulation methods as described here will always be advantageous.
• non-linear two-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 two-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 caused to move 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, it is important to also carefully consider the role of these contrast agents in non-linear diagnostic imaging.
• chromophoric agents may be useful for diagnostic 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 two- 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 may be 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. Contrast 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.
• any contrast agent that is useful for single-photon excitation can be used with two-photon excitation, where the enhanced control over 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. For example,
• phthalocyanine derivatives including chloroaluminum-sulfonated phthalocyanine [CASPc]; chloroaluminum phthalocyanine tetrasulfate [AlPcTS]; mono-, di-, tri- and tetra-sulphonated aluminum phthalocyanines [including AlSPc, AlS2Pc, AlS3Pc and AlS4Pc]; silicon phthalocyanine [SiPc IV]; zinc(II) phthalocyanine [ZnPc]; bis(di-isobutyl octadecylsiloxy)silicon 2,3- naphthalocyanine [isoBOSINC]); and Ge(IV)-octabutoxy-phthalocyanine;
• photoactive 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.
• Biogenic contrast agents in two-photon excited imaging Under ideal conditions, standard 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. In fact, it is desirable to have an improved method for increasing specificity in the targeting of agent destination. A means for achieving 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.
• photo-active moieties such as FITC
• 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 means for diagnosis of disease and infection.
• Additional means for achieving biospecificity in agent targeting include, but are not limited to, use of ligands, haptens, carbohydrate, lipid, or protein receptors or complexing agents, chelators, and encapsulating vehicles, such as liposomes, fullerenes, crown ethers,and cyclodextrins.
• the NIR Source 108 produces a beam of NIR radiation 110 consisting of a rapid series of high peak power pulses of NIR radiation.
• standard commercially available mode-locked titanium: sapphire lasers are capable of outputting mode-locked pulses with durations ⁇ 200 fs and pulse energies of about
• the pulse train emitted by the NIR source 108 constitutes a beam of NIR radiation 110 that is easily focussed using standard optical means, such as reflective or refractive optics 112.
• the focused NIR beam 114 can then be directed onto a specimen 116 to be imaged.
• Simultaneous two-photon photo-activation of the diagnostic imaging agent will be substantially limited to the confocal region 118 of the focused beam 114 due to the high instantaneous irradiance level that is only present at the focus.
• Excitation light that is scattered 120 by the specimen 116 will not have a sufficient instantaneous irradiance level for significant excitation of any diagnostic imaging agent that may be present in areas outside of the confocal region 118.
• Light emitted 122 by diagnostic imaging agent molecules present in the confocal region 118 will exit the confocal region 118 in a substantially isotropic manner.
• a portion of the emitted light 124 is captured by a detection means 126, such as a photomultiplier tube, that is mounted at a position inside or outside of the specimen 116.
• This detection means 126 is fitted with a wavelength selection means 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 means 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 means 126 is captured by a processor means 132, the primary purpose of which is to record emission response from diagnostic imaging agent as a function of location of the confocal region 118.
• a complete image of the specimen 116 may be obtained by examining the contents of the processor means 132 as a function of location of the confocal region 118. This image may be used to identify zones of interest 134, such as subcutaneous tumors or other diseased areas.
• a modulation means may be incorporated into the general embodiment shown in FIGURE 11; such modulation means 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 two-photon excited emission at the second harmonic, or to facilitate detection of emitted light using phase photometric approaches.
• FIGURE 12 shows that a modulator means 136, such as an electro-optic or acousto- optic modulator, a chopper, or other means, located so as to interact with the beam of NIR radiation 110 emitted by the NIR source 108 can be used to encode the beam of NIR radiation 110 with a modulation pattern that is registered to the output of a modulator driver 138 that provides a drive signal 140 to the modulation means 136.
• the modulated beam of NIR radiation 142 thereby produced is then directed onto the specimen 116 as described previously for FIGURE 11.
• the two-photon excited emitted light 144 thereby produced will exit the confocal region 118 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 means 126, such as a photomultiplier tube, that is mounted at a position inside or outside of the specimen 116.
• This detection means 126 is fitted with a wavelength selection means 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 means 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 means 126 is captured by a processor means 150.
• the processor means 150 serves two primary purposes, firstly to demodulate the modulated signal thus issued 148 from the detection means 126 using a demodulation reference output 152 issued by the modulator driver 138, and secondly to record demodulated emission response from the diagnostic imaging agent as a function of location of the confocal region 118.
• a complete image of the specimen 116 may be obtained by examining the contents of the processor means 150 as a function of location of the confocal region 118. This image may be used to identify zones of interest 134, such as subcutaneous tumors or other diseased areas.
• an unfocused beam of NIR radiation may be used to illuminate superficial features of a specimen to provide a direct imaging means of detection.
• a NIR source such as the mode-locked titanium: sapphire laser
• the NIR Source 108 produces a beam of NIR radiation 110 consisting of a rapid series of high peak power pulses of NIR radiation.
• This beam is modulated using a modulator means 136 located so as to interact with the beam of NIR radiation 110 emitted by the NIR source 108.
• This modulator means 136 encodes the beam of NIR radiation 110 with a modulation pattern that is registered to the output of a modulator driver 138 that provides a drive signal 140 to the modulation means 136.
• the modulated beam of NIR radiation 142 thereby produced is then defocused using standard optical means, such as reflective or refractive optics 154, to produce a divergent excitation beam 156 that is directed onto a specimen 116 to be imaged.
• Simultaneous two-photon photo- activation of diagnostic imaging agent present on or near the surface of the specimen 116 produces modulated two-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 means 158, such as a charge coupled device array, that is mounted at a position outside of the specimen 116.
• This imaging detection means 158 is fitted with a wavelength selection means 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 means 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 means 158 is captured by a processor means 162.
• the processor means 162 serves two primary purposes, firstly to demodulate the modulated signal thus issued 160 from the imaging detection means 158 using a demodulation reference output 152 issued by the modulator driver 138, and secondly to record demodulated emission response from the diagnostic imaging agent as a function of location of emission.
• this alternate embodiment enables direct videographic imaging of surface features 164, such as skin cancer lesions, to be performed based on spatial differences in two-photon excited emission across the illuminated surface of the specimen 116.

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• 1996
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