The development of laser scanning fluorescence microscopy will be outlined. The focus will be technical instrumentation applied to solve biological problems through dynamic, high- resolution imaging. Laser scanning confocal microscopy will be presented first, followed by two-photon excitation fluorescence microscopy. Ideal imaging modes for two-photon imaging will be highlighted: deep tissue imaging and live cell imaging. Contributions from selected pioneers over the last decade of multi-photon imaging field will be highlighted in specific biological applications areas where two-photon imaging has already been established as the best (or only) option: intact tissues, developing embryos, and whole animal studies. The specific, unifying thread will focus in the quest for the observation of microtubule dynamics during the first few, asymmetric cell divisions in Caenorhabditis elegans embryos.

Nonlinear microscopy may be broadly divided into two classes, the first involving resonant multi-photon absorption, and the second involving non-resonant multi-harmonic generation (MHG). We present here the general theory behind MHG microscopy, with emphasis on nonlinear scattering cross-sections, and radiation powers and angular distributions. We also illustrate some specific applications of 2HG microscopy in membrane imaging.

In order to study complex cellular interactions in the developing somite and nervous system, we have been refining techniques for labeling and imaging individual cells within the living vertebrate embryo. Most recently, we have been using MPLSM to analyze cellular behaviors, such as cell migration, filopodial extension, cell process collapse, and neuron pathfinding using time-lapse microscopy in 3-dimensions (3-d). To enhance the efficiency of two-photon excitation in these samples, we have been using a Zeiss LSM 510 NLO fiber delivery system with a Grating Dispersion Compensator (GDC). This system not only offers the convenience of fiber delivery for coupling our Ti:Sapphire laser to the microscope, but also affords us precise control over the pulsewidth of the mode- locked beam. In addition, we have developed a novel peptide/non-cationic lipid gene delivery system to introduce GFP plasmid into somite cells. This approach has allowed us to generate detailed 3-d images of somite cell morphologies at various stages of somite development in a way that best preserves the vitality of the cells being imaged.

Advances in histopathology and immunohistochemistry have allowed for precise microanatomic detail of tissues. Two Photon Confocal Microscopy (TPCM) is a new technology useful in non-destructive analysis of tissue. Laser light excites the natural florophores, NAD(P)H and NADP+ and the scattering patterns of the emitted light are analyzed to reconstruct microanatomic features. Guinea pig skin was studied using TPCM and skin preparation methods including chemical depilation and tape striping. Results of TPCM were compared with conventional hematoxylin and eosin microscopy. Two-dimensional images were rendered from the three dimensional reconstructions. Images of deeper layers including basal cells and the dermo-epidermal junction improved after removing the stratum corneum with chemical depilation or tape stripping. TCPM allows good resolution of corneocytes, basal cells and collagen fibers and shows promise as a non-destructive method to study wound healing.

The spectroscopy and photochemistry of protoporphyrin IX (PpIX) in ethanol and in Triton X-100 micelle solution and Verteporfin in methanol and Triton X-100 micelle solution have been examined using nea infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. For PpIX, we have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense two-photon excitation fluorescence spectra, which allows their detection at low relative concentrations. Verteporfin displays photodynamic behavior in methanol similar to that of PpIX in ethanol. However, in micelle solution Verteporfin exhibits photodynamic behavior indicative of two sensitizer populations, excimers and monomers. Photochemical models are presented.

A new optical interference filter deposition technology demonstrated that provides the deep blocking and extended transmission regions required for multiphoton fluorescence applications. This technology allows for the deposition of high phase thickness coatings with many more layers than was previously possible. Theoretical blocking at a level greater than optical density 9 is achieved. We present examples of shortpass edge filters and bandpass filters with high transmission and deep blocking. A dichroic mirror with high reflection in the near infrared and an extended region of high transmission throughout the visible is also presented.

The development of practical multi-photon microscopy has dramatically changed optical filter design. While most microscopy still utilizes one photon excitation, more and more emphasis is being placed on multi-photon optics. This has created completely new opportunities for the filter designers, but it has also created incredible challenges.

This paper reviews the design process underlying development of a second generation multi-photon laser scanning microscope (MPLSM) system and the methods used to characterize its performance. The purpose is to show how each of several elements of the design present complex design choices which must be resolved in the overall interests of instrument performance combined with ease of use in difficult experiments involving live samples. The paper is intended to stimulate discussion of how close we are to making MPLSM accessible as a routine microscopy method, as opposed to a specialized, user engineered technique. The issue of detector design in MPLSM poses the well known problem of how to collect as much as possible of the emitted fluorescence as its escapes from the sample. The challenge in design of a commercial system is to combine the highest possible detection sensitivity with the requirements of laser safety, ease of use and adaptability to different microscope platforms. This paper will present a 'hybrid' approach to MPLSM detection where the user can select one of several detection strategies according to the nature of the particular sample. Proposals will be presented how detector performance can be compared between systems of different design.

Over the past decade, scanning fluorescence microscopy based on two-photon excitation has become an important branch of microscopic bio-imaging. Compared to traditional scanning techniques, two-photon microscopy offers a number of distinct advantages. First, scanning of the point-like excitation spot used for imaging results in images with excellent axial depth discrimination. In addition, the limited extent of the excitation volume also limits specimen photo-damage to the focal volume. Finally, the long, near-infixed wavelengths used for sample excitation allow in-depth, non-invasive imaging of optically turbid biological samples. For in-depth imaging, the microscopic objective and often optically heterogeneous biological specimen forms a complex system. To optimize imaging quality in two-photon microscopy, an understanding of the point-spread-function (PSF) is essential. In this work, we attempted to characterize the two-photon PSF by two methods: direct imaging of 0.1 ym fluorescent microspheres and multicolor imaging of 2 ym green fluorescent microspheres in a uniform blue fluorescent background. In both measurements, the turbidity of the surrounding medium was varied by changing the concentration of Liposyn III, a scattering component in the specimen. We found that at discrete Liposyn III concentrations between 0 and 2%, the PSF widths were not affected by the amount of scatterers present. However, the imaged contrast continued to degrade as a function of the amount of scatter. This suggests that the broadening of the tail region of the PSF can be the cause of image contrast loss. We will also discuss the possibility of using blind- deconvolution as a method to obtain PSF information in complex biological specimen.

Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one- photon systems. Our Institute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two- photon laser scanning microscopy (TPLSM) to understand the real time cellular transport of the chemotherapeutic agent, Luteinizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the integration of two-photon and nanosized technologies. Nanoclinics (20 - 30 nm silica bubbles) can be fabricated to contain these two photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.

Luminescence-based oxygen sensors, particularly those based on platinum-group complexes are of growing analytical importance. Commercial applications include aerodynamic studies of cars and aircraft in wind tunnels, monitoring of oxygen concentration during fermentation processes and in bioreactors, measurement of biological oxygen demand, and fluorescence detection and imaging of oxygen in blood, tissue, cells and other biological samples. Significant problems in the design and manufacture of polymer-supported, luminescence-based oxygen sensors include the observed non-linearity of the Stern-Volmer calibration plot and the multiexponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule within the polymer matrix. It will be shown that conventional, confocal, and two-photon fluorescence microscopy are invaluable tools with which microcrystals of the sensor molecule can be detected within sensor films. The design of the imaging systems, the measurement methods, and the results will be compared for the three approaches. As a result of the reduction in blur intensity and the minimization of photobleaching, two-photon microscopy provided the easiest and most effective method of microcrystal detection. The implications of the results in the rational design and mass production of luminescence-based oxygen sensors is discussed.

The non-linear nature of multi-photon fluorescence excitation, SHG and THG restricts the signal detecting volume to the vicinity of the focal point. As a result, the technology has intrinsic optical sectioning capability. The use of multi-photon fluorescence excitation also allows micro-fluorometry at high spatial resolution. Under high intensity illumination, biological specimen not only emits fluorescence, but also generates harmonic emissions. Conventional ultra-fast Ti-sapphire laser allows efficient excitation of most biologically important fluorescent probes and SHG in the deep blue range. In contrast, the use of ultra-fast Cr-forsterite laser makes possible simultaneous detection of two- and three-photon fluorescence, SHG and THG

We wish to understand how neural systems store, recall, and process information. We are using cultured networks of cortical neurons grown on microelectrode arrays as a model system for studying the emergent properties of ensembles of living neurons. We have developed a 2-way communication interface between the cultured network and a computer- generated animal, the Neurally Controlled Animat. Neural activity is used to control the behavior of the Animat, and 2- photon time-lapse imaging is carried out in order to observe the morphological changes that might underlie changes in neural processing. The 2-photon microscope is ideal for repeated imaging over hours or days, with submicron resolution and little photodamage. We have designed a computer-controlled microscope stage that allows imaging several locations in sequence, in order to collect more image data. For the latest progress, see: http://www.caltech.edu/~pinelab/PotterGroup.htm.

Multi-photon imaging has generated intense interest from investigators using fluorescent imaging assays in live cells. It can be argued that this technique is rapidly overtaking confocal microscopy as the method of choice for three- dimensional fluorescent imaging of in vivo preparations. Because of the cost involved in purchasing a commercial multi- photon imaging microscope, many investigators have elected to build their own system by adapting in-house confocal laser scanning microscopes. One of the components used for this adaptation involves the construction of an external pulse compressor. Pulse compressors are used to add negative dispersion to pulsed radiation, which undergoes group velocity dispersion when traversing optical elements. In this chapter, we describe a practical guide to building an external pulse compressor.

Tissue remodeling is associated with both normal and abnormal processes including wound healing, fibrosis and cancer. In skin, abnormal remodeling causes permanent structural changes that can lead to hypertropic scarring and keloid formation. Normal remodeling, although fast and efficient in skin, is still imperfect, and a connective tissue scar remains at the wound site1. As a result, methods are needed to optimize tissue remodeling in vivo in all cases of wound repair. Since fibroblast-mediated contraction of engineered 3-D collagen based tissues (RAFTs) represents an in vitro model of the tissue contraction and collagen remodeling that occurs in vivo, RAFT tissue contraction studies combined with two-photon microscopy (TPM) studies are used to provide information on ways to improve tissue remodeling in vivo. In the RAFT models discussed here, tissue contraction is modulated either by application of exogenous growth factors or photodynamic therapy. During tissue contraction, TPM is used to image changes in Collagen Type I fibers in the RAFT skin models. Tissues are imaged at depth at day 15 after modulation. TPM signal analysis shows that RAFT tissues having the highest collagen density have the fastest rate of decay of fluorescent signal with depth.

Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

Conventional epifluorescence microscopy coupled with chronic animal window models has provided stunning insight into tumor pathophysiology, including gene expression, angiogenesis, interstitial transport, and drug delivery. However, the findings to date have been limited to the tumor surface (<150 microns). This is an important drawback because the internal architecture of tumors is known to be heterogeneous, with a collagenous tumor/host interface, highly vascularized outer regions, and poorly vascularized inner necrotic regions. Here we present the first application of the multiphoton laser-scanning microscope (MPLSM) to monitoring drug delivery in tumors, phenotypic tumor cell behavior, and tumor-induced gene promoter activity in vivo. Furthermore, we show that the MPLSM can be used in living tumors to quantify physiological parameters such as vascular density, blood flow velocity, leukocyte/endothelial interactions, and single vessel permeability. These measurements are performed with high three-dimensional resolution up to depths of several hundred microns, thus providing novel insights into the internal milieu of tumors. These findings will allow the development of drug therapeutic strategies that not only affect the tumor surface, but also internal regions.

Using confocal and multiphoton microscopy we have mapped the three-dimensional arrangements of chromosomes, microtubules and gamma tubulin during cell division in plants. We have also for the first time imaged diivision in living, intact plant tissue. These results are preliminary, but exciting and we anticipate considerable further progress will be possible with advances in hardware which are now becoming available.

Apoptosis is a physiological process of cell death resulting from an intricate cascade of sequential protein-protein interactions. Using donor and acceptor mutant GFP fusion constructs, we have monitored the interaction between specific pro- and anti-apoptotic members of the Bcl-2 family with each other as well as proteins located in the outer mitochondrial membrane, as current hypotheses regarding apoptosis suggest that interaction of Bcl-2 family members with each other, or with other mitochondrial membrane proteins, regulates apoptosis. Our data indicate that specific interactions between pro- and anti-apoptotic Bcl-2 family members do occur in situ in the mitochondrial membrane, are altered during apoptosis and regulate cellular sensitivity to apoptosis. These findings are the first to demonstrate real time protein-protein interactions in situ at the level of individual mitochondria.

Cells respond to environmental cues or developmental programs by modifying protein complexes in the nucleus to alter patterns of gene transcription. Recent advances in digital imaging coupled with the development of new fluorescent probes provide the tools to begin to study where and when changes in protein interactions take place in the nucleus of the living cell. Here, we describe the application of fluorescence resonance energy transfer (FRET) using both wide-field and 2-photon (2P) microscopy to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. The efficiency of FRET will be improved if the overlap of the donor emission spectra with the absorption spectra for the acceptor is increased. The trade off for this improved efficiency, however, is that there will be an increase in the background signal from which the weak sensitized acceptor emission must be extracted. Here, we compare and contrast the FRET signals obtained from dimerized C/EBP(alpha) proteins fused to several different color variants of the jellyfish green fluorescent protein (GFP). We use both wide-field and 2P FRET microscopy to characterize the spectral cross-talk and FRET signals for each of the donor and acceptor pairs.

We investigated the potential of two-photon excitation microscopy for the imaging in large blood vessels. Experiments were carried out on isolated rat aorta, labeled with a DNA/RNA dye. Images of the vessel wall indicated that a penetration depth of more than 200 micrometers could be reached. Moreover, blood cells and platelets inside blood vessels could be imaged through the vessel wall. Fluorescence Lifetime Imaging (FLIM) was used as a contrast mechanism for discrimination of autofluorescence from fluorescence of labeled blood cells. We were able to observe labeled blood cells through the vessel wall and identify them by their morphology and characteristic fluorescent lifetimes.

Fluorescence resonance energy transfer (FRET) microscopy provides a tool to visualize the protein with high spatial and temporal resolution. In multi-photon FRET microscopy one experiences considerably less photobleaching compared to one-photon excitation since the illumination is the diffraction limited spot and the excitation is infrared-pulsed laser light. Because of the spectral overlap involved in the selection of the fluorophore pair for FRET imaging, the spectral bleed-through signal in the FRET channel is unavoidable. We describe in this paper the development of dedicated software to correct the bleed-through signal due to donor and acceptor fluorophore molecules. We used living cells expressed with BFP-RFP (DsRed)-C/EBP(alpha) proteins in the nucleus. We acquired images of different combinations like donor alone, acceptor alone, and both acceptor and donor under similar conditions. We statistically evaluated the percentage of bleed-through signal from one channel to the other based on the overlap areas of the spectra. We then reconstructed the images after applying the correction. Characterization of multi-photon FRET imaging system taking into account the intensity, dwell time, concentration of fluorophore pairs, objective lens and the excitation wavelength are described in this paper.

Most multiphoton imaging has been undertaken using tuneable femtosecond Ti:Sapphire lasers which are large, expensive and require a level of laser expertise to operate. Although new commercial, computer controlled systems are becoming available they are still complex instruments. We report on the development of a range of novel laser sources for multiphoton microscopy based upon optically pumped semiconductor materials.

We construct a high-energy short-pulse Cr⁴⁺:forsterite oscillator and apply it for nonlinear optical imaging. We show that the unique wavelength of Cr⁴⁺:forsterite laser is perfectly suitable for deep subsurface imaging utilizing simultaneously several different imaging techniques. We also demonstrate that longer wavelength is more appropriate for non-invasive imaging of living cells.

Temporally decorrelated multifocal arrays eliminate the spatial interference of adjacent foci that occurs in multifocal arrays and allow multifocal imaging with the diffraction-limited resolution of a single focus even with closely spaced foci. To date, we have produced 1-D temporally decorrelated multifocal arrays using low throughput etalons, which limited the efficiency of the arrays. In this work, we demonstrate a 2-D high-efficiency cascaded-beamsplitter array for producing the beamlets. Using the cascaded beamsplitters, we split the 800-nm light from an ultrashort-pulsed Ti:Al₂O₃ laser into a 2-D array of beamlets in which the pulses arrive at a plane perpendicular to the propagation direction at different times. We then overlap the collimated beams with slightly different angles at the entrance aperture to a 1.25 NA oil-immersion objective and produce 2-dimensional array of foci that are temporally decorrelated. This allows multiphoton imaging with diffraction-limited focusing, even for pulses as short as 20-fs. This new method of imaging will make it possible to completely overlap the foci and eliminate the need for scanning. This makes highly efficient use of the power available from typical ultrafast lasers, increasing the frame rate in multiphoton microscopy and the throughput in micromachining applications.

In this review of our work, we describe the application of multiphoton-excited fluorescence as a detection strategy for biological molecules fractionated in micrometer-diameter electrophoresis channels. By tightly focusing a modelocked titanium:sapphire laser beam at the outlet of such channels, spectroscopically similar components can be differentiated in analysis times that range from milliseconds to minutes. Moreover, the ability to excite different chromophores through the combined energies of different numbers of photons (e.g., two and three near-infrared quanta) provides a means to analyze species that are spectroscopically diverse. Finally, we demonstrate that multiphoton photochemistry can be used as a rapid 'photoderivatization' technique for hydroxyindoles and potentially other biological species, in some cases significantly improving the mass detectability of these analytes.

In a novel application of two-photon scanning fluorescence microscopy (TPM), three-dimensional spatial distributions of the hydrophilic and hydrophobic fluorescent probes, sulforhodamine B (SRB) and rhodamine B hexyl ester (RBHE), in excised full-thickness human cadaver skin were visualized and quantified. These findings utilizing TPM demonstrate that, in addition to providing three-dimensional images that clearly delineate probe distributions in the direction of increasing slun depth, the subsequent quantification of these images provides additional important insight into the mechanistic changes in transdermal transport underlying the visualized changes in probe distributions across the slun.

A most-important-variables analysis of practical, successful multiphoton excitation fluorescence microscopy is presented. The key strength of multiphoton imaging -- localization of the excitation volume -- helps to decouple the excitation from the emission; the emission no longer needs to be imaged. The presentation starts with maximizing the detected signal and proceeds to the laser source considerations. The main goal is to match the instrumentation to the biological problem. The main aspects covered are... Emission collection: potential signal; Focal plane mismatch: chromatic aberration; Light detected: actual signal; Live cell imaging: preservation of biological function; Deep imaging: preservation of image contrast; Signal production: potential limitations.

We developed a 3-D image cytometer based on two-photon scanning microscopy. The system keeps the inherent advantages from two-photon scanning microscopy: (1) The ability of imaging thick tissue samples up to a few hundred micrometers, (2) The ability to study tissue structures with subcellular resolution, (3) The ability to monitor tissue biochemistry and metabolism, and (4) The reduction of specimen photobleaching and photodamage. Therefore, 3-D image cytometer has the ability to characterize multiple cell layer specimens, in contrast with 2-D image cytometer where only single cell layer samples can be imaged. 3-D image cytometry increases its frame rate by adapting a polygonal mirror scanner and high-speed photomultiplier tubes. The current frame rate is 13 frames per second. High throughput rate is achieved by imaging multiple cell layer specimens in 3-D at a high frame rate. The throughput rate of this system is dependent on the choice of objective lenses, specimen properties, and the speed of computer-controlled specimen stage. It can be up to approximately 100 cells per second which is comparable with that of 2-D image cytometers. With the high throughput rate and deep tissue imaging capability, 3-D image cytometer has the potential for the detection of rare cellular events inside living, intact tissues. A promising application of this 3-D image cytometer is the study of mitotic recombination in tissues. Mitotic recombination is a mechanism for genetic change. Therefore it is one of causes for carcinogenesis. However, the study of this process is difficult because recombination event is rare and it occurs at a rate of one cell in 10⁵ cells. The new method for the study is (1) to engineer transgenic mice whose cells will express fluorescence in the presence of mitotic recombination, (2) to detect cells which have undergone mitotic recombination with 3-D image cytometry. The estimated time required to quantify spontaneous recombination rate is approximately within a few hours in the case that the mutation occurs at a rate of 1/10⁵. The ability of this 3-D image cytometer to resolve tissue structures at video rate was demonstrated in the study of ex vivo human skin dermal structure. 25 X 25 section images were taken by shifting the acquisition region with computer- controlled specimen stage. Wide area images were reconstructed by combining each image sections. The size of complete wide area images is approximately 25 mm X 25 mm. We further performed experiments to verify this cytometer's ability for population statistics measurement. We prepared cell cultures containing a mixture of cells expressing cyan and yellow fluorescent proteins. These cell cultures with mixing ratio ranging from 1/10 up to 1/10⁵ were imaged. Experimental results show that the presence of a few rare cells in large pool of the other cells can be quantitatively measured. We also imaged a punched ear specimen from a transgenic mouse which carries green fluorescent protein. The data demonstrates that this system can resolve a single green fluorescent cells in the tissue.

In laser-induced fluorescence diagnosis and photodynamic therapy of cancer the applied photosensitizers (PS) are often covalently derivatized with macromolecules to improve their selective accumulation in the cancerous tissue, while maintaining its single-photon excited photophysical properties. In this contribution methoxy-polyethylene glycol (MPEG, MW ~5 kDa) and human serum albumin (HSA, MW ~60 kDa) are used as PS carriers. Multiphoton (MP) excitation of the PS is favorable as compared to single-photon excitation because the penetration depth of the laser light is improved (>5 mm) due to the longer wavelength of the ~200 fs laser pulses used in this case (700-1050 nm). In this study cotton fibers and silica gel beads (<20 mm) were stained with various PS and multiphoton-induced fluorescence was detected with a MP laser scanning microscope. The slopes of the log-log plots of the detected fluorescence intensity versus the laser excitation intensity vary between 1.8-2.6 for the various PS investigated. The excitation wavelength dependence of the MP-induced fluoresence indicates that the excitation cross section maxima can be shifted substantially relative to twice the wavelength of the one-photon absorption maxima. Some PS (photofrin II, purpurin, mTHPC-[MPEG]2 and diaminoanthra-quinone) do not exhibit multiphoton-induced fluorescence. Some derivatized PS (sulforhodamine B, erythrosin B, purpurin) exhibit MP-induced fluorescence, although no single-photon absorption band exists in the spectral region around half the excitation wavelength

Photodynamic Therapy has emerged as a new modality in the treatment of various nonmalignant and malignant diseases. It involves the systemic administration of tumor specific photo-sensitizers with the subsequent application of visible light. This combination causes the generation of cytotoxic species, which damage sensitive targets, producing cell injury and tumor destruction. Although, photofrin is the only photosensitizer currently approved for PDT and tumor detection, its concomitant cutaneous photosensitization poses a significant problem. Hence, δ-aminoleuvulinic acid (δ-ALA) a precursor for the endogenous production of Protoporphyrin IX, through heme biosynthesis pathway, has gained significant importance in the Photodynamic Therapy. Though δ-ALA is present naturally in the cells, exogenous δ-ALA helps to synthesis more of PpIX in the tumor cells, as the fast growing tumor cells take up the administered δ-ALA more than the normal cells. Based on these facts, many invasive studies have been reported on the kinetics of δ-ALA at cellular level by chemical extraction of PpIX from the cells. In the present study we have studied the kinetics of δ-ALA induced PpIX fluorescence from Hela cells by perchloric/Methanol extraction method. However, the amount of PpIX synthesized in the cells at different point of incubation time by noninvasive methods has not been reported. Hence we have also used a noninvasive technique of measuring the kinetics δ-ALA induced PPIX fluorescence from Hela, an epithelial cell derived from human cervical cancer by both single photon (steady state) and multi photon excitation. From the studies it is observed that the δ-ALA induced PpIX is more at 2 hours incubation time for 2 mM of δ-ALA concentration. Further, it is observed that with steady state fluorescence imaging method, the excitation light itself cause the Photodynamic damage, due to the prolonged exposure of the cells than in multi photon excitation, leading to the rounding of cells. This may be due to the activation of PpIX in cells by the excitation light source.

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.

Image correlation spectroscopy (ICS) was developed as an imaging analog of fluorescence correlation spectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. Ics was first implemented on a confocal laser scanning microscope (CLSM) and entails spatial autocorrelation analysis of fluorescence fluctuations within an image sampled from an area of the cell. Spatial and temporal autocorrelation analysis of image time series enables measurement of both the molecular dynamics and aggregation state of the imaged molecules. The parallel nature inherent in the collection of multiple fluctuations in an imaging scheme improves the signal to noise ratio of the correlation analysis, which enhances dynamic measurements for slowly moving species in membrane systems. We outline our development of two-photon ICS and describe recent applications of the method for measurements of flow, diffusion and aggregation behavior of green fluorescent protein/integrin receptor constructs in living cells. We also describe the use of two-photon excitation to perform two-color image cross-correlation spectroscopy to measure the dynamics and colocalization of non-identical species labeled with different fluorophores.

We present a system for in-vivo skin imaging consisting of a two-photon fluorescence and a confocal reflectance video microscope. The two devices share one microscope objective, but have separate light sources, detectors, scan-units and control electronics. This makes it possible to image a region of the skin using the two different modalities, while exploiting the specific advantages of each method. In the images we clearly distinguish several skin layers, i.e., stratum corneum, viable epidermis, basal layer and upper dermis. Close to the skin surface individual keratinocytes in the stratum corneum are visible with the two-photon microscope, while in reflectance images the texture is much more uniform. Slightly deeper in the skin the smooth cellular structure of the epidermis becomes visible for both imaging modalities. Below the basal layer, which marks the boundary between epidermis and the dermis, fibrous structure appears which can be attributed to capillary vessels and dermal collagen. We use the combined imaging device for studying the effect of occlusion on human skin. From the fluorescence images we observe swelling of the stratum corneum, while the reflectance microscope shows changes in the scattering properties due to hydration. We show that by combining two microscopes in one we can obtain images that contain complementary information, thereby enhancing the potential for each individual modality.

Deep tissue imaging may have important biomedical applications in the areas of skin disease diagnosis, wound healing, and tissue engineering. For the study oftissue physiology with microscopic resolution, we used two-photon microscopic imaging based on the excitation of endogenous fluorophores. While autofluorescence is observed ubiquitously in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. The different fluorescent species are expected to have different fluorescence excitation and emission spectra. Self-modeling curve resolution (SMCR) can be applied to extract spectroscopic components from two-photon images. In ex vivo human skin, we were able to acquire a four-dimensional data set (3D space + excitation spectra). We extracted the major spectral components from this data set using multivariate curve resolution and correlated these species with known tissue structures. From the SMCR analysis, it was determined that there are approximately seven factors that contribute to most of the autofluorescence from human skin. This analysis provides us with the concentration ofthe species at different depths within the skin and also with a reconstructed image of the skin due to each single factor alone. Several ofthese chemical components have been identified, such as collagen, elastin, and NAD(P)H. In addition to providing insight into tissue physiology, we are able to optimize the excitation wavelength for each biochemical species for skin imaging applications.

Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated frame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ~5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules are occasionally observed.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced production of reactive oxygen species (ROS) in JAR cells by using fluorescent probe 2',7'-dichlorofluorescin diacetate with confocal laser scanning microscopy. TCS-induced ROS showed dependence on the increase in intracellular calcium and on the presence of extracellular calcium. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-indued increase in intracellular calcium monitored using fluo 3-AM, suggesting that TCS-induced ROS might mediate by the increase in intracellular Ca2^PLU concentration. Simultaneous observation of the nuclear morphological changes and production of ROS in JAR cells with two-photon laser scanning microscopy and confocal laser scanning microscopy revealed that ROS involved in the apoptosis of JAR cells, which was confirmed by that antioxidant (alpha) -tocopherol prevented TCS-induced ROS formation and cell death. The finding that calcium-dependent TCS-induced ROS involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.

Virtually all laser based microscopy imaging methods involve a single laser, with ultrafast lasers emerging as the enabling tool for a variety of methods. Two-photon fluorescence is a high sensitivity method with selectivity depending on a chromophore that is either added or produced by genetic engineering. While there are fundamental advantages over white light or other fluorescence microscopies, there are unavoidable limitations such as bleaching, photoinduced damage to the cell, and the inability to label some major constituents of the cell, particularly the abundant species. Raman imaging affords chemical selectivity but application is limited due particularly to its low sensitivity and unavoidable fluorescence background. Adding a second laser beam, shifted from the first laser by a molecular vibrational frequency, increases the detected Raman signal by many orders of magnitude and in addition shifts the detected signal to the high energy (blue) side of both lasers, removing fluorescence artifacts. Signal levels sufficient to acquire high signal-to-noise ratio images of 200 by 200 pixels in one minute requires sub-nanojoule pulse energy. A convenient, tunable source of the Stokes-shifted beam is provided by an Optical Parametric Amplifier (OPA), which requires an amplified laser. 250-kHz sources have ample energy and in addition keep the average sample power on the order of 0.1 mW, a level that even sensitive biological systems tolerate at the focal spot diameter of 0.3 micrometers . Long-term viability of mammalian cells has been demonstrated during dozens of scans in a single plane. Two-photon fluorescence provides a useful complimentary data channel that is acquired simultaneously with the Raman image. Several dyes and green fluorescence protein have been used for this purpose. Interpretation of images, acquiring three dimensional images, and identification of cellular features are ongoing activities.

We derive an exact steady state solution for a diffusion-coupled reaction localized to a small but open spherical sub-volume in a fluid reaction medium. We show that this leads to a fluorescence-based method for measuring the diffusion constant and the photochemical properties of fluorophores that is simpler and more robust than existing techniques such as fluorescence correlation spectroscopy (FCS). We use this method to study the photobleaching of rhodamine-B labeled protein molecules under different illumination intensities. Together with complementary data provided by FCS, this determines the average number of photons emitted by the fluorophores before photobleaching (~5x10⁴). We demonstrate that this technique can be easily implemented on any confocal or multiphoton microscope or spectrometer and thus it should be adaptable to a variety of biological and chemical problems.

Two-photon excitation can excite physiological ultraviolet (UV) chromophores at visible wavelengths and holds significance for applications such as non-linear microscopy, micro-pharmacology, localized heating and tissue ablation. However, little quantitative data is available on the absolute two-photon absorption cross sections ((sigma) ₂) of these molecules. Their low two-photon absorbance and limited solubility implies that the sensitivity required for such measurements is much higher than that available from standard techniques. We employ the recently developed generalized z-scan technique to measure the absolute value of the (sigma) ₂ of tryptophan, the most ubiquitous of the physiological near-UV chromophores, and obtain the value of 32.0 +/- 1.2 mGM (1 GM equals 10^-50 cm⁴sec/photon/molecule) at 532 nm. This is the first determination of the absolute (sigma) ₂ of any biological UV chromophore, and can be used to calibrate the previously reported relative two-photon excitation spectra of a number of such molecules.

An important ingredient in improving Multi Photon Laser Scanning Microscopy, MPLSM, is the development of efficient exogenous two-photon fluorescent (TPF) probes. Here we report on a new class of two-photon fluorophores, specifically designed in order to maximize their efficiency in potential MPLSM applications. The fluorophores possess a symmetric Donor-Acceptor-Donor (D-n-A-n-D) structure with varying conjugation length and have strong donors and acceptors. We have studied the two-photon excitation (TPE) properties of these fluorophores and found the following properties: (1) Very large two-photon absorption coefficients (6 > 1000 GM); (2) Peak TP excitation wavelength which are strongly shifted to the red ((lambda) 1 micrometer); (3) Large fluorescence quantum efficiency; (4) Large Stokes shifts of the fluorescence bands. These properties make them particularly suitable for imaging thicker samples, relying on the large improvement in TPE cross-sections and the reduced attenuation at both the excitation and emission wavelengths. We also describe TPE fluorescence anisotropy experiments revealing the tensorial shape of the fluorophores.

Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC₅. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Spines also undergo rapid morphological changes, although the function of this motility is still unclear. We have investigated the effect of spine movement on spine calcium dynamics with two-photon photobleaching of enhanced Green Fluorescent Protein (EGFP) and calcium imaging of action potential-elicited transients in spines from layer 2/3 pyramidal neurons in mouse visual cortex slices. The elongation or retraction of the spine neck during spine motility alters the diffusional coupling between spine and dendrite and significantly changes calcium decay kinetics in spines. Our results demonstrate that the spine's ability to compartmentalize calcium is constantly changing.

The physics and chemistry of fluorescent resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years, but only with recent technical advances has it become feasible to apply FRET in biomedical research. FRET microscopy is a better method for studying the structure and localization of proteins under physiological conditions than are X-ray diffraction, nuclear magnetic resonance, or electron microscopy. In this study, we used four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides 2-D spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleed through and photo bleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. We will discuss the advantage of fluorescence lifetime methods to measure FRET signals at the moment of the protein-protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution.

Here, we use fluorescence resonance energy transfer (FRET) imaging techniques to assay and study the organization and dynamics of endosomes in epithelial cells. We use polarized epithelial MDCK cells stably transfected with polymeric IgA-receptor (pIgA-R) to analyze the co-localization, within 10 -100A of pIgA-R-ligand complexes labeled with different fluorophores in the apical endosome. When internalized at 17 degree(s)C for four hours, these complexes co-localize in the apical endosome, which is located underneath the apical plasma membrane. While the transport pathways crossing are thought to be understood, the actual morphology of this endosome has not been completely characterized. Here, we compare the ability of laser scanning confocal and Two-Photon FRET microscopy to image the co-localization of differently labeled (donor: Alexa488, acceptor: Cy3) receptor-ligand complexes in the apical endosome. While the preliminary results are broadly similar, we have found that Two-Photon FRET microscopy possesses significant advantages over laser confocal microscopy FRET. In confocal microscopy FRET, the actual FRET signal in the acceptor channel, following donor excitation, is contaminated by the excitation of the acceptor molecule by the donor wavelength as well as by the cross-talk between donor and acceptor emissions in the acceptor channel. We are in the process of testing an algorithm that will correct this problem. In Two-Photon microscopy, we were able to prevent the excitation of the acceptor molecule by the donor wavelength. Furthermore, we have developed a method to manipulate the Two-Photon FRET data post-acquisition to remove the remaining contaminating signal, i.e. the cross-talk between donor and acceptor emissions in the acceptor channel. Since Two-Photon microscopy avoids out-of-focus bleaching which allows repeated scanning in multiple focal planes, a more detailed picture of intracellular events can be obtained. Therefore, we should be able to use this technology to assay the FRET signal along the vertical axis of polarized epithelial cells, such as MDCK cells. In summary, our results indicate that the Two-Photon FRET microscopy is a powerful tool to assay intracellular protein-protein co-localization/interaction events.

We have set-up a two-photon excitation fluorescence microscope based on a compact commercial confocal laser scanning head and a mode-locked Ti:Sapphire infrared pulsed laser, directly coupled in air to the scanning head We report about the architecture and the imaging capabilities of this microscope using high numerical aperture objectives, in terms of its spatial resolution and excitation volume. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical path in air guiding the IR laser beam (two-photon/ TPE). In both cases, an optical fiber delivers the emitted fluorescence to the control unit. Under TPE regime, the resolution (Full Width at Half Maximum intensity - FWHM) was 210 nm (x-y) and 700 nm axially at 720 nm excitation. Due to the inherent spatial selectivity of the TPE we are evaluating the utilization of the instrument for micro patterning using the property of photobleaching and the characteristics of photobleaching in comparison with the single-photon mode. Moreover, attention is given to pulse broadening as function of penetration depth. The system is mainly used for biological applications related to cell and tissue imaging and dynamic cellular processes.

In the past scientists have had difficulties visualizing cellular processes in thick tissue specimens. With conventional imaging techniques, fluorescence signals from above and below the focal plane create background noise that significantly degrades the resultant images. Today we have two-photon excitation microscopy, which surpasses other imaging systems in its ability to see further and more clearly into thick tissue. The two-photon system has an infrared pulsed laser light illumination that hits only at the focal plane, thus reducing autofluorescence and photobleaching. Although this technique is superior to others in fluorescence imaging, the two-photon images can still have a considerable amount of background glow deep down in the tissue. Due to this problem, we propose that digital deconvolution be used to improve these signals by eliminating the background noise in the two-photon images. The purpose of deconvolution is to undo the degradation of the image that was created by convolution. Deconvolution uses the entire fluorescent signal and digitally reverses it through the use of a point spread function. We have developed a model system for measuring the depth penetration of our two-photon system integrated with the Biorad MRC 600. With this system the deconvolved signal at different depths shows a better signal-to-noise (S/N) of the two-photon images than those without deconvolution. The biological applications of this process will be discussed in this paper.

We describe design and installation of a multi-mode microscopy core facility in an environment of varied research activity in life-sciences. The experimentators can select any combination of a) microscopes (upright, upright fixed-stage, inverted), b) microscopy modes (widefield, DIC, IRDIC, widefield epifluorescence, transmission LSM, reflection and fluorescence CLSM, MPLSM), c) imaging techniques (direct observation, video observation, photography, quantitative camera-recording, flying spot scanning), d) auxiliary systems (equipment for live specimen imaging, electrophysiology, time-coordinated laser-scanning and electrophysiology, patch-clamp). The equipment is installed on one large vibration-isolating optical table (3m X 1.5m X 0.3m). Electronics, auxiliary equipment, and a fiber-coupled, remotely controlled Ar⁺-Kr⁺ laser are mounted in a rack system fixed to the ceiling. The design of the shelves allows the head of the CSLM to be moved to any of the microscopes without increasing critical cable lengths. At the same time easy access to all the units is preserved. The beam of a Titanium-Sapphire laser, controlled by means of an EOM and a prism GVD, is coupled directly to the microscopes. Three mirrors mounted on a single precision translation table are integrated into the beam steering system so that the beam can easily be redirected to any of the microscopes. All the available instruments can be operated by the educated and trained user. The system is popular among researchers in neuroanatomy, embryology, cell biology, molecular biology - including the study of protein interactions, e.g. by means of FRET, and electrophysiology. Its colocalization with an EM facility promises to provide considerable synergy effects.

We have combined a confocal laser scanning head modified for TPE with some spectroscopic modules to study single molecules and molecular aggregates of rhodamine 6G and labeled proteins on glass substrates. The fluorescence intensity of the spots occurs at definite values that are multiples of a reference signal and extrapolate to the background level measured on the images. These properties suggest that these spots arise from single rhodamines. The discrete character of the intensity distribution can therefore be used as a simple, quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots. These studies have been performed by using a combined confocal and spectroscopic architecture realized for two-photon excitation.

A new Time-Correlated Single Photon Counting (TCSPC) imaging technique delivers combined intensity-lifetime images in a two-photon laser scanning microscope. The sample is excited by laser pulses of 150 fs duration and 80 MHz repetition rate. The microscope scans the sample with a pixel dwell time in the +s range. The fluorescence is detected with a fast PMT at the non-descanned port of the laser scanning microscope. The single photon pulses from the PMT and the scan control signals from the scanning head are used to build up a three-dimensional histogram of the photon density over the time within the decay function and the image coordinates x and y. Analysis of the recorded data delivers images containing the intensity as brightness and the lifetime as colour, images within selected time windows or decay curves in selected pixels. The performance of the system is shown for typical applications such as FRET measurements, Ca imaging and discrimination of endogenous fluorophores or different dyes in living cells and tissues.