This PDF file contains the front matter associated with SPIE-OSA Proceedings Volume 6630, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The method of Spectral Precision Distance Microscopy (SPDM) has been used to determine distances between two FISH (Fluorescence-in situ-Hybridization)-labeled gene regions on chromosome 9. To this end we applied methods to correct for chromatic aberrations of the microscope optics alone and also of the sample induced aberrations due to mismatch of the refractive indices. Using a confocal microscope and a threshold based position determination algorithm, positions could be measured with an accuracy of about 65 nm inside of fixed cell nuclei. Distances obtained from the measurements have been verified using a 3D computer model of the cell nucleus. In principle, this SPDM approach could be combined with novel fluorescence microscopes to obtain structural information well below the optical resolution. At present the precision limit of the distance measurements is set by variations of the refractive index throughout the specimens.

In this study, we present the detailed imaging of the nematode Caenorhabditis elegans (C. elegans) at microscopic level by performing Two-Photon Excitation Fluorescence (TPEF), Second-Harmonic Generation (SHG) and Third Harmonic Generation (THG) measurements. Due to their inherent advantages in comparison with the conventional microscopy (increased resolution, ability to section deep within tissues, minimization of photodamage and photobleaching effects), the non-linear microscopy techniques comprise a unique and extremely powerful tool for the extraction of valuable and unique information from biological samples. We developed a compact, reliable, inexpensive non-linear imaging system, utilizing femtosecond laser pulses (1028nm) for the excitation of biological samples. The use of 1028nm wavelength as excitation source minimizes photodamage effects and unwanted heating (due to the water absorption) of the biological specimens. The emitted THG signal lies in the near UV part of the spectrum (343nm). Detailed and specific structural and anatomical features of the worm were collected by recording THG signals. Consummative, unique information concerning the morphology and the functions of the nematode was obtained by implementing the combination of THG, SHG and TPEF image contrast modalities on the same microscope.

The intrinsically ordered arrays of proteins in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very powerful tool in the investigation of structural dynamics occurring in muscle during active production of force. A full characterization of the polarization-dependence of the SHG signal represents a very selective information on the orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber axis. Optimizing the setup for accurate polarization measurements with SHG, we developed a line scan imaging method allowing measurement of SHG polarization curves in different physiological states. We demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.

In this work we use multiphoton microscopy to observe the post surgery structure variation of rabbit cornea after photorefractive keratectomy (PRK). In addition, we added mitomycin C (MMC) to the post surgery rabbit cornea in order to investigate the effect of MMC treatment on the postoperative regeneration.

Conductive keratoplasty (CK) is a new refractive surgery for presbyopia and hyperopia patients. By applying radio frequency current at the peripheral regions of cornea, collagen, the most abundant composition of corneal stroma, shrinks due to the heat generated. The shrinkage at the periphery alters the corneal architecture and achieves clearer focus for near vision. In this work we use multiphoton microscopy to observe the post surgery structure variation at both submicron resolution and over a large region within the tissue. Since collagen can be induced to generate strong second harmonic generation (SHG) signal, multiphoton excitation provide direct visualization of collagen orientation within corneal stroma. In addition, since the SHG intensity of collagen tissue deteriorates with increasing thermal damage [1-3], our methodology can be used to characterize the extent of corneal stroma damage from the CK procedure. Finally, the influence of CK on the morphology and distribution of keratocytes can also be investigated by detecting multiphoton excited autofluorescence from the cells.

The last decade has witnessed momentous advances in fluorescence microscopy. The introduction of novel fluorescent markers, together with the development of original microscopy techniques, made it possible to study biomolecular interactions in living cells and to examine the structure and function of living tissues. The emergence of these innovative techniques had a remarkable impact on all the life sciences. However, many biological and medical applications involve the detection of minute quantities of biomolecules, and are limited by the signal weakness in common observation conditions. Here, we show that silver and gold-coated microscope slides can be used as mirror substrates to efficiently improve detection sensitivity when fluorescence microscopy is applied to micrometer-thick biological samples. We report a fourfold enhancement of the fluorescence signal and a noticeable strengthening of the image contrast, when mirror substrates are used with standard air microscope objectives. We demonstrate that metal-coated substrates provide the means to get sensitivity-enhanced fluorescence detection with dry optics, while keeping a wide field observation and a large depth of field. This is a crucial advantage for automated and high-throughput applications to cell and tissue diagnostic analysis.

New approach to acquisition, analysis and reconstruction of Microscopic Fluorescence Lifetime Images (FLIM) and Hyper Spectral Images (HSI) is presented. Spatial selectivity is obtained with a Digital micro-Mirror Device Illuminator (DMDI) combined with a fluorescence microscope. More specifically spatially selective illumination is achieved by tilting the relevant group of micro-mirrors to reflect the excitation light from a UV picosecond laser diode towards chosen regions on the sample. In the first step, the whole field fluorescence image is collected by a color CCD camera for further qualitative spectral analysis and sample segmentation. In the next step fluorescence of the sample is excited segment by segment and acquired with a single detector (e.g. photomultiplier in photon counting mode for FLIM, CCD spectrophotometer for HSI) from the whole field of view. The acquired fluorescence is analyzed in following step for further FLIM or HSI image reconstruction. This can be facilitated by either raster scanning over the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow to Globally Analyze (GA) the sample and supply on-line good initial values for fitting algorithms associated with the subsequent raster-scanning, which in turn decreases the computation time needed to obtain a satisfactory quality-of-fit. FLIM/HIS images acquired on phantoms and on biological samples demonstrate the possibilities for temporal and spectral "unmixing".

Total internal reflection fluorescence microscopy (TIRFM) is a powerful optical technique to observe fluorescence close to surfaces. Associated with fluorescence lifetime imaging, TIRFM enables to measure contrasts independent of fluorophores concentration and reveal intracellular activity with subwavelength axial resolution. We developed an original setup which allows, thanks to a wide-field time resolved detection, to measure nanoseconds fluorescence lifetimes of membrane receptors in order to apprehend their signalisation pathway.

Two-photon microscopy has been successfully applied in biological and material sciences since 1990. However, it is known that the resolution of two-photon imaging is adversely affected from the index mismatch induced spherical aberrations. Spherical aberration increases the focal volume and causes degradation of image resolution as one images deeper into the specimen. In this work, we propose to use this intrinsic artifact to measure the refractive index in specimens of uniform refractive indices. Using the intensity profiles of standard refractive liquids as our reference, we can compare the intensity profile of an unknown specimen of uniform refractive index with that of the reference to determine the refractive index of the unknown specimen.

Single molecule detection methods, in particular those based on fluorescent labels offer the possibility to gain not only qualitative but also quantitative insight into the function of complex biological systems. Fluorescence Correlation Spectroscopy (FCS) is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness in the pico- to nano-Molar concentration range, with broad applications in Biology and Chemistry. Although FCS in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts FCS to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by FCS-curve fitting, a FCS dilution series and confocal bead scanning. The results are compared in the view of quantitative FCS measurement and analysis.

The theoretical development of a new iterative method based on a diffraction imaging model for the computation of a specimen's complex transmittance function (magnitude and phase) from DIC images is presented. This new method extends our initial work (RD method presented by Preza¹) which was based on the assumption that the specimen does not absorb light and thus only the specimen's phase function or optical path length (OPL) distribution was computed from rotationally-diverse (RD) DIC images. In this paper, we quantify this approximation by modeling the magnitude of the synthetic object as a deviation from unity by a small perturbation. Synthetic, noiseless DIC data are generated from these test objects and processed with the RD method. Our results show that although for weakly absorbing objects the RD method may be adequate for some applications, in general the results can be quantitatively unacceptable. This supports the development of the new alternating minimization method presented in this paper. Preliminary results from the current implementation of the AM method show that the discrepancy measure utilized in the method goes to zero as iterations increase but a constrain on the estimated magnitude is necessary in order to obtain quantitative specimen properties.

The goal was to develop an upright microscope platform for the screening of slides employing one- and two-photon laser scanning techniques. A highly compact, vibration damping unit was created, which combines novel concepts for moving a slide in three dimensions, keeping it focused while doing so, scanning a laser-focus over the sample using novel galvanometer-control concepts, combining and separating excitation and emission beam and spectrally dispersing the emitted light by a linearized prism-spectrograph. Spectral detection is achieved by turning a 128 x 128 back-thinned EMCCD detector in a continuously reading spectral point-detector. To make the unit even ore versatile, it can be turned into a conventional wide field fluorescence microscope, enabling rapid routine observation to select regions of the sample for a subsequent, more detailed confocal analysis.

Here we present a multiphoton excitation microscopy setup extending the excitation wavelengths far beyond one micron. A synchronously pumped fs-OPO (OPO PP-Automatic, APE) pumped by a fs-Ti:Sapphire oscillator is used as the light source. The biological relevant wavelength range from <1050 to >1350 nm can be covered with a fixed pump frequency and a single optics set through hands free, automated tuning. Together with the Ti:Sapphire pump laser (Coherent Chameleon) excitation wavelengths from 680 to 1600nm are achieved. Two separate scanners (LaVision BioTec) are optimized for Ti:Sapphire and OPO wavelength ranges respectively including dispersion compensation for maintaining the short pulses at the sample site. An overall transmission of 30-38% up to 1400 nm was verified. Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of the penetration depths, strongly reduced photo damage, a 30fold increased excitation efficiency of red fluorescent dyes, including RFP and Cy5.5. Excitation at 1100 nm further leads to a 4fold decrease in autofluorescence, resulting in a significantly improved signal-to-noise ratio. The resolution is slightly reduced in comparison to Ti:Sapphire excitation, which corresponds well to the longer excitation wavelength used. Phototoxicity and photobleaching is reduced by 80- 95%. An OPO pump wavelength around 800nm opens up the possibility to use the Ti:Sapphire laser to pump the OPO and to excite the sample simultaneously giving the opportunity to excite dyes such as GFP with the pump laser and red shifted fluorophores (for instance RFP) with the OPO at the same time.

Inline holography with a pinhole is extended by using a multi-spot source and a CCD. The arrangement is aimed on microscopic imaging. By using a pinhole array as a multi-spot source the size of observable samples can be extended. The intensity of the illumination of samples can be increased up to several orders. It enables the increase of the distance to the CCD to get an enlarged, pixel adapted hologram pattern. Very small pinholes are applicable and the useable aperture can be increased, too.

Inline holographic microscopy is known as a technique for absolute phase retrieval. Any distortion of the needed reference wave, e.g. from the surfaces of a test slide, causes reconstruction errors. We apply a multi plane detection technique, which generates the same information as a hologram but does not use any reference wave. The technique was demonstrated for microscopic resolution in case of amplitude objects [1], [2]. This paper focuses on imaging and reconstruction of phase objects. The technique is experimentally applied to a Pleurosigma test chart and a phase test standard. Objects with a phase step of π/3 and lateral structure sizes of 2 μm are well resolved.

Refractive-index mismatch in conventional confocal microscopy produces severe degradation on axial resolution of sectioning image because the spherical aberration is generated in specimen. In this study, we propose a polarized photon-pairs confocal laser scanning microscope (PCLSM) in which a two-frequency linear polarized photon-pairs (LPPPs) laser beam is produced. The common-path propagation of LPPPs integrated with optical heterodyne technique not only can reduce the spherical aberration but also decreases scattering effect in specimen at same time. Therefore, the better axial and lateral resolutions of the sectioning image are produced simultaneously. In the experiment, a verification and comparison between PCLSM and conventional confocal laser scanning microscope (CLSM) on the ability of cancellation of spherical aberration induced by cover glass are demonstrated experimentally. Finally, the ability of PCLSM which can decrease the spherical aberration based on the common-path propagation of LPPPs associated with polarization gating, spatial coherence gating and spatial filtering gating is discussed.

We describe a method of optical refocusing for high numerical aperture systems that is particularly relevant for confocal and multiphoton microscopy. Crucially the method avoids spherical aberration that is common to other optical refocussing systems. Further refocusing is implemented remotely from the specimen.

We demonstrate the use of gold nanorods as molecularly targeted contrast agents for two-photon luminescence (TPL) imaging of cancerous cells 150 µm deep inside a tissue phantom. We synthesized gold nanorods of 50 nm x 15 nm size with a longitudinal surface plasmon resonance of 760 nm. Gold nanorods were conjugated to antibodies against epidermal growth factor receptor (EGFR) and labeled to A431 human epithelial skin cancer cells in a collagen matrix tissue phantom. Using a 1.4 NA oil immersion objective lens, we found that excitation power needed for similar emission intensity in TPL imaging of labeled cells was up to 64 times less than that needed for two-photon autofluorescence (TPAF) imaging of unlabeled cells, which would correspond to a more than 4,000 times increase in emission intensity under equal excitation energy. However, the aberrations due to refractive index mismatch of the immersion oil and the sample limit imaging depth to 75 µm. Using a 0.95 NA water immersion objective lens, we observe robust two-photon emission signal from gold nanorods in the tissue phantoms from at depths of up to 150 µm. Furthermore, the increase in excitation energy required to maintain a constant emission signal intensity as imaging depth was increased was the same in both labeled and unlabeled phantom, suggesting that at the concentrations used, the addition of gold nanorods did not appreciably increase the bulk scattering coefficient of the sample. The remarkable TPL brightness of gold nanorods in comparison to TPAF signal makes them an attractive contrast agent for early detection of cutaneous melanoma.

A new, broadly tuneable synchronously pumped picosecond optical parametric oscillator (OPO) for Coherent anti-Stokes Raman Scattering (CARS) microscopy is presented. It is based on a non-critically phase-matched LBO crystal, pumped by the second harmonic (532 nm) of a mode-locked Nd:Vanadate laser. The tuning range covers 680 nm to 990 nm (Signal beam) and 1150 nm to 2450 nm (Idler beam), thus completely substituting picosecond - Ti:Sapphire lasers. By using the Signal and Idler as pump and Stokes beams for CARS microscopy, this translates into a vibrational frequency range of ~1350 - >10.000 cm^-1. Both beams are extracted from the same cavity mirror and therefore propagate collinearly. Due to the mechanism of their generation, Signal and Idler are optically synchronized, and thus, perfectly overlap in space and in time with no jitter. The 5 ps pulses generated are close to transform limited and of excellent beam quality (M² < 1,1) and show a high pointing stability. The output power for Signal and Idler is about 2 W @ 4 W pump power leading to 50% overall conversion efficiency. The perfect spatial and temporal overlap, stable operation, and broad tuneability makes the described OPO an ideal and nearly hands-free laser source for CARS microscopy. The longer operational wavelength range results in higher penetration depths and lower sample photodamage than previously reported systems. Thus, our CARS source is optimized to image highly heterogeneous tissue samples, as will be shown in several applications. The latest methods for further sensitivity improvements will be presented.

Lipid droplets have become a major research topic recently, as they are found to be involved in obesity related diseases. Most of this research has been focused on the localization of the proteins playing a role in lipid droplet formation or breakdown. The role of different lipid species however remains unclear because it is difficult to distinguish different fatty acids with the present microscopy techniques. Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue of spontaneous Raman scattering. Multiplex CARS microscopy can provide quantitative, chemical and physical information, making it an excellent tool to study the composition and thermodynamic phase of lipid droplets. To investigate the potential of CARS in this field, we have incubated HeLa cells with four different fatty acids, varying in saturation. The fatty acids were internalized by the cells and stored as lipid droplets, which we imaged with multiplex CARS microscopy. We were able to distinguish either of the fatty acids as such in lipid droplets inside the cells. Furthermore, we found that solid phase fatty acids were fluidized when present in lipid droplets. This illustrates the potential of CARS microscopy to elucidate the possible role of the chemistry of fatty acids in lipid droplet regulation.

The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the monitoring of molecular changes and distribution of biomolecules and in particular of low molecular weight markers as they occur during the differentiation of neuroblastoma cells.

Multiphoton microscopy is an interesting optical technique, which allows for non-invasive imaging of highly light scattering media such as human skin. Recent reports have showed the potential of applying this technique for 3D visualisation of cell structures of biological tissue without previous sectioning of the tissue samples. In this study, we have applied two-photon microscopy on excised lesions of human non-melanoma skin cancer ex vivo in order to find diagnostic criteria using this technique. The skin samples have been investigated by a multiphoton microscopy system based on a fs-pulsed Ti:sapphire laser connected to a confocal microscope. The autofluorescence of the skin was detected using excitation at 780 nm. The cell nuclei distribution turned out to be one important parameter, which can be used for discriminating between tumour and normal tissue. We are now developing a technique for automatic detection and characterisation of tissue, based on an image analysis algorithm. The detection of cell nuclei has been found crucial for this purpose. The goal is to develop a fast characterisation algorithm that can be used on line in connection to in vivo investigations. This would allow for a true non-invasive biopsy technique in the future.

In vivo simultaneous collagen and elastin measurements using the multiphoton tomograph DermaInspect have been performed in skin dermis. We showed it was possible to get simultaneous measurements of autofluorescence (AF) and Second Harmonic Generation (SHG) with a newly developed device using 2 PMTs for time-correlated single photon counting. Unlike elastin (AF), collagen structures are able to generate second harmonics (SHG). Comparing the images and SHG / AF ratios recorded in the depth of the outer and inner sides of the forearm of two European female volunteers (31 and 60 years old, respectively) shows differences in collagen and elastin fibres density. It decreases with depth in the 60 years old volunteer compared to the younger one, and the skin of younger volunteer shows more collagen.

In this investigation, we used in vivo nonlinear optical microscopy to image normal and carcinogen DMBA treated skin tissues of nude mice. We acquired two-photon autofluroescence and second harmonic generation (SHG) images of the skin tissue, and applied the ASI (Autofluorescence versus SHG Index) to the resulting image. This allows us to visualize and quantify the interaction between mouse skin cells and the surrounding connective tissue. We found that as the imaging depth increases, ASI has a different distribution in the normal and the treated skin tissues. Since the DMBA treated skin eventually became squamous cell carcinoma (SCC), our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy. We envision this approach to be effective in studying tumor biology and tumor treatment procedures.

Transdermal drug delivery provides a non-invasive route of drug administration, and can be a alternative method to oral delivery and injection. The stratum corneum (SC) of skin acts as the main barrier to transdermal drug delivery. Studies suggest that depilatory enhances permeability of drug through the epidermis. However, transdermal delivery pathway and mechanism are not completely understood. Previous studies have found that depilatory changes the keratinocytes of epidermis, and cause the protein in combination with lipid extraction of SC to become disordered. Nevertheless, those studies did not provide images of those processes. The aim of this study is to characterize the penetration enhancing effect of depilatory agent and the associated structural alterations of stratum corneum. Fresh human foreskin is treated by a depilatory agent for 10 minutes and then subjected to the treatment of fluorescent model drugs of hydrophilic rhodamine and hydrophobic rhodamine-RE. The penetration of model drugs is imaged and quantified by multiphoton microscopy. Our results showed that the penetration of both hydrophilic and hydrophobic agents can be enhanced and multifocal detachment of surface corneocytes is revealed. Nile red staining revealed, instead of a regular motar distribution of lipid around the brick of corneocytes, a disorganized and homogenized pattern of lipid distribution. We concluded that depilatory agents enhance drug penetration by disrupting both the cellular integrity of corneocytes and the regular packing of intercellular lipid of stratum corneum.

The trans-cutaneous pathway for drug delivery is of particular interest since it allows a simple and non-invasive administration of pharmaceutically relevant compounds. As the skin is an effective barrier for many of these compounds, various strategies have been developed to enable and control the trans-cutaneous transport. Here we discuss, how multiphoton microscopy and spectral imaging can be valuable tools for the analysis of the penetration pathways of topically applied drugs. A time dependent study of the cutaneous penetration of a fluorescent drug model released from a nano-particular carrier is presented. The localization of single nano-particles in human skin (ex vivo) and the discrimination of different fluorescent compounds, as the drug model, the particle's label and the cutaneous endofluorescence by spectral imaging and selective excitation is shown. Multiphoton imaging techniques were found to be excellent methods for the non-invasive evaluation of cutaneous drug delivery strategies and analysis of dermal penetration pathways down to the sub-cellular level.

The advantage of confocal fluorescence microscopy is the ability to acquire high resolution images of fluorescent specimens non-invasively. On the other hand, the advantage of Raman microscopy is the ability to provide the chemical characteristics of specimens from spectroscopy. To obtain simultaneously high resolution images and chemical characteristics of specimens, fluorescence signals from stained cell and Raman spectrum from cell itself should be separated. By separating two kinds of signals, confocal fluorescence image and Raman spectrum are acquired simultaneously at the same position. In this paper, we demonstrate a confocal fluorescence microscopy combined with the Raman microscopy. And we propose a method that eliminates Raman spectrum of fluorophore itself from Raman spectrum of the stained cell and that obtains simultaneously confocal fluorescence image from stained cell and Raman spectrum of cell itself without replacing a cell.

A new technique for improving the axial resolution of confocal microscope is proposed. Based on the interference between two different frequency beams, which are separated axially, a frequency domain field confined focal spot is generated. The effective region made by the interference makes the point-spread function (PSF) of confocal microscope sharper. The three-dimensional imaging equations are derived. The intensity distribution of frequency domain field confined focal spot is proportional to the absolute value of the product of two fields. Three-dimensional intensity pointspread function (IPSF) is calculated numerically. The farther two beams are separated axially, the sharper IPSF is obtained. The numerical results show that the full width half maximum (FWHM) of the IPSF is improved by factor of 1.78 maintaining the strength of side lobe at 0.5 relative to main lobe. Also simulations for two-point resolution show the same improvement in the axial resolution.

Liver is the chemical factory in body responsible for important functions such as metabolism and detoxification. When liver can not be regenerated in time to amend damages that has occurred, failure of hepatic functions can result. Traditionally, the study of liver pathology has depended on histological techniques, but such methods are limited to ex-vivo observation. In order to study hepatic metabolism in vivo, we have designed a hepatic imaging chamber made of biocompatible titanium alloy (6V4Al-Ti, ELI grade). In combination with multiphoton and second harmonic generation microscopy, our approach allows the intravital observation of hepatic intravital activities to be achieved. Processes such as hepatic metabolism and disease progression can be studied using this methodology.

We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets, resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8x8 multi anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and orange peel cells.

We demonstrate an alternative light source for CARS microspectroscopy based on a fiber laser and a photonic crystal fiber. The light source simultaneously delivers a picosecond pump pulse at 1033.5 nm and a frequency shifted femtosecond Stokes pulse, tunable from 1033.5 nm to 1400 nm. This corresponds to a range 0 - 2500 cm^-1, so that Raman-active vibrations in this frequency range can be probed. The spectral resolution is 5 cm^-1, given by the spectral width of the pump pulse. The frequency range that can be probed simultaneously is 200 cm^-1-wide, given by the spectral width of the Stokes pulse. The achievable average powers are 50 mW for the pump and 2 mW for the Stokes pulse. The repetition rate is 35 MHz. We demonstrate the capability of this light source by performing CARS microspectroscopy and comparing CARS spectra with Raman spectra.

Fluorescence correlation spectroscopy (FCS) is widely used for investigation of concentration, diffusion coefficients and dynamics of single molecules. To introduce spatial resolution in FCS measurement, we develop a novel FCS system, which uses an electron-multiplying charge-coupled device (EM-CCD) to get FCS data at each pixel. We tested 3 samples, which have different concentrations of fluorescent beads, and successfully investigated the difference of correlation coefficients of FCS signal. In addition, we introduce a new illumination method for EM-CCD based FCS measurement, to limit depth of a observation volume. Although a evanescent field has a nature of limited penetration depth, the penetration depth which is 50 to 200nm in typical, is short in comparison with the resolution in the lateral direction. As a result FCS measurement becomes too sensitive in the depth direction, but worse in lateral direction. So we introduce a novel illumination method, in which a laser beam is incident with an angle slightly smaller than the critical angle to illuminate fluorescent molecule (critical-angle illumination). The depth of observation volume can be controlled with the angle of incidence. We expect this method to be applied to a measurement of local diffusion coefficient of molecules in living cells.

In the biology field there is a high demand for three-dimensional (3D) microscopy techniques owing to the need for obtaining depth information. Confocal microscopy is the reference method but it has the drawback of slow image acquisition time. Different configurations were developed to obviate this such as spinning-disk that basically consists in a set of parallel confocal microscopes and line scanning that uses line illumination so the scanning in the fast axis is not required. Amongst other 3D techniques there is the digital microscopy that normally uses a wide-field microscope to acquire 2D images (optical sections) of the specimen containing substantial contributions from out-of-focus portions. To remove this, a computational method is used that is based on the definition of a model for the process of image formation and recording. A compromise should be taken between a more accurate model with best results or a simplified one with a lower computational processing time. This work is based on a laboratory prototype of a wide-field microscope with white-light illumination using a CMOS linear image sensor. It aims at the application of one simplified model that takes advantage of a new method for the determination of experimental point spread function (PSF) that uses a specimen consisting of a set of lines in two-perpendicular directions due to the anisotropy that results from detector geometry. The 1D and 2D PSF were built. A simulation of the application of the model was developed using Matlab ((Mathworks ©) using different simulated specimens. Its preliminary results will be presented.

We describe the characterisation of a hyperspectral fluorescence lifetime imaging microscope that exploits high-speed time-gated imaging technology and a tunable continuum source for 6-D fluorescence imaging. This line-scanning confocal microscope can record the full spectral-temporal (i.e. excitation-emission-lifetime) fluorescence matrix at each pixel in a three dimensional (x-y-z) sample. This instrument has been applied to biological samples including model membranes and live cells labelled with the phase-sensitive membrane dye di-4-ANEPPDHQ, for which significant variation of lifetime with emission wavelength is observed.

The dendritic processes of neurons have been shown to possess active and dynamic properties that give them the ability to modulate synaptic integration and shape individual synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly light scattering brain tissue requires an imaging/recording mechanism with high spatio-temporal resolution as well as optical sectioning and random access site selection capabilities. Our lab has made significant steps in developing such a system by combining the spatial resolution and optical sectioning ability of advanced imaging techniques such as confocal and multi-photon microscopy with the temporal resolution and random access capability provided by acousto-optic laser scanning. However, all systems that have been developed to date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which many neurons can be studied since they represent complex three-dimensional (3D) structures. We have previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of acoustooptic deflectors and does not require axial movements of the objective lens. We have also shown how, when used with the ultra-fast laser pulses needed in multi-photon microscopy, this scheme inherently compensates for the spatial dispersion which would otherwise significantly reduce the resolution of acousto-optic based multi-photon microscopy. We have now coupled this scanning scheme to a modified commercial research microscope and use the combined system to effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are in the low microsecond (μs) range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D structures such as neuronal dendrites at several thousand volumes per second.

Due to the unique optical properties, gold nanoparticles (NPs) can play a useful role in biological cellular imaging as biological probes. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) system, we recorded the images of Karpas 299 cells incubated without, or with gold NPs, and ACT1 antibodies conjugated with gold NPs. From the FLIM, we can easily discriminate the difference among different experiment conditions due to the distinct lifetime between cells and gold NPs. Our results present that nonconjugated gold NPs are accumulated inside cells, but conjugated gold NPs bind homogeneously and specifically to the surface of cancer cells. For single Karpas 299 cells, the signal is very week when the excitation power is about 10mw; while the power is approximately 28 mw, a very sharp cell imaging can be obtained. For the Karpas 299 incubated with ACT1 conjugated gold NPs, while the excitation power is 10mw, gold NPs have clear fluorescence signal so that the profile of cells can be detected; Signal of gold NPs is very strong when the power arrived in 20mw. These results suggest that the multiphoton lifetime imaging of antibody conjugated gold NPs can support a useful method in diagnosis of cancer.