Computer-controlled, repeatable ultra-short pulse lasers and a broad wavelength range autocorrelator have opened up practical spectral investigations of the group delay dispersion in laser scanning microscope systems. The laser output pulse duration was measured via intensity-based (two-photon absorption) autocorrelation and the laser spectral bandwidth was measured via spinning spectrometer. The separate measurements provided the ~0.39 timebandwidth product for two different Coherent ultra-short pulse lasers. The laser-scanning system pulse durations were measured at the sample plane with high numerical aperture objective lenses. The pulse broadening of ultra-short laser pulses through a laser-scanning multi-photon system has been characterised experimentally from 720nm to 950nm. The pulse spreading of individual laser-scanning system components was estimated from Gaussian pulse dispersion with data acquired using a standard, characterized objective lens and an external GaAs:P diode signal. The objective lens was found to produce the most pulse broadening, and most apochromatic objective lenses showed two-fold increases in the magnitude of dispersion values. All spectra of measured components followed the same shape and trend, as expected for normal dispersion. During this study, the average power incident on the Carpe autocorrelator was kept below ~50mW (130fs and 800nm), but this limitation has been removed in current versions of the autocorrelator. Dielectric broadband reflector mirrors introduce significant dispersion, which changes sign with wavelength; this can reduce or increase the system pulse spreading, but will certainly make component spectral measurements challenging.

Histological analysis is the clinical standard for assessing tissue health and the identification of pathological states. Its invasive nature dictates that its use should be minimized without compromising diagnostic accuracy. A promising method for minimally invasive histological analysis is optical biopsy, which provides cross sectional or 3D images without any physical sectionings. Optical biopsy method based on multiphoton excitation microscopy can image cross-sectional image for deep tissue structures with subcellular resolution based on tissue endogenous fluorescence molecules. Despite its suitability for tissue imaging, multiphoton microscopy has not been used for in vivo clinical applications due to both compactness and imaging speed problems. We are developing a high-speed, handheld, miniaturized multifocal multiphoton microscope (H²M⁴) as an optical biopsy probe to enable optical biopsy with subcellular resolution. We incorporate a compact raster scanning actuator based on optimizing a piezo-driven tip tilt mirror by increasing its bandwidth, and reducing its nonlinearity. For flexible light delivery, we choose a photonic bandgap crystal fiber, which transmits ultrashort pulsed laser delivery with reduced spectral distortion and pulse width broadening. We further demonstrate that this fiber produces minimal spatial mode distortion and can achieve comparable image point spread function (PSF) as free space delivery. We further investigate the applicability of multiphoton microscopy for clinical dermal investigation by imaging ex vivo human skins with both normal and abnormal physiologies. This demonstrates the performance of H²M⁴ and the possibility of optical biopsy for diagnosing skin diseases.

Despite the fact that laser scanning confocal microscopy (LSCM) has become an important tool in modern biological laboratories, it is bulky, inflexible and has limited field of view, thus limiting its applications. To overcome these drawbacks, we report the development of a compact dual-clad photonic-crystal-fiber (DCPCF) based multiphoton scanning microscope. In this novel microscope, beam-scanning is achieved by directly scanning an optical fiber, in contrast to conventional beam scanning achieved by varying the incident angle of a laser beam at an objective entrance pupil. The fiber delivers femtosecond laser pulses for two-photon excitation and collects fluorescence back through the same fiber. Conventional fibers, either single-mode fiber (SMF) or multimode fiber (MMF), are not suitable for this detection configuration because of the low collection efficiency for a SMF and low excitation rate for a MMF. Our newly invented DCPCF allows one to optimize collection and excitation efficiency at the same time. In addition, when a gradient-index (GRIN) lens is used to focus the fiber output to a tight spot, the fluorescence signal collected back through the GRIN lens forms a large spot at the fiber tip because of the chromatic aberrations of the GRIN lens. This problem prevents a standard fiber from being applicable, but is completely overcome by the DCPCF. We demonstrate that this next generation scanning confocal microscope has an extremely simple structure and a number of unique features owing to its fundamentally different scanning mechanism: high flexibility, arbitrarily large scan range, aberration-free imaging, and low cost.

We developed a set up consisting of an Optical Tweezers plus linear and non-linear micro-spectroscopy system to add the capabilities of manipulation and analysing the captured object. For the confocal micro-spectrometer we used a 30 cm monochromator equipped with a cooled back illuminated CCD. The spectroscopic laser system included a cw and a femtosecond Ti:sapphire lasers that allowed us to perfom raman, hyper-raman, hyper-rayleigh and two-photon excited (TPE) luminescence in trapped particles with an Nd:YAG cw laser. With the cw Ti:sapphire laser we obtained raman spectra of a single trapped polystyrene microsphere and red blood cells and silicon to evaluate the performance of our system. The femtosecond Ti:sapphire laser was used to observed hyper-rayleigh and hyper-raman peaks of SrTiO₃ with 60s integration time only. In the past, hyper-raman measurements required integration times of few hours, but the huge intensity together with the 80 MHz repetition rate of the femtosecond laser decreased this time for the seconds range. The sensitiveness of our system also permitted to observe more than 14 Mie resonance peaks in the TPE luminescence of a single stained trapped microsphere, which agrees well with the calculations. This system opens up the possibility to perform spectroscopy in a living trapped micro-organism in any desired neighbourhood and dynamically observe the chemical reactions and/or mechanical properties change in real time.

The ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning. Our method relies on temporal focusing of the illumination pulse. The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration at the focal plane, before stretching again beyond it. Combining temporal focusing with line-scanning microscopy results in an enhanced depth resolution, equivalent to that achieved by point scanning. Both the scanningless and the line-scanning techniques are applied to obtain depth-resolved two-photon excitation fluorescence (TPEF) images of drosophila egg-chambers.

Nonlinear Raman spectroscopy is employed to study structural transformation of collagen (type I) in solution. Relatively strong signal and polarization background suppression reveal significant changes of the vibrational spectrum of collagen, which are attributed to the formation of cholesteric globules. Circular dichroism spectroscopy confirms the concentration dependent structural transformation. Excellent axial resolution achieved in nonlinear Raman microscopy also allows to study for the first time the effect of large molecule adhesion on the surface.

Optical microscopy that can visualize the molecular vibration with a nanometric spatial resolution has been realized by a combination of near-field optics and coherent anti-Stokes Raman scattering (CARS) spectroscopy. A metallic probe with a sharp tip is used to strongly enhance optical near-field in the local vicinity of the tip owing to the excitation of local surface plasmon polariton. CARS signals of molecules in the local area can be strongly induced by the plasmonic field. We have visualized DNA molecules and single-walled carbon nanotubes (SWNTs) with a spatial resolution far beyond the diffraction limit by the tip-enhanced near-field CARS microscopy.

Diagnostic imaging trends are progressing toward the molecular level with the advent of molecular imaging techniques. Optical molecular imaging techniques that utilize targeted exogenous contrast agents or detect endogenous molecular signatures will significantly extend our ability to detect early pathological changes in biological tissue, and treat diseases early when they are most amenable to be cured. We have developed a technique called Nonlinear Interferometric Vibrational Imaging (NIVI) that takes advantage of the coherent nature of coherent anti-Stokes Raman scattering (CARS) processes and the coherent optical ranging and imaging capabilities of optical coherence tomography (OCT). OCT uses interferometry and heterodyne detection in the time or spectral domain to localize reflections of near-infrared light deep from within highly-scattering tissues. OCT has found wide biological and medical applications, and recently, molecular imaging methods have been developed. By utilizing the molecular-sensitivity of CARS, NIVI performs optical ranging and multi-dimensional molecular imaging with OCT-like optical systems, enabling the retrieval of not only χ⁽³⁾ [chi⁽³⁾] amplitude but also phase information, the rejection of problematic non-resonant background four-wave-mixing signals, enhanced sensitivity from heterodyne detection, and a relaxation of the high-numerical aperture focusing requirements present in CARS microscopy. We present recent progress and advances in NIVI, including depth-ranging capabilities that extend significantly deeper than current CARS microscopy methods and are potentially more suitable for cross-sectional deep-tissue in vivo imaging.

Refractive index mismatch induced spherical aberration (RIMISA) is a ubiquitous problem for in-depth optical biological imaging and it is an effect most researchers like to avoid. Associated with RIMISA is a broadening of the point-spread-function (PSF) which leads to a degradation of image resolution and the loss of specimen signal. It is these features that render RIMISA to be useful for one important application in biological application, the determination of sample refractive index. We will show how RIMISA under multiphoton and confocal microscopy can be used to determine the refractive indices of uniformly luminescent specimens and a comparison will be made between the multiphoton and confocal approaches.

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two-channel detection and two-photon excitation flow cytometry (T³FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo.

The intrinsic optical sectioning, reduced light-scattering, and reduced photodamage of multiphoton laser-scanning microscopy (MPLSM) has generated great interest for this technique in experimental Neuroscience, as it enables to study both structure and function of fine neuronal processes within living brain tissue. At present, virtually all MPLSM systems employ galvanometric beam positioning. Due to this inertia-limited approach, single-dimension line scans are employed to achieve frame rates sufficient for functional imaging. Although such line scans allow adequate sampling rates (≤1kHz), two significant drawbacks remain. First, the majority of scan time is wasted by illuminating regions of no interest, while sacrificing signal integration time at sites-of-interest. Second, the sites from which signals can be recorded are limited to those along a single line. Alternatively, acousto-optic (AO) beam positioning with high-resolution TeO₂ deflectors allows inertia-free skipping between arbitrary sites within the field-of-view in <15μs. This achieves high sampling rate recording at multiple, non-adjacent sites quasi-simultaneously (1-5kHz frame rate, 12-60 sites). Such a multi-site optical recording system would greatly advance studying complex neuronal function, by enabling membrane potential or calcium transients to be observed throughout the complex geometry of neuronal dendrites. This paper presents images and functional recordings from living neurons within brain slices, acquired with AO-MPLSM. Our novel imaging system allows a user to collect structural images first and subsequently select sites of interest for fast functional imaging. To demonstrate the system’s power, we present high-speed recordings (1kHz) from >10 sites within the dendrites of pyramidal neurons in acute brain slices, at signal-to-noise ratios comparable to line-scan systems.

We use dissociated cultures of E-18 rat cortical neurons to study how they process the information. To correlate electrophysiological data with corresponding network structure, we observe effects of the stimuli on structural changes in this culture using multiphoton microscopy. To keep our 2D and 3D cultures alive for long-term studies, it is necessary to protect them against photodamage. At the same time, we need a flexible microscope design to accommodate our multielectrode array (MEA) electrophysiological station. We have constructed a custom-designed multiphoton microscope based on design of Tsai et al. The microscope is optimized for two-photon imaging to collect maximum possible fluorescent signal using minimum excitation laser intensity. Special attention is paid to get uniformly illuminated images and the ability to use the entire bandwidth of the pulsed laser (700-1000 nm) with the same set of optical components. Flexibility of the design will allow us to easily change or incorporate other optical components suitable for different experimental needs. This microscope will allow us to do electrophysiology and imaging concurrently while maintaining the optimum temperature and CO2 levels.

Many biochemical reactions and processes are regulated by proteins associated with cellular membranes. Trans-membrane proteins play an important role in many aspects of cellular development, cellular migration and signaling, and many diseases. Quantitative measurement of protein dynamics under various experimental conditions can give insights into the mechanisms of interaction and the functionality of the protein. Fluctuation techniques, such as fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS), have been used for such dynamic measurements in membranes. However, FCS is limited to fast dynamics, and ICS works best on a flat 2-dimensional area. We present an alternative way to measure protein transport in spherical (non flat) living cells that combines laser scanning microscopy and image correlation methods: ring correlation spectroscopy (RCS). The RCS analysis is performed on CLSM or two-photon cross-sectional images of labeled proteins in the cell membrane, where the optical sectioning gives a “ring” of fluorescence in the images. We present computer simulations of two dimensional diffusion confined to the surface of a spherical shell, where the RCS analysis can extract the set input parameters from the simulation. As well, we present RCS analysis of two-photon microscopy images of Pre-B leukocytes cells expressing CD44 labeled with EGFP.

Most fluorescence fluctuation experiments use a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. Scanning of the laser beam in a circular pattern collects the fluorescence signal from a moving observation volume. The fluctuations contain now information about temporal and spatial properties of the sample. Synchronization between beam scanning and data collection allows us to evaluate the fluctuations for every position along the scanned trajectory. We present the theory of position-sensitive scanning fluorescence fluctuation spectroscopy and experimentally verify the theory. This technique is useful for detecting and characterizing directed transport processes in the presence of diffusion.

Fluorescence correlation spectroscopy (FCS) could provide a more useful tool for intracellular studies and biological sample characterization if measurement times could be reduced. While an increase in laser power can enable an autocorrelation function (ACF) with adequate signal-to-noise to be acquired within a shorter measurement time, excitation saturation then leads to distortion of the ACF and systematic errors in the measurement results. An empirical method for achieving reduced systematic errors by employing a fitting function with an additional adjustable parameter has been previously introduced for two-photon FCS. Here we provide a unified physical explanation of excitation saturation effects for the three cases of continuous-wave, pulsed one-photon excitation, and two-photon excitation FCS. When the time between laser pulses is longer than the fluorescence lifetime, the signal rate at which excitation saturation occurs is lower for pulsed excitation than for cw excitation, and due to the disparate timescales of the photophysical processes following excitation, it is lower still for two-photon excitation. We use a single-molecule description of FCS to obtain improved analytical ACF fitting functions for the three cases. The fitting functions more accurately account for saturation effects than those previously employed without the need for an additional empirical parameter. Use of these fitting functions removes systematic errors and enables measurements to be acquired more quickly by use of higher laser powers. Increase of background, triplet photophysics, and the cases of scanning FCS and fluorescence cross-correlation spectroscopy are also discussed. Experimental results acquired with a custom built apparatus are presented.

We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This procedure offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 to 800 nm for excitation which are coupled to the microscope via a single mode fiber. The corresponding emission is guided to a fibre coupled photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC system, via the unique Time-Tagged Time-Resolved (TTTR) data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently create the full fluorescence lifetime image (FLIM). The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows for example to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for advanced Forster Resonance Energy Transfer (FRET) analysis. In this paper, we present some selected results acquired with standard laser scanning microscopes upgraded towards temporal resolution.

Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

Reef-building corals are dependent on dinoflagellate algal symbionts (zooxanthellae). Within the range of habitats of any one coral species there can be huge variations in light intensities, so there is a risk of photoinhibition from excess light. In extremes of light and heat, senescent algae are expelled en masse, a phenomenon known as coral bleaching. In freshly isolated tissue the chlorophyll fluorescence has a lifetime of ~1.1ns. 6 hours and 15 hours after isolation the zooxanthellae looked visually healthy, but the lifetimes had increased to 2ns after 6 hours and 2.2ns after 15 hours. Zooxanthellae which were visibly damaged or necrotic had a mean lifetime of 3ns. Lifetime of chlorophyll fluorescence is thus a sensitive indicator, revealing effects in cell metabolism before any structural changes are evident. The occurrence of FRET between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504 and amilFP593 (names indicate emission peaks) were 2.8ns, 2.9ns and 2.9ns respectively. In the coral sample, imaging the entire emission spectrum from 420nm, the mean lifetime was reduced to 1.5ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3ns, supporting this interpretation.

FLIM/FRET is an extremely powerful technique that can microscopically locate nanometre-scale protein-protein interactions within live or fixed cells, both in vitro and in vivo. The key to performing sensitive FRET, via FLIM, besides the use of appropriate fluorophores, is the analysis of the time-resolved data present at each image pixel. The fluorescent transient will, in general, exhibit multi-exponential kinetics: at least two exponential components arise from both the interacting and non-interacting protein. We shall describe a novel method and computer program for the global analysis of time resolved data, either at the single level or through global analysis of grouped pixel data. Kinetic models are fitted using the Marquardt algorithm and iterative convolution of the excitation signal, in a computationally-efficient manner. The fitting accuracy and sensitivity of the algorithm has been tested using modelled data, including the addition of simulated Poisson noise and repetitive excitation pulses which are typical of a TCSPC system. We found that the increased signal to noise ratio offered by both global and invariance fitting is highly desirable. When fitting mono-exponential data, the effects of a ca. 12.5 ns (ca. 80 MHz) repetitive excitation do not preclude the accurate extraction of populations with lifetimes in the range 0.1 to 10 ns, even when these effects are not represented in the fitting algorithm. Indeed, with global or invariance fitting of a 32x32 pixel area, the error in extracted lifetime can be lower than 0.4% for signals with a peak of 500 photon counts or more. In FRET simulations, modelling GFP with a non-interacting lifetime of 2.15 ns, it was possible to accurately detect a 10% interacting population with a lifetime of 0.8 ns.

Various problems arising during molecular imaging of different fluoroprobes and metabolites used in photodynamic therapy could be circumvented by focusing on time-resolved detection. For this, an interesting new method seems to be time-correlated single photon counting, where a time-to-amplitude converter determines the temporal position and a scanning interface connected to the scanning unit of a laser microscope determines the spatial location of a signal. In combination with spectral resolved detection (spectral lifetime imaging) the set-up achieves the features of highly sophisticated lifetime imaging systems. The photoactive substance on which 5-ALA PDT is based, is protoporphyrine IX which is synthesized in mitochondria. Alternatively, other metabolites from 5-ALA could be involved. Subcellular differentiation of those metabolites without extensive extraction procedures is not trivial, because of highly overlapping spectral properties. Measuring the fluorescence lifetime on a subcellular level could be a successful alternative. To record lifetime images (τ-mapping) a setup consisting on a laser scanning microscope equipped with detection units for time-correlated single photon counting and ps diode lasers for short-pulsed excitation was implemented. The time-resolved fluorescence characteristics of 5-ALA metabolites were investigated in solution and in cell culture. The lifetimes were best fitted by a biexponential fitting routine. Different lifetimes could be found in different cell compartments. During illumination, the lifetimes decreased significantly. Different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In addition cells were coincubated with the nuclear staining dye DAPI, in order to investigate the cell cycle. Using appropriate filtering or alternatively spectral lifetime imaging the time-resolved fluorescence of DAPI could be very well distinguished from 5-ALA-metabolites. In contrast to ALA, the lifetime of DAPI, which was best fitted monoexponentially did not change during photobleaching, making this dye a perfect internal standard.

Multiphoton imaging has developed into an important technique for in-vivo research in life sciences in the last few years. A near-infrared laser beam is focused into a sample such that multiphoton-absorption can be generated which stimulates a fluorescence signal as well as second harmonic generation (SHG). Recently it has been shown that it is possible to image the epidermis in vivo with a resolution of about 1 μm. It was possible to produce 3-dimensional autofluorescence maps of the investigated tissue. However, the depth range of this technique is limited through the working distance of the focusing optics mostly to the epidermal part of the skin. Gradient index lenses offer possibilities to expand the imaging depth into the dermal layer. With typical diameters of up to 2mm and lengths between 2 and 6 cm they are capable of transmitting the laser, fluorescence and SHG radiation and to be integrated into an imaging system. First results of the applicability of gradient index lenses for imaging of skin are presented.

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, with many advantages over existing one- or two-photon fluorescence techniques. A non-linear phenomenon employing mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry. Many important structural proteins such as collagen and cellulose show intrinsic SHG, thus providing access to sub-resolution information on symmetry. However, we are particularly interested here in "resonance-enhanced" SHG from styryl dyes. In general SHG is a combination of a true second-order process and a third-order process dependent on a static electric field, such that SHG from membrane-bound dyes depends on a cell's trans-membrane potential. With simultaneous patch-clamping and non-linear imaging of cells, we have found that SHG is a sensitive probe of trans-membrane potential with sensitivities that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging. With the sensitivity of SHG to local electric fields from other sources such as the membrane dipole potential as well as the quadratic dependence of SHG on concentration, we have found that SHG imaging of styryl dyes is also a powerful technique for the investigation of lipid phases and rafts and for the visualization of the dynamics of membrane-vesicle fusion following fertilization of an ovum.

An intrinsic cellular emission allied with second harmonic signals are promising in-vivo clinical diagnostic tools for corneal abnormalities, cancer and wound healing. The extent of corneal damage in K14-DN-Clim mice will be addressed.

Optical coherence tomography (OCT) provides micrometer scale structural imaging by coherent detection of light backscattered from a sample. The significance of OCT would be greatly enhanced by the capability to measure molecular specific signals. Observation of fluorescent markers is unfortunately not possible using OCT because fluorescence is not a coherent process. Instead, the methods being researched to extend OCT to include molecular contrast are, amongst others: transient absorption (pump-probe imaging), Coherent Anti-Stokes Raman Scattering (CARS), and second harmonic generation. With any of these techniques, the quality of the images obtained is limited by the conversion efficiency of the nonlinear process, which is inevitably much less than 1 and thus has resulted in low SNR and long acquisition times in previously reported work. Recent publications have demonstrated a sensitivity advantage of 20-30dB for spectral domain (SD) techniques in OCT over conventional time domain acquisition. The increased sensitivity of SD OCT systems stands to benefit the small signal powers observed in molecular contrast OCT. We have constructed a prototype spectral domain second harmonic OCT system utilizing a 130 femtosecond Nd:Glass laser and a pair of custom spectrometers for simultaneous acquisition of the fundamental and second harmonic signals. We report a 10dB increase in sensitivity while imaging 100 times faster than in initial reports of second harmonic OCT using time domain systems.

Harmonics optical microscopy (HOM) provides a truly “noninvasive” tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α₆β₄ integrin, laminin 5 and α₃β₁ integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the “clinical latent phase” that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.

Diseases associated with the liver, a major internal organ, can lead to serious health problems. In this work, we present multiphoton images of normal and diseased liver specimens and we will characterize the changes to pathological liver specimens. In particular, we will focus on the physiological changes associated with liver fibrosis. Our results show that multiphoton microscopy is a useful technique for distinguishing normal and diseased liver tissues and that it has potential applications for in vivo diagnosis of liver diseases.

Animal embryo development exhibits a complex ensemble of cell movements that are tightly regulated by developmental gene expression. It was proposed recently that mechanical factors may also play an important role during development. Investigating these dynamical processes is technically challenging and requires novel in vivo investigation methods. We show that multiphoton microscopy can be used for both perturbing and analyzing morphogenetic movements in vivo. (i) nonlinear microscopy is well adapted for the sustained imaging of early Drosophila embryos despite their highly scattering nature; (ii) femtosecond pulse-induced ablation can be used to process specific tissues in vivo. Combining this approach with multimodal microscopy (two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG)), we report the successful quantitative modulation of morphogenetic movements in vivo. Our data provides insight to the issue of morphogenesis regulation.

Transgenic lines carrying a specific tissue tagged by green-fluorescence-protein (GFP) have been a powerful tool to developmental biology because they encapsulate the expression of endogenous genes. Traditionally with two-photon fluorescence microscopy based on a femtosecond Ti:sapphire laser (with a wavelength between 700-980nm), green fluorescence can be excited by simultaneous absorption of two photons for high-resolution three-dimensional (3D) optical imaging. However for in vivo biological applications, Ti:sapphire-laser based optical technology presents several limitations including finite penetration depth, strong on-focus cell damage, and phototoxicity. For high optical penetration and minimized photodamages, two-photon imaging based on light sources with an optical wavelength located around the biological penetration window (~1300nm) is desired, where unwanted light-tissue interactions including scattering, absorption, and photodamages can all be minimized. Previous experiments around the optical penetration window indicated inefficient green fluorescence excitation of GFP through three-photon absorption. Red fluorescence protein is thus highly desired for future non-invasive in vivo two-photon imaging. Screening from embryos injected with DNA fragment containing a heart-specific regulatory element of zebrafish cardiac myosin light chain 2 gene (cmlc2) fused with HcRed gene, we generate a zebrafish line that has strong two-photon red fluorescence expressed in cardiac cells based on a 1230nm femtosecond light source working in the biological penetration window. Combined with its nonlinearity, high penetration depth, and minimized photodamages, this method provides superb imaging capability compared with the traditional GFP based two-photon microscopy, offering deep insight into the noninvasive in vivo studies of gene expression in vertebrate embryos.

In this work we used our set up consisting of an optical tweezers plus non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited (TPE) luminescence of captured single cells conjugated with quantum dots of CdS and CdTe. The CdS nanocrystals are obtained by our group via colloidal synthesis in aqueous medium with final pH = 7 using sodium polyphosphate as the stabilizing agent. In a second step the surface of CdS particles is functionalized with linking agents such as Glutaraldehyde. The CdTe quantum dots are functionalized in the its proper synthesis using mercaptoacetic acid (AMA). We used a femtosecond Ti:sapphire laser to excite the hyper Rayleigh or TPE luminescence in particles trapped with an Nd:YAG cw laser and a 30 cm monochromator equipped with a cooled back illuminated CCD to select the spectral region for imaging. With this system we obtained hyper Rayleigh and TPE luminescence images of macrophages and other samples. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.

Remarkable advances have been made in studying the dynamic events of protein molecules in living cells and tissues using advanced light microscopy imaging techniques and green fluorescent proteins (GFPs). Identification of the interacting protein partners is critical in understanding its function and place in the biochemical pathway, thereby establishing its role in important disease processes. FLIM-FRET microscopy technique, allow the study of proteins in multiple ways including what proteins are expressed, where they are expressed- and where they move over time. It has been observed that the eCFP-eYFP FRET pair may not be that suitable to localize the association of protein molecules since the eCFP has two-components lifetime. The new Cerulean green fluorescent protein appears to have only one-component lifetime. We describe the extensive investigation of eCFP and Cerulean to study the dimerization of the transcription factor CCAAT/enhancer binding protein alpha in GHFT1-5 living cell nucleus using the time-correlated single photon counting (TCSPC) FLIM-FRET microscopy.

Multiphoton microscopy (MPM) and optical coherence tomography (OCT) are two important techniques for non-invasive and high-resolution imaging of highly scattering biomedical tissues. MPM and OCT provide complementary information about tissues as the detected signals originate from different substances and mechanisms. Combining MPM and OCT can simultaneously provide rich functionality and morphology information about biomedical tissues. We report a novel system which combines multiphoton and optical coherence microscopy. A femtosecond Ti:Sapphire laser is used as the light source which has a pulse width of 10 fs and a spectral bandwidth of 100 nm. The ultrafast Ti:Sapphire laser provides the short pulse and the broad bandwidth required by high-resolution MPM and OCT, respectively. By matching the resolution and the imaging volume of MPM and OCT, we achieved co-registration between the MPM and OCT images. Dispersion compensation is critical in this MPM/OCT system and its effect is discussed. Representative images of co-registered MPM/OCT are presented.

In this paper we present experimental and theoretical study of limitations on deep imaging of biological tissue structure using multiphoton fluorescence microscopy (MFM), that are imposed by scattering of short NIR pulses in optically turbid medium. The time stretching of the laser pulse and its transversal widening limit the maximum imaging depth of MFM since the magnitude of fluorescence response from the medium is the power function of energy flux density in the excitation laser beam. Increasing the imaging depth by raising the laser power may result in phototoxic damage of biological objects. We have theoretically and experimentally studied temporal structure of collimated femtosecond laser pulse scattered in an optically turbid model medium with controlled concentration of micron-sized spherical beads. In parallel we investigated phototoxic effect on biological liquid at high-intense femtosecond NIR pulse irradiation for various expositions by structural analysis of dehydrated blood plasma droplet. The irradiation regimes were found with complete recovery of the droplet structure, as well as with its slight and severe stable modification.

Two-photon autofluorescence and second harmonic generation (SHG) microscopy are useful imaging techniques for studying tissue components. In this work, we apply this imaging modality to study porcine eye. In particular, we use SHG microscopy to investigate the structural changes to excised porcine corneas and found that this technique is useful for studying its structural changes under thermal treatment.

We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. When applied to Forster resonance energy transfer (FRET) measurements, these setups are capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.

Confocal microscopy is the method of choice in biological 3D-imaging, however, the axial resolution is limited to ~500 nm. During the last decade it has been successfully demonstrated that the axial resolution can be substantially improved with 4Pi microscopy. We report a 4Pi microscope realized as a fast beam scanning system consisting of a 4Pi-module linked to a state-of-the-art confocal microscope. As a result, the advantages of the confocal system such as scanning, sensitive multicolor detection and imaging speed are combined with the superior resolution of 4Pi microscopy. This novel microscope is eminently suited for biological applications. It is designed both for single-photon and for two-photon picosecond excitation, and also enables joint coherent illumination and detection, i.e. 4Pi type C. The superior PSF and OTF of the system enable 80 nm axial resolution in cells mounted in aqueous media. We present the optical design of the system and demonstrate an up to 7-fold improved optical sectioning in live cells.

A scheme for fast three-dimensional laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counter-propagating acoustic waves that are both chirped and offset in their frequencies, it is possible to simultaneously scan both in the axial and lateral directions. This scheme also has the added benefit of inherently compensating for spatial dispersion when ultra-fast laser pulses are used in acousto-optic multi-photon microscopy.