Fluorescence anisotropy, a measure of the polarization state of fluorescence emission, is a sensitive measure of molecular rotational motion and of resonance energy transfer (RET). We report here the formalism and application of dynamic and static fluorescence anisotropy measurements primarily intended for implementation in imaging systems. These include confocal lasre scanning microscopes (CLSM) as well as wide-field instruments, in the latter case adapted for anisotropy-based dynamic frequency domain fluorescence lifetime imaging microscopy (FLIM), a method we denote as rFLIM. Anisotropy RET is one of the modalities used for fluorescence RET (FRET) determinations of the association, and proximity of cellular proteins in vivo. A requirement is the existence of intrinsic or extrinsic probes exhibiting homotransfer FRET (in our nomenclature, energy migration or emFRET) between like fluorophores. This phenomenon is particularly useful in studies of the activation and processing of transmembrane receptor tyrosine kinases involved in signal transduction and expressed as fusions with Visible Fluorescence Proteins (VFPs).

Photochromic FRET (pcFRET), a member of the family of acceptor depletion FRET techniques (adFRET), embodies a general conceptual and experimental scheme based on a coupled system of a fluorescent donor and a photchromic acceptor. The procedure involves the reversible and cyclic spectroscopic depletion of the acceptor, and was initially conceived for the determination of FRET efficiency on a continuos, pixel-by-pixel basis in the microscopy of living cells. However, the modulation of donor fluorescence in pcFRET has implications for a wide range of applications. We present the formalism for quantitative interpretations of photostationary and kinetic data, from which the relevant kinetic rate constants and quantum uields for the cyclization and cycloreversion reactions of the photochromic acceptor can be derived. The scheme was applied to a model system consisting of a fluorescent donor (Lucifer Yellow) covalently bound to a diheteroarylethene acceptor. In a Perspectives section, we discuss photochromic probes, instrumentation issues, and the potential of pcFRET for analyizing chemical equilibria and kinetics, in the latter case with a new technique we have denoted Photochromic Relaxation Kinetics (pcRelKin).

In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5•10⁶s^-1. A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 10⁷ photons per second and records double-exponential FLIM data within less than 10 seconds.

Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPa), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPa was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPa was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.

FRET-based assay has been used to determine the organization of transferrin-receptor bound to holo-transferrin in basolateral endocytic membranes and compare it to the previously characterized clustered organization of polymeric IgA-receptor (pIgA-R) bound to pIgA-R ligand in apical endocytic membranes. In polarized MDCK-PTR cells, we have internalized holo-transferrin from the basolateral plasma membrane - labeled with donor and acceptor fluorophores. Transferrin-receptor-holo-transferrin complexes were imaged in the basolateral endocytic compartment using FRET confocal laser scanning microscopy in fixed and live MDCK polarized cells. A two-parameter FRET assay demonstrates whether complexes are randomly distributed or clustered: Acceptor's positive impact on E% signifies random distribution; E% being independent of acceptor fluorescence levels indicates clusters. A second parameter for clustering is E% being negatively dependent on D:A ratios. Our results indicating a clustered organization of transferrin-receptor-holo transferrin complexes fit the well-known homodimeric structure of transferrin-receptor.

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. A devastating element of TBI is the diffuse and widespread injury of axons in a process known as traumatic axonal injury (TAI). TAI is a difficult entity to study as gross protein or molecular analyses have been largely precluded due to the requisite for specifically localizing protein changes to axons rather than the supporting glia or neuronal soma. As such, much of the mechanistic insight into TAI pathogenesis thusfar has come through histochemical and immunohistochemical analyses. In order to address the next generation of questions in TAI pathogenesis it has become critical to develop methodologies that allow for direct examination of protein-protein interactions in relation to sites of TAI. In the current communication, we report on a modified method of multiphoton Fluorescent Resonance Energy Transfer (FRET) microscopy that allows for the direct assessment of protein-protein interactions in aldehyde fixed tissue sections through the use of a conventional dual-label immunofluorescent approach. In the utilization of this technique, we explored whether the bcl-2-related proteins BAD and Bcl-xL heterodimerize in a pro-apoptotic fashion within traumatically injured axons following TBI. Adult SD rats were subjected to impact acceleration TBI and euthanized at multiple time points. Vibratome sections derived from adult SD rats that had previously undergone impact acceleration TBI were processed for immunohistochemical double labeling with anti-BAD 1° antibody/Alexa 488 2° antibody, followed by anti-Bcl-xL 1° antibody/Alexa 555 2° antibody. Images were processed for spectral bleedthrough, and efficiency/distance calculations were performed. BAD/Bcl-xL heterodimerization was examined in relation to cytochrome c release and caspase-3 activation, by employing a third immunofluorescent label visualized with an Alexa 647 dye. Multiphoton FRET microscopy was carried out upon 40 micron thick sections. Further, to determine if FRET analysis could be performed in thick tissue specimens, 100 and 200 micron thick sections were examined as well. At 6h postinjury, swollen axons in medial lemniscus demonstrated a mean energy transfer efficiency greater than 20% indicating formation of Bad-Bcl-xL complexes. Thick tissue specimens up to 200 microns thick likewise demonstrated FRET efficiencies greater than 20%. Specimens positively labeled for either cytochrome c or caspase-3 demonstrated FRET efficiencies greater than 10%. The current investigation demonstrates FRET microscopy can be employed to assess protein-protein interactions in aldehyde-fixed tissue sections through multi-label immunofluorescent methodologies. It is believed that the current approach will have widespread applicability as examination of protein-protein interactions within in vivo systems may now be assessed on the cellular and subcellular level within aldehyde fixed tissue sections.

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile plaques in the brain. Clinical techniques are just becoming available for detecting plaques, allowing a definitive diagnosis of the disease. Using multiphoton microscopy and transgenic mouse models that develop senile plaques as they age, we have demonstrated chronic, in vivo imaging of these neuropathological lesions. We have used these tools to evaluate contrast agents with high affinity and specificity for senile plaques that would be suitable for non-invasive imaging with PET scanning if appropriately radiolabeled. These imaging tools should translate into early diagnostic procedures, as well as end-points for clinical trials aimed at clearing senile plaques from the brain. We have also developed FLIM for FRET determinations in vitro and in vivo between appropriate donor and acceptor fluorophores to examine the proximity of domains within a single protein. These results indicate that FRET measurements using FLIM can determine interactions of proteins on the nanometer scale, facilitating an understanding of both static and dynamic protein assemblies in neuropathological diseases.

Five-dimensional (5D) multiphoton measurements with submicron spatial resolution, 270 ps temporal resolution and 5 nm spectral resolution have been performed on living cells and tissues at 750 nm - 850 nm laser excitation. A compact (65x62x48 cm³) multiport laser scanning microscope TauMap (JenLab GmbH) equipped with fast PMT and CCD camera, SPC 830 time-correlated single photon counting board and Sagnac interferometer was used. Laser excitation radiation was provided by a tuneable MaiTai Ti:sapphire femtosecond laser as well as by a 405 nm 50 MHz picosecond laser diode. The spectral and temporal fluorescence behaviour of intratissue chloroplasts of water plant leafs, of a variety of exogenous fluorophores as well as of fluorescent proteins in transfected brain cells have been studied. When calculating fluorescence lifetime images (FLIM) we found differences in intracellular twophoton fluorescence lifetimes vs. one-photon fluorescence lifetimes. Multiphoton FLIM-FRET and multiphoton spectral FRET studies have been performed in living HBMEC brain cells using CFP and YFP fusion proteins. It was shown that FLIM-FRET data depend on laser power due to photodestructive multiphoton effects. This has to be considered in long-term fluorescence resonance energy transfer studies of dynamic protein-protein interactions.

In Fluorescence Lifetime Imaging (FLIM) the ns fluorescence decay is used for imaging. It yields information about the local environment of the fluorescent molecule and is very well suited for quantitative imaging. FLIM was introduced more than one decade ago now. Most of the implementations of FLIM require comparatively long acquisition times on the order of ten seconds or more. This hampers the use of FLIM for the study of dynamic processes. In FLIM at least one order of magnitude more signal is required than in conventional intensity imaging. Therefore, fast FLIM acquisition rates require efficient detection schemes and detectors. We evaluated the count rate limitation in time gated and TCSPC FLIM of a number of single photon counting detectors. In particular we looked at the performance of a conventional fast head-on PMT (R1894), a GaAs photocathode PMT (H7422P-40) and a single photon counting avalanche photo diode (SPCM-AQR14). The high quantum efficiency GaAs photocathode PMT and avalanche photo diode detectors show lifetime shifts in both time gated detection and in TCSPC starting at a detection count rate of 1 - 2 MHz. The conventional PMT shows lifetime shifts starting at a detection count rate of about 2.5 MHz in TCSPC and at about 6 MHz for the time gated detection system. The detection efficiency of the TCSPC based system goes down rapidly above about 1 MHz due to the dead time of the detection electronics. The time gated detection system shows little or no reduction of the detection efficiency up to detection count rates of 10 MHz with the conventional fast PMT. The time gated-detection system was coupled to a multi-photon excitation microscope. Calcium transients were recorded in cardiac rat myocytes at a 1 Hz frame rate. The system operated at the full repetition rate of the Ti:Sa laser. Here, the frame rate was limited by the maximum count rate of the PMT.

Fluorescence detection is the dominant technology for cellular imaging, clinical diagnostics, DNA analysis and drug discovery. In fluorescence microscopy, the fluorophores are subject to photobliching and detectability is limited by cellular autofluorescence. We describe new approach to develop fluorescent probes that display long emission wavelength, long decay times, and high quantum yield and high fluorescence brightness. These luminophores are covalently linked pairs of long-lifetime fluorophores (like metal-ligand complexes) and a short-fluorescence-lifetime and high quantum yield dyes. Using resonance energy transfer (RET) it is possible of obtaining desirable spectral properties and long fluorescence lifetime in covalently linked pairs. The long-lifetime donor results in a long-lived fluorescence component in the acceptor decay. Importantly the emission spectrum of the luminophore is that of the acceptor and quantum yield of the luminophore approaches that of the higher quantum yield acceptor. Such luminophores are suitable for fluorescence measurements in biological samples with the use of real time background suppression to eliminate autofluorescence. We discuss experimental examples based on long lived metal-ligand-complexes and long wavelength acceptors like Texas Red. The emission maxima (spectra) and decay time of such RET tandems can be readily adjusted by selection of the donor, acceptor and distance between them. Such luminophores with long-wavelength emission and adjustable long lifetime can have numerous applications in one-photon and multi-photon cellular and tissue imaging with the use of off-gating the excitation pulse and sample autofluorescence.

Two photon microscopy is a powerful tool for cells or tissues imaging. However it presents the drawback of being a laser-scanning technique leading to long acquisition time for fluorescence lifetime imaging. Thus it is commonly limited to intensity images that only give indications on location of fluorophores but hardly reports physico-chemical properties and interactions between cells components. To preserve biological samples from too long experiments and provide a more complete spectroscopic tool we developed a time-resolved multifocal multiphoton microscope. This setup allows us to speed up the acquisition while keeping the possibility to measure both intensity and lifetime images for all multifocal points.

Fluorescence resonance energy transfer (FRET) is a fluorescence microscope imaging process involving nonradiative energy transfer between two fluorophores (the donor and the acceptor). FRET is used to detect the chemical interactions and, in some cases, measure the distance between molecules. Existing approaches do not always well compensate for bleed-through in excitation, cross-talk in emission detection and electronic noise in image acquisition. We have developed a system to automatically search for maximum-likelihood estimates of the FRET image, donor concentration and acceptor concentration. It also produces other system parameters, such as excitation/emission filter efficiency and FRET conversion factor. The mathematical model is based upon a Poisson process since the CCD camera is a photon-counting device. The main advantage of the approach is that it automatically compensates for bleed-through and cross-talk degradations. Tests are presented with synthetic images and with real data referred to as positive and negative controls, where FRET is known to occur and to not occur, respectively. The test results verify the claimed advantages by showing consistent accuracy in detecting FRET and by showing improved accuracy in calculating FRET efficiency.

Overexpression of HER2 alters the cellular behavior of EGF receptor (EGFR) and itself, with great implications on cell fate. To understand the molecular interactions underlying these alterations, we quantified the association between the two receptors by looking at efficiency changes in fluorescence resonance energy transfer (FRET) between a small number of molecules at the membrane of living cells. Human mammary epithelial (HME) cells expressing varying degrees of HER2 were studied, to identify and compare the degree of receptors interactions as a function of HER2 overexpression. A high resolution wide-field laser microscope combined with a high sensitivity cooled CCD camera was used to capture simultaneously donor and acceptor emissions. Alternating between green and red lasers every 80 msec, donor, FRET, and acceptor images were acquired and were used to calculate FRET efficiency. Automated image analysis was developed to create FRET efficiency maps from overlapping donor, acceptor and FRET images, and derive FRET efficiency histograms to quantify receptor-receptor interactions pixel by pixel. This approach enabled us to detect subtle changes in the average distance between EGFR molecules, and between EGFR and HER2. We found pre-existing EGFR homoassociations, and EGFR-HER2 heteroassociations in cells overexpressing HER2, and identified the changes in these interactions with ligand stimulation. These observations demonstrate the power of FRET measurements between small numbers of molecules in identifying subtle changes in molecular interactions in living cell.

Excitation saturation and other photophysical dynamics can have a dramatic influence on the effective imaging point spread function (psf) in fluorescence microscopy. Specifically, saturation leads to increased fluorescence observation volumes and altered spatial profiles for the psf. These changes have important implications for both fluorescence correlation spectroscopy (FCS) and imaging applications. A detailed characterization of these changes is required for accurate interpretation of FCS measurements. We here introduce a method to calculate molecular excitation profiles that represent the true fluorescence observation volume under the influence of excitation saturation in two-photon microscopy. An analytical model that accounts for pulsed excitation is developed to calculate the influence of saturation at any location within the excitation laser profile, and the overall saturation influenced molecular excitation profiles are evaluated numerically. Fluorescence signals measured with a solution of Rhodamine 6G are presented, showing good agreement with these calculations.

The combination of dual-color fluorescence correlation spectroscopy (FCS) and two-photon excitation is a powerful tool for probing protein-protein interactions. The submicron resolution and single molecule sensitivity of the technique make it attractive for in vivo applications. However, the strong spectral cross talk between the two emission channels of most fluorescent dye mixtures provides a challenge for the analysis of dual-color FCS experiments. We describe a new technique, dual-color photon counting histogram (PCH) analysis that overcomes some of the challenges associated with spectral cross talk. Dual-color PCH is an extension of regular PCH that simultaneously analyses the photon counts of two detection channels. We demonstrate that dual color PCH quantitatively resolves protein mixtures in vitro. We also apply dual-color PCH to study proteins in biological cells. The fluorescent proteins ECFP and EYFP, which are commonly used for dual-color studies in cells, have significant spectral cross talk. We will discuss the resolvability of these fluorescent proteins and present data that successfully resolve the protein mixtures in vitro and in vivo. Our results show that dual color PCH is a promising technique for the characterization of protein-protein interactions in intact cells.

Fluorescence correlation spectroscopy(FCS) normally employs measured fluctuations in molecular fluorescence from within a defined observation volume created by a laser focus to extract information about dynamic processes associated with those molecules. In order to extract quantitative information about these processes, the laser focal volume must be well characterized. We show in this work, similar to others before, that a description of the focal volume in terms of vector diffraction theory allows quantitative analysis of FCS sufficient to resolve the diffusion coefficients of small molecules-Rhod 6G and Alexa 546, here-in the diffraction-limited volume of a high numerical aperture objective lens. We subsequently demonstrate the utility of a “reverse” FCS experimental paradigm in which the focal volume itself is characterized for a known molecular sample. Using a combination of wide-field and localized detection, we characterize the focal volume location and relative size as the beam collimation and correction collar are varied. We find that Gaussian beam theory can predict the focal volume location over a wide range of collimation conditions. It can also predict the relative focal volume size for converging and collimated beams, provided that spherical aberration is corrected for each collimation condition using the correction collar.

Two- and three-photon excitation was used to investigate the properties of two fluorescent DNA base analogs: 2-aminopurine and 6-methylisoxanthopterin. 2-aminopurine is a widely used fluorescent analog of the DNA base adenine. Three-photon excitation of 2-aminopurine is achievable by using intense femtosecond laser pulses in 850-950 nm spectral region. Interestingly, the three-photon excitation spectrum is blue-shifted relative to the three-times-wavelength single-photon excitation spectrum. The maximum of the absorbance band in the UV is at 305 nm, while the three-photon excitation spectrum has a maximum at around 880 nm. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2-aminopurine for DNA-protein interaction studies. However, due to relatively small three-photon absorption cross-section, a good signal-to-noise fluorescence correlation curves take very long time to obtain. Fluorescence properties of 6-methylisoxanthopterin, the fluorescent analog of guanine, were investigated using two-photon excitation. This molecule has the lowest energy absorption band centered around 350 nm, thus, two-photon excitation is attainable using 700 to 760 nm output of Ti-sapphire laser. The excitation spectrum of this molecule in the infrared well matches the doubled-wavelength single-photon excitation spectrum in the UV. The high fluorescence quantum yield of 6-methylisoxanthopterin allows efficient fluorescence correlation measurements and makes this molecule a very good candidate for using in in vitro DNA-protein interaction studies.

Integrins are an important and highly conserved class of extracellular matrix binding membrane bound receptors that provide a functional linkage between cells and their environment. The excellent spatial resolution and short observation time of a modern fluorescence correlation spectroscopy (FCS) instrument is uniquely suited to the study of quantitative differences in integrin behavior on different parts of a single cell. We hypothesize that the application of FCS to the study of these integrin dynamics on the membranes of nerve cell growth cones will give previously unavailable quantitative insight into nerve cell guidance. Accordingly, we have characterized the application of two integrin-binding small peptide-based FCS analytes to primary rat dorsal root ganglion cells. It results that neither the RGD-based nor the SIKVAV-based analyte exhibited specific association with primary rat dorsal root ganglion growth cone membranes in a range that is accessible to FCS observation. However, the techniques and instrumentation developed for these experiments will be useful for evaluating additional integrin ligands.

Tissue patterns appear to be specified by gradients of morphogens. Although it has been established that morphogens are indeed distributed in gradients (morphogenetic gradients), how these gradients arise is not well understood. Two main types of mechanisms have been proposed: (1) diffusion-based, and (2) transcytosis; a third "bucket-brigade" mechanism has also been proposed. The diffusion model is based on the assumption that a morphogen diffuses from a region of origin to form the gradient. The other model proposes that morphogens instead are taken up by one cell and transit that cell to be released on the other side. A third model proposes a “bucket brigade” mechanism in which receptor-bound morphogens on one cell move by being handed off to receptors on an adjacent cell. To provide insight into the mechanism of the formation of the morphogen gradients, we conducted fluorescence correlation spectroscopy (FCS) measurements of morphogen Dpp-EGFP fusion protein in intact Drosophila imaginal disks with two-photon excitation at 850 nm. The FCS results are analyzed with a two-species model. The first species can be attributed to Dpp-EGFP in Brownian motion, while the second species could result from either a large complex involving Dpp-EGFP formed through biochemical reactions, or from anomalous diffusion of Dpp, presumably due to its transport along the cell membranes. Our studies demonstrate for the first time that FCS is capable of investigating molecular dynamics in intact tissues.

We have investigated the kinetics of the conformational changes of a new type of molecular beacons called tripartite molecular beacons. The rate constants corresponding to the opening and closing of the beacons have been obtained from fluorescence correlation spectroscopy experiments. We found that both rate constants are larger for the tripartite molecular beacon than for the corresponding molecular beacon. This paper outlines the importance of using very low excitation intensities for this type of measurements, and of considering the fact that the beacon still emits a residual background fluorescence in its closed form. We also report on the exploration of several strategies to improve the precision of the measurements by increasing the characteristic time associated with the diffusion of the beacons so that it would not be confused with the relaxation time associated with the conformational changes.

In multiplex CARS microscopy the generated anti-Stokes signal is generated and detected simultaneously over a significant part of the vibrational spectrum. The signal-to-noise ratio of the thus detected spectra is limited only by shot-noise. This principle is demonstrated using a dilution series of 2-propanol in water. It is derived theoretically and shown experimentally that for low solute concentrations - in contrast to methods that suppress the non-resonant background - the CARS signal strength from a particular vibrational mode depends linearly on its concentration. Furthermore, excellent agreement is shown between the experimental data and fits to the theory. It is shown that this approach permits rapid (20 ms acquisition) detection of a single lipid mono-layer, with sufficient signal-to-noise to determine the order parameter for the acyl chain packing. Also it is demonstrated that this detection scheme provides an absolute measure of the solute concentration.

A high level of fluorescence background signal rejection was achieved for solid and powder samples by using a combination of simple low-resolution spectrograph and ultrafast intensified/gated CCD camera. The unique timing characteristics of CCD camera match exceptionally well characteristics of Ti:sapphire oscillator allowing fast gated light detection at a repetition rate of up to 110 MHz, making this approach superior in terms of duty cycle in comparison with other time-resolved Raman techniques. The achieved temporal resolution was about 150 ps under 785 nm Ti:sapphire laser excitation. At an average excitation power up to 300 mW there was no noticeable sample damage observed. The strong Hexobenzocoronane (HBC) fluorescence with a lifetime about 2.1 ns was efficiently rejected and Raman spectrum revealed. The combination of spectrometer and ultrafast gated CCD camera allows simultaneous study of spectral and temporal characteristics of emitted light for the fluorophores with a fluorescence lifetime in nanosecond range. It is particularly important in biomedical spectroscopy, since the majority of endogenous fluorophores has a relatively short lifetime of about 1-5 ns. This capability opens an exciting possibility to build a universal instrument for solving multitask problems in applied laser spectroscopy.

We report on a very cost-effective (<$50,000) nonlinear Raman microscopy system utilizing coherent anit-Stokes Raman scattering (CARS). By using narrowband picosecond radiation at 1064 nm and a synchronized broadband continuum radiation in the range of 1150-1400 nm both the broadband spectral imaging and high spectral resolution are achievable in a microscopic set-up. We also suggest and experimentally demonstrate a novel scheme to selective Raman imaging of cellular structures, such as collagen and cellular membranes.

Coherent Anti-Stokes Raman Scattering (CARS)-microscopy has in recent years developed as a promising microscopical technique for label-free microscopy of living cells. We propose a new concept, spectral focusing, for highly efficient coherent anti-Stokes Raman scattering (CARS) microscopy. It allows optimal use of the excitation energy of femtosecond laser pulses in terms of generated CARS signal against a low background. This is accomplished by introducing a linear chirp in the excitation pulses. The temporal delaying of the excitation pulses can be used to record vibrational spectra of a sample. Despite the inherently broad spectral width of the excitation pulses, the technique enables resolution of spectral features 60 times narrower than the bandwidth of the probe light. First applications of this technique are exemplified with CARS of micron sized crystallites of sodium nitroprusside, a commonly used hypotensive agent.

A novel technique for achieving high spectral resolution with a femtosecond laser system is presented. Transform-limited 800 nm, 90 femtosecond (fs) pulses pass off two gratings, stretching the pulse in time to a pulse width of several picoseconds due to an induced linear temporal chirp directly proportional to the grating separation. This chirped pulse is the degenerate pump (ω_P) and probe (ω_p) pulse for the CARS experiment. When overlapped in time with the 1050 nm, 90 fs transform-limited Stokes (ω_S) pulse, only a fraction of the chirped ω_p pulse generates the CARS signal, thereby creating a temporal slit that defines the spectral resolution of the technique. Spectra for liquid methanol and liquid isooctane are presented, with ~6 cm^-1 spectral resolution achieved for isooctane. Resonance enhancement and the mechanism of achieving high spectral resolution are shown by adjusting the ω_S wavelength and ω_p delay relative to the ω_S pulse.

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1050- 1300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.

We report on the integration of active optical elements in a multiphoton microscope to improve the resolution and overall image quality when imaging deeply into biological samples. Optical models were generated of sample systems and these have been compared with the performance of the complete imaging system. The active elements used were commercially available flexible membrane mirrors controlled by custom, home written, software. Significant improvements in image quality have been demonstrated using a range of optimisation routines based on the analysis of the images produced by the system, rather than with a wavefront sensor. A three-fold increase in the resolution 100μm into the sample was achieved.

For the last decade, multiphoton excitation fluorescence microscopy has found numerous applications in biology. Multiphoton microscopy provides several advantages over conventional fluorescence microscopy, including increased penetration depth, improved signal-to-background ratio, and reduced photodamage. Despite its suitability for tissue imaging, multiphoton microscopy has not been used for in-vivo clinical applications due to its lack of portability and its slow imaging speed. Multiphoton microscopy has recently been improved with the development of high speed imaging systems and handheld devices. High speed imaging has been achieved by simultaneously exciting multiple foci in the specimen, known as multiphoton multifocal microscopy (MMM). Compact devices have been developed by combining fiber optic delivery and miniaturized scanning devices. We have developed a handheld device for high speed multiphoton microscopy based on optical fiber delivery, multifoci excitation/detection and a novel scanner. Our system is designed to be sufficiently compact such that it can be used for in vivo clinical imaging, or optical biopsy with potential applications in dermal, cervical and colorectal cancer diagnosis. The power available from a typical Ti:sapphire laser is fully utilized by using multifoci excitation; this results in reduced image acquisition time. Femto-second pulses from a Ti:sapphire laser are delivered to our system through conventional optical fiber. We realize multifoci excitation with a microlens array, and multifoci detection with a multi-anode PMT. A high bandwidth tip tilt mirror is further used as the scanning element for high speed imaging. The feasibility of this handheld MMM is demonstrated by measuring the performance of major components individually. This work is supported by NIH R33 CA091354.

High-speed two-photon imaging based on multi-foci excitation requires the use of spatially resolved detectors, such as charge coupled device (CCD) cameras, instead of single channel photomultiplier tube (PMT). The performance of systems based on both a PMT and a CCD in turbid medium was evaluated by measuring the image point spread function (PSF) and the image contrast as a function of depth and scattering coefficient with single point scanning. We found no significant change in the full-width at half maximum of the point spread function (PSF) for depth up to 100 μm. However, the CCD lost contrast significantly faster as a function of depth and increase scattering. This discrepancy is resolved by measuring a low amplitude but broad tail in the PSF distribution. The tail of the PSF distribution can be up to 200 μm in diameter. We further evaluate scattering effects in the imaging of GFP neurons in a mouse brain slice.

There is an increasing amount of interest in functionalized microstructural, microphotonic and microelectromechanical systems (MEMS) for use in biological applications. By scanning a tightly focused ultra-short pulsed laser beam inside a wide variety of commercially available polymer systems, the flexibility of the multiphoton microscope can be extended to include routine manufacturing of micro-devices with feature sizes well below the diffraction limit. Compared with lithography, two-photon polymerization has the unique ability to additively realize designs with high resolution in three dimensions; this permits the construction of cross-linked components and structures with hollow cavities. In light of the increasing availability of multiphoton imaging systems at research facilities, femtosecond laser manufacturing becomes particularly attractive in that the modality provides a readily accessible, rapid and high-accuracy 3-D processing capability to biological investigators interested in culture scaffolds and biomimetic tissue engineering, bio-MEMS, biomicrophotonics and microfluidics applications. This manuscript overviews recent efforts towards to enabling user accessible 3-D micro-manufacturing capabilities on a conventional proprietary-based imaging system. Software which permits the off-line design of microstructures and leverages the extensibility of proprietary LCSM image acquisition software to realize designs is introduced. The requirements for multiphoton photo-disruption (ablation) are in some ways analogous to those for multiphoton polymerization. Hence, “beam-steering” also facilitates precision photo-disruption of biological tissues with 3-D resolution, and applications involving tissue microdissection and intracellular microsurgery or three-dimensionally resolved fluorescence recovery after photobleaching (FRAP) studies can benefit from this work as well.

Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.

For the purpose of functional third harmonic optical microscopy, it is necessary to find a method to locally enhance third harmonic generation at specific cellular site. We have demonstrated that by matching the third harmonic generation frequency of a Cr:forsterite laser and the surface plasmon resonance frequency of <50-nm silver nanoparticles, localized enhancement of third harmonic intensity of more than 100-folds can be achieved both in phantom and in real biological tissues. This strongly enhanced third harmonic signal can then be applied to specific molecule imaging by attaching the nanoparticles to the target molecule with the advantages of noninvasiveness and deep penetration capability.

We have applied a new, 1030 nm wavelength, infrared diode-pumped femtosecond laser source to multiphoton microscopy, and present comparative results on the efficiency of fluorescence generation versus wavelength for several fluorophores. It is shown that an emission wavelength of 1030 nm is optimal both for GFP and DsRed excitation.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α₆β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α₃β₁-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α₃β₁ integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α₃β₁ integrin, like α₆β₄ integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.

We describe and apply two raster scanning algorithms that are suited for fast imaging and for spectroscopy in a two photon microscope. Imaging can be performed at a rate of 1-100 Hz per line with a closed loop piezo-actuator. In order to reach the single molecule sensitivity and to study the dynamics of the fluorescence emission, the detection is performed via avalanche photodiodes. In a slow scanning algorithm we have implemented photon counting histogram and lifetime analysis on the image. In this way we are able to discriminate between local concentration and molecular brightness and to measure lifetimes on extended samples.

Novel polymeric materials carrying a drug depot have been developed which are suitable for fabrication of photochemically modulated drug delivery devices. In order to avoid uncontrolled drug release the drug is covalently attached to the polymer backbone using a photo-active linker. Controlled drug release from the polymer can be accomplished either via single-photon excitation or by two-photon absorption (TPA). In particular the second possibility is of interest for applications where exposure to day light or UV light may not be omitted. One example are polymeric intraocular lenses (IOL), which are implanted instead of the opaque natural lens during cataract surgery. Secondary cataract formation is quite often observed after implantation of polymeric IOLs. In this study the well known cell toxic agent 5-fluorouracil (5FU) attached to a methylmethacrylate-based polymer was investigated as an IOL which can upon photochemical excitation release 5FU in order to treat or to prevent secondary cataract formation. The photochemical cleavage of the linker molecule was analyzed with single- and two-photon excitation. UV/VIS spectroscopy and HPLC analysis confirmed the release of 5FU form the polymer backbone. The diffusion of the drug precursor out from the polymer as well as the hydrolysis of the drug precursor which leads to 5FU formation were investigated in vitro.

This study explores the feasibility of using cytotoxic derivatives of cyclohexanone/piperidone with strong two-photon absorption (TPA) simultaneously for fluorescent labeling of cancer cells and their subsequent biochemical destruction. Recent studies have shown that these compounds have an efficient two-photon excited fluorescence when pumped with infrared laser radiation. Their molecular cross-section of TPA can be as high as 30.0x10-48cm4s/photon. A cytotoxic two-photon absorbing compound will attach itself selectively to cancer cells and act as a fluorescent label of the cancer cell at harmless levels of excited infrared radiation. Also, when the process of chemical destruction of the cancer cell is over, the fluorescence changes its rate and optical spectrum. In this case, fluorescent labeling could yield more accurate information, not only on the location and distribution of cancer cells, but also on their evolution in time. Compounds combining both properties (cytotoxicity and two-photon excited fluorescence), which are now carried by different chemical agents, are expected to improve the efficiency of the cancer treatment and lower the cost.

The application of second harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterisation of biologically and commercially important plant polysaccharides. We have found that imaging of cellulose requires rather high laser powers which are above optimal values for live cell imaging. Starch, however is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong 3-photon excited fluorescence which may be enhanced by resonance effects.

We report on the novel application of nonlinear optics to study molecular assemblies. By using the third-harmonic generation in solution we were able to determine both the third-order nonlinear susceptibility of collagen and the size of fibrils. The developed approach also helped us in revealing the helix-coil transformation of collagen in solution.

An interesting area of applying multiphoton fluorescence microscopy is in investigating the delivery of fluorescent nanoparticles (quantum dots) across biological barriers such as the skin. Fluorescence nanoparticles are nanometer in size and understanding the delivery mechanisms of these materials across the skin can be important in understanding the delivery of important biological macromolecules for therapeutic purposes. In addition, pathological diagnosis can be performed with the successful delivery of fluorescent nanoparticles.

Photochemically controlled drug release systems require quite special linker systems for the photocleavable attachment of a drug to a polymer backbone. Such linkers must be photochemically cleavable exactly at that point where a detachment of the drug molecule is possible without any added chemical groups in order not to influence its therapeutic properties. The need for such special linker systems is a bottleneck. The photochemical cleavage of chemical bonds requires energies in the UV and may be accomplished either by single-photon or two-photon absorption. We found that dimers of drugs like 5-fuorouracil (5-FU) show significant changes in their physico-chemical properties like solubility in polymer matrices compared to their monomers. Due to this differences they may be used as drug depot forms without the need of any linker system at all. Upon photochemical cleavage of the dimer, either by single-photon or two-photon absorption, the active monomer form of the drug is released without any residual groups attached. Synthesis, chemical and photochemical characterization of dimeric 5-FU is reported and potential applications in polymer systems for medical applications are outlined.

Near-IR ultrafast pulse laser and confocal microscope are combined to create a multiphoton multichannel non-linear imaging technique, which allows in situ 3-D characterization of nonfluorescent nanoparticles in biological systems. We observed intense CARS signals generated from various metal oxides due to their high third-order nonlinear susceptibilities (Chi(3)), which do not depend on the vibrational resonance but on the electronic resonance. We show that fine and ultrafine particles of metal oxides in alveolar macrophage cells may be imaged in vitro using CARS and multiphoton fluorescence microscopy with highest optical resolution for extended periods without photobleaching effects. The advantage of the epi-detection over the forward detection for imaging sub-micron particles has been investigated.

The polarization dependence of the second harmonic emission of purified in-vitro reconstituted fibrils of collagen has been examined. The results confirmed the quasi-hexagonal crystalline structure within the fibrils. Interesting different polarization behaviours were seen between collagen types I and II, which can be utilized as an experimental technique for differentiation.

Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that absorbs strongly at UV wavelengths and fluoresces following multiphoton excitation. BaP is enzymatically converted to hydroxylated metabolites, which are further modified to more polar conjugates. To determine whether fluorsecent signal from parent compound and metabolites could be differentiated by MPLSM, multiphoton excitation spectra were determined from 730-880 nm using a tunable Ti:Sapphire laser. BaP-3-hydroxy (BaP-3-OH) was the most fluorescent and the two conjugated metabolites, BaP-3-sulfate and BaP-3-glucuronide, exhibited fluorescence intensity intermediate between BaP and BaP-3-OH. For example, at 760 nm the fluorescence of conjugated metabolites was four-fold greater, while BaP-3-OH exhibited 16-fold greater fluorescent intensity than BaP. At wavelengths longer than 830 nm there was no excitation of BaP above background. Spectral differences at the longer wavelengths were used to detect the presence of the primary metabolite BaP-3-OH in the presence of parent or conjugated metabolites in fish egg homogenates. Thus, multiphoton excitation spectral characteristics can provide a means to follow the tissue distribution of parent and metabolite in developing fish.

Fouling of contact lenses is often due to tear protein diffusion into and aggregation within the contact lens material. These processes can diminish water and oxygen diffusion and create optical cloudiness of the lens. In order to understand the interactions between proteins and hydrogel contact lens materials a study was designed to measure the diffusivity of two model proteins within hydrogel films of varying composition using fluorescence correlation spectroscopy (FCS). Diffusion of human serum albumin (HSA) and apoferritin (aFER) was examined in a range of ~20 μm thick poly(acrylamide) (pAA) and poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels. Protein diffusivity was measured as a function of depth position within each hydrogel film. The characteristic diffusion time for two proteins in pHEMA hydrogels increased relative to both their diffusivity in solution and in pAA hydrogels, indicating that the protein-pHEMA interaction rather than the degree of hydrogel crosslinking is responsible for the observed effects. The resulting spatial representation of the molecular diffusion of proteins into and interaction with hydrogel materials builds a basis on which to conduct similar studies using commercial contact lens samples.

The femtoliter excitation volume of multiphoton microscopy renders all emitted photons useful in detecting fluorescence signals. We have previously demonstrated the viability of transmitting the emitted fluorescence via multimode optical fibers onto a photomultiplier tube (PMT) in a full-field arrangement. A custom MPLSM, based on a commercially available confocal microscope was developed to readily switch between the regular descanned path for confocal microscopy and our non-descanned pathways (direct detection) supporting fiber-coupled detection. We now wish to demonstrate the efficacy of fiber-based detection using a side-by-side comparison of our fiber-coupled paradigm to the traditional method of directly focusing the fluorescence, through air, onto a PMT. To effectively compare the two methods, we have incorporated a second direct detection epi-fluorescence pathway for air-coupling onto a PMT that does not affect the performance of the fiber-based MPLSM. We found that fiber-based detection compares favorably against traditional direct detection. We demonstrate the viability of fiber-based detection for high-resolution neuronal brain slice imaging.

FRET imaging is widely used in biology to map protein interactions in living cells. A number of recent technical developments promise to provide more accurate and quantitative measurement FRET efficiencies and protein bonding ratio. Using a reference FRET construct based on a doubly labeled DNA, one can estimate the accuracy of different techniques like spectral or lifetime imaging to resolve FRET. Further improvement have lead to the possibility of measuring in a single detector both spectral and lifetime information from the fluorescence emitted by the sample. We are currently building such a system based on an intensity modulated multi-anode PMT and have developed a global fitting algorithm to extract valuable information from combined spectral and lifetime imaging data sets.

Intensity based FRET visualizes protein molecules in living cells and tissues. Lifetime techniques, however, will demonstrate the dynamic functional activity of the protein molecules, because its signal does not depend on changes in fluorophore concentration or excitation intensity. If a laser pulse excites a large number of similar molecules with a similar local environment, and as long as no energy is transferred to another molecule, the lifetime is the “natural fluorescence lifetime”. If energy is transferred, however, the actual fluorescence lifetime is less than the natural lifetime, because an additional path for de-excitation is present. With the occurrence of FRET, strong energy transfer results in extreme quenching of the donor fluorescence and a decrease in the fluorescence lifetime. In this paper we will explain the development of the two-photon FLIM-FRET microscopy with our existing multiphoton microscopy and demonstrate the change in donor lifetime by photobleaching the acceptor molecules in living cells.

Endogenous fluorophores, such as NAD(P)H/FAD and collagen/elastin, have been regarded as in vivo quantitative fluorescence biomarkers for precancerous changes of epithelial tissue. However, the fluorescence signal measured by conventional spectroscopy is a mixture of autofluorescence from the epithelium and deep structures. The dominant fluorescence of collagen/elastin from connective tissue in deep layers creates serious challenge for extracting the epithelial fluorescence of NAD(P)H/FAD that is weak, but important for the characterization of tissue pathology. In this work, we instrumented a confocal fluorescence spectroscopy system and a two-photon excited fluorescence spectroscopy system to measure the depth-resolved single- and two-photon fluorescence spectra from the rabbit esophageal tissues. The excitation wavelengths were 349 nm and 735 nm, respectively. Both systems provided good optical sectioning. The information obtained from depth-resolved fluorescence was generally consistent with the histology of the examined tissue sample. The NAD(P)H signals from epithelial layers were clearly separated from the collagen signal from deep layers. In addition, strong second harmonic generations given by collagen fibers were observed. This work demonstrates that depth-resolved fluorescence spectroscopy may produce more accurate information on the diagnosis of tissue pathology.