Living pancreatic cancer tissues grown subcutaneously in nude mice are studied by in vivo Raman spectroscopy and autofluorescence imaging. Comparing the same point spectra of alive pancreatic cancer tissue to that of the dead tissue, it is found that they are different each other. The results suggest that the spectral changes reflect the protein conformational changes in the tumor tissue with death of the host animal. From the result of autofluorescence study, in vivo autofluorescence imaging has potential as a method to assign the histological elements of the pancreatic cancer tissue without any staining. These results strongly suggest that combination of these techniques is very important to study biological tissue.

Nonlinear optical (NLO) microscopy provides a minimally invasive optical method for fast molecular imaging at subcellular resolution with 3D sectioning capability in thick, highly scattering biological tissues. In the current study, we demonstrate the imaging of arterial tissue using a nonlinear optical microscope based on photonic crystal fiber and a single femto-second oscillator operating at 800nm. This NLO microscope system is capable of simultaneous imaging extracellular elastin/collagen structures and lipid distribution within aortic tissue obtained from coronary atherosclerosis-prone WHHLMI rabbits (Watanabe heritable hyperlipidemic rabbit-myocardial infarction) Clear pathological differences in arterial lumen surface were observed between healthy arterial tissue and atherosclerotic lesions through NLO imaging.

Raman microspectroscopy is applied for the first time to study the effect of high-power light irradiation on isolated melanosomes and melanosomes in retinal pigmented epithelium cells.

Histopathologic recognition is the gold standard in breast cancer diagnoses and is a primary determinant tool for cancer research. Unfortunately, the manual nature of histopathologic recognition leads to low throughput analysis, delays in decision-making and errors. Here, we present an automated means to accurate histologic recognition using mid-infrared molecular spectroscopy. Fourier transform infrared (FT-IR) spectroscopic imaging is combined with statistical pattern recognition and high throughput sampling to provide automated tissue segmentation into constituent cell types. The method does not need dyes or probes and dispenses with human input. Results demonstrate that the technique is capable of accurate histologic segmentation that can potentially become competitive with that attained by conventional immunohistochemical analyses.

Confocal reflectance full-pupil and divided-pupil line-scanning microscopes provide optical sectioning and image nuclear detail in skin. Line-scanning with linear detectors is a simpler alternative to point-scanning for imaging weakly scattering epidermis and the oral epithelium. With illumination of 830 nm, a water immersion lens of numerical aperture 0.9 and slit width three times smaller than the diffraction-limited line width, the instrumental full width at half maximum (FWHM) optical sectioning (linespread function) for the full-pupil design is 1.4 +/- 0.07 μm, which degrades through fullthickness human epidermis to 2.8 +/- 0.78 μm. The lateral resolution is 0.7±0.10 μm, which degrades to 1.6±0.28 μm through human epidermis. The divided-pupil design demonstrates instrumental optical sectioning of 1.7 μm, which degrades to 7.6 μm through human epidermis. The lateral resolution is 1.0 μm, which degrades to 1.7 μm. Heavy scattering in the dermis decreases contrast. Images of skin in-vivo show nuclear detail as expected with the predicted and experimentally verified sectioning. However, pixel crosstalk and speckle artifact degrade image quality in strongly scattering and aberrating tissues. The sources of degradation (aberration and scattering) are evaluated for the two design to assess the feasibility of these techniques for in vivo imaging.

A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBD-cholesterol) as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.

Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing loss are commonly available for research. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes the study of their hair cells difficult. We developed a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae were harvested, left intact within their otic capsule bone, and glued upright in a recording chamber. The bone overlying the region of the cochlear epithelium to be studied was opened and Reissner's membrane was incised. A fluorescent indicator was applied to the preparation to image intracellular calcium. A custom-built upright two-photon microscope was used to image the preparation using three dimensional scanning. We were able to image about 1/3 of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we found that outer hair cells demonstrated increased fluorescence compared with surrounding supporting cells. Thus, this methodology can be used to visualize hair cell calcium changes and mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are reduced.

Evanescent wave phase profiling is incorporated with digital holography into a new surface imaging technique termed total internal reflection holographic microscopy (TIRHM). Quantitative images of tissue structures and profiles of cellular membranes are presented to demonstrate the method's performance capabilities. Applications of this technique include measurements of cellular membranes and their transport processes without the addition of fluorophores. The angular spectrum method to compensate for tilt anamorphism due to the inclined TIR plane is also discussed.

Tumors are highly metabolically active and thus require ample oxygen and nutrients to proliferate. Neovasculature generated by angiogenesis is required for tumors to grow beyond a size of about 1-2mm. Functional tumor vasculature also provides an access point for development of distant metastases. Due to the importance of the microvasculature for tumor growth, proliferation, and metastasis, the microvasculature has emerged as a therapeutic target for treatment of solid tumors. We employed spectral imaging in a rodent window chamber model to observe and measure the oxygen transport function of tumor microvasculature in a human renal cell carcinoma after treatment with a fast acting vascular disrupting agent. Human Caki-1 cells were grown in a dorsal skin-fold window chamber in athymic nude mice. Spectral imaging was used to measure hemoglobin saturation immediately before, immediately after and also at 2, 4, 6, 8, 24 and 48 hours after administration of the tubulin binding agent OXi4503. Up to 4 hours after treatment, tumor microvasculature was disrupted from the tumor core towards the periphery as seen in deoxygenation as well as structural changes of the vasculature. Reoxygenation and neovascularization commenced from the periphery towards the core from 6 - 48 hours after treatment. The timing of the effects of vascular disrupting agents can influence scheduling of repeat treatments and combinatorial treatments such as chemotherapy and radiation therapy. Spectral imaging can potentially provide this information in certain laboratory models from endogenous signals with microvessel resolution.

Ocular uveal melanosomes contain both eumelanin and pheomelanin. The ratio of these two melanins has been discussed in relation to the epidemiological data for skin cancer rates, with increased incidence observed for increased relative concentrations of pheomelanin. Recent studies suggest that a similar trend exists underlying the epidemiology of uveal melanomas. In the present study, the biomolecular organization of human iridal melanosomes from different colored irises were examined to determine if the photoreactivity changes with the altered eumelanin:pheomelanin ratio, and whether such changes can account for epidemiological results. Specifically, photoemission electron microscopy (PEEM), a unique surface-sensitive, direct-imaging technique capable of providing chemical information not obtained by other electron microscopies, was used in combination with Duke University's tunable UV free electron laser (FEL) to determine the surface electrochemical properties of melanosomes from blue and dark brown irides. The results demonstrate that the melanins are organized such that pheomelanin is encased by eumelanin. This "casing model" is consistent with kinetic information available on the early steps of melanogenesis and provides new insights into molecular mechanisms underlying the epidemiology of uveal melanoma.

A Raman spectroscopic technique enables to observe intracellular molecules without fixation or labeling procedures in situ. We demonstrated a classification of human lung cancer cells with Raman spectroscopy and principal component analysis. Normal lung cell-lines and 4 pathological types of cancer cell-lines were seeded on culture dishes and examined. It was as a preliminary study for direct Raman imaging spectroscopy, which could be available for clinical use, to diagnose cancer. The result suggests that Raman spectroscopy could be a complementary method for immunohistology study. We also constructed a new direct Raman imaging system consisting of a high sensitive CCD image sensor, narrow band pass-filters, and a background-free electrically tunable Ti:Sapphire laser. The observation wavelengths can be switched immediately for the purpose of malignancy rapid diagnosis or real time measurement for apoptotic cells. The potential ability of the direct Raman imaging system is supposed to evaluate apoptosis by UV irradiation and anticancer drug-treatment for living lung cancer cells.

We demonstrated cell imaging without any stain by far-field 2-color infrared (IR) super-resolution microscopy, combining laser fluorescence microscope and picosecond transient fluorescence detected IR (TFD-IR) spectroscopy. TFD-IR spectroscopy detects IR absorption by monitoring fluorescence due to an electronic transition from a vibrational excited level by an additional visible light. By using the IR microscopy based on TFD-IR spectroscopy, the spatial resolution of the image can be increased to the visible diffraction limit of sub-μm, i.e., the IR is super-resolved. Cell auto-fluorescence due to flavin molecules was monitored for label-free detection of the cellular components. The fluorescence image of an A549 cell was obtained by introducing both an IR light at 3300 nm and a visible light at 560 nm. The spatial resolution of the image was estimated to be 1.6 μm. This is about 2.5-times higher resolution than the diffraction limit of IR light. The fluorescence intensity of the images at 3448 nm was smaller than that at 3300 nm, corresponding to the smaller IR absorption. Therefore, IR spectral imaging of a single cell was achieved with superresolution.

Raman spectroscopy has been utilized to investigate properties of biomolecules due to its capability of detecting molecular vibrations that represent molecular species, structures and environmental conditions. In this research, we used Raman scattering to image dynamics of molecules in living cells. By combining the 532 nm excitation and a slitscanning detection technique, we observed dynamics of molecular distributions of cytochrome, protein beta sheets, and lipids in an unstained HeLa cell during mitosis with a frame rate of 5 minutes.

The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for establishing the ability of HPLC-LIF protein profiling technique for discrimination, using hard clustering and Fuzzy clustering methods. The clustering algorithms have quite successfully classified the profiles as belonging to normal, cancer of cervix, and oral cancer conditions.

The fluorescence spectra, delayed luminescence (DL) spectra and DL decay dynamics of human serum were studied by fluorescence and time resolved emission spectrum technology under different excitation conditions in this paper. The results we obtained are shown as follows: (1) the DL spectrum is similar to the time resolved fluorescence spectrum within 50ns after Ps laser pulse excitation. (2) The intensity and decay time of DL from the serum samples are dependent on excitation power and irradiation time. Under fixed excitation power, the longer irradiation time is, the higher the DL intensity; after the excitation energy reaches about 200mJ, the DL intensity is nearly unchanged. The change of DL decay time follows the similar regulation to that of DL intensity. (3) As the excitation energy increases, the spectral distribution of the relative intensities exhibits an observable change. The higher the excitation energy is, the stronger the relative intensity at short wavelength region. The results show that the delayed luminescence of human serum is mainly originated from its delayed fluorescence, phosphorescence, and induced bio-photon emission. These results may be also useful for the development of serum diagnosis technology.

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Due to increasing availability of pharmaceuticals from many sources, a need is growing to quickly and efficiently analyze substances in terms of the consistency and accuracy of their chemical composition. Differences in chemical composition occur at very low concentrations, so that highly sensitive analytical methods become crucial. Recent progress in dispersive spectroscopy with the use of 2-dimensional detector arrays, permits for signal integration along a long (up to 12 mm long) entrance slit of a spectrometer, thereby increasing signal to noise ratio and improving the ability to detect small concentration changes. This is achieved with a non-scanning, non-destructive system. Two different methods using P&P Optica high performance spectrometers were used. High performance optical dispersion Raman and high performance optical absorption spectroscopy were employed to differentiate various acetaminophen-containing drugs, such as Tylenol™ and other generic brands, which differ in their ingredients. A 785 nm excitation wavelength was used in Raman measurements and strong Raman signals were observed in the spectral range 300-1800 cm^-1. Measurements with the absorption spectrometer were performed in the wavelength range 620-1020 nm. Both Raman and absorption techniques used transmission light spectrometers with volume phase holographic gratings and provided sufficient spectral differences, often structural, allowing for drug differentiation.

Small-molecule microarrays composed of tens of thousands of distinct synthetic molecules, natural products, and their combinations/modifications provide a high-throughput platform for studying protein-ligand interactions. Immobilization of small molecule compounds on solid supports remains a challenge as widely varied small molecules generally lack unique chemical groups that readily react with singly or even multiply functionalized solid support. We explored two strategies for immobilizing small molecule compounds on epoxy-functionalized glass surface using primary-aminecontaining macromolecular scaffolds: bovine serum albumin (BSA) and amine-modified poly-vinyl alcohol (PVA). Small molecules with N-hydroxysuccinimide (NHS) groups were conjugated to BSA or amine-modified PVA. Small-molecule-BSA conjugates and small-molecule-PVA conjugates were subsequently immobilized on epoxy-functionalized glass slides through amine-epoxy reactions. Using an oblique-incidence reflectivity difference (OI-RD) scanning microscope as a label-free detector, we performed a comparative study of the effectiveness of BSA and PVA as macromolecular scaffolds for anchoring small molecule compounds in terms of conjugation efficiency, surface immobilization efficiency, effect of the scaffold on end-point and kinetics of subsequent binding reactions with protein probes.

We describe a new oblique-incidence reflectivity difference (OI-RD) scanning microscope for high-throughput screening, in microarray format on functionalized glass slides, small-molecule compound libraries for protein ligands. The microscope employs a combination of scan mirror for y-scan and single-axis translation stage for x-scan. For a printed microarray with over 10,000 features, each of 100 μm in diameter and distinct small molecule targets, we can acquire an end-point image of the microarray in 90 minutes with pixel resolution of 20 μm × 20 μm. The microscope is also capable of measuring binding kinetics of over 10,000 protein-ligand reactions simultaneously. We also describe a number of strategies for immobilizing small molecule compounds on functionalized glass slides: (1) conjugating the compounds (through a chemically inert linker) with a lysine residue so that the primary amine on the lysine serves as the anchor to epoxy-functionalized glass surface; (2) conjugating the compounds (through a linker) with a biotin residue so that the biotin serves as the anchor to streptavidin-functionalized glass surface; (3) immobilizing small molecule compounds without modification on isocyanate-functionalized glass surface through non-specific reaction of nucleophilic molecular motifs on most bioactive compounds with isocyanate groups. We present preliminary measurements of protein-small molecule binding reactions using the new microscope and the surface immobilization strategies.

The technical objective of this study has been to design, build and validate biocompatible hollow fiber implants based on fluorescence with integrated biophotonics components to enable in fiber kinetic cell based assays. A human osteosarcoma in vitro cell model fiber system has been established with validation studies to determine in fiber cell growth, cell cycle analysis and organization in normal and drug treated conditions. The rationale for implant development have focused on developing benchmark concepts in standard monolayer tissue culture followed by the development of in vitro hollow fiber designs; encompassing imaging with and without integrated biophotonics. Furthermore the effect of introducing targetable biosensors into the encapsulated tumor implant such as quantum dots for informing new detection readouts and possible implant designs have been evaluated. A preliminary micro/macro imaging approach has been undertaken, that could provide a mean to track distinct morphological changes in cells growing in a 3D matrix within the fiber which affect the light scattering properties of the implant. Parallel engineering studies have showed the influence of the optical properties of the fiber polymer wall in all imaging modes. Taken all together, we show the basic foundation and the opportunities for multi-modal imaging within an in vitro implant format.

We used laser nanodissection to study the nature, magnitude and distribution of forces at cell-cell contacts during tissue morphogenesis in live Drosophila embryos. We designed two set-ups coupling a near infrared femtosecond laser to either an inverted fluorescence microscope or a spinning-disk confocal microscope. We show that the developed tools are able to locally disrupt the acto-myosin network while preserving the integrity of the membranes. With these systems, we could explore the redistribution of cortical elements and relaxation of cell-cell contacts after local ablation.

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to cell biology. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce clones by photoablative dilution. The LEAP instrument performs automated fluorescence imaging and real-time image analysis to classify cells. The instrument also features a pulsed laser that gives it the ability to purify a sample by eliminating unwanted cells via laser ablation or UV-induced apoptosis. In photoablative dilution, rare cells are deposited into a multiwell plate at 10 cells per well. Then one cell is chosen to clone, and the other cells present in the well are eliminated by laser ablation. We have successfully used LEAP to produce single cell clones in 95% of wells (originally containing 5±2.1 cells/well). While photoablative dilution is a very effective way of producing clonal cultures, it has a fundamental limitation in the low number of cells that can be processed. This can be overcome by performing a pre-enrichment to increase the frequency of the rare cells to be cloned. Another enrichment strategy is flow cytometry based cell sorting. Flow sorting can provide greater than 104 fold enrichment and cells can be sorted directly into a multiwell plate. With pre-enrichment, photoablative dilution can be used to clonally isolate rare cells. This is especially important in cases where the total number of potentially rare cells recovered by first stage enrichment sorting is only 10-200 cells. Such a situation which would normally preclude second pass sorting for purity by the high-throughput first stage cell separation technology.

Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with sub-wavelength accuracy. Here, we report a quantitative phase laser microsurgery system, which takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system would enable absolute quantitation of localized alteration/damage to transparent phase objects, such as the cell membrane or intra-cellular structures, being exposed to the laser microbeam, which was not possible using conventional phase-contrast microscopy.

During cell adhesion and migration, a cell forms focal adhesion, which connects cytoskeleton with extracellular matrix (ECM) through integrin, and applies cytoskeletal force to the ECM through focal adhesion. In the initial phase of cell adhesion (initial adhesion), protein related to cell adhesion recruits other components to reinforce adhesion force and grows to focal complex. To study the mechanism of cell adhesion, we focused on relationship between variation of mechanical property of cell adhesion and related protein for cell adhesion. Especially, we approached by understanding mechanical property of initial adhesion. To measure this property, we developed a "cell palpation system", which utilizes optical tweezers to apply mechanical stimulus to a cell and to investigate reactive force. As below, this system gives information on the mechanical property (membrane support tension) and a time course of the property by using an optically manipulated microbead through an analysis based on mechanical model of this microbead. To create cell adhesion between the microbead and cell surface, the microbead was coated with collagen and we investigated the mechanical property of initial adhesion. And we analyzed the processes in relation to maturation of initial adhesion at a single molecular level.

All multi-cellular living organisms are very complex microfluidics systems, which are assembled 'inside-out', as the result of a complex 'tug-of-war' process comprising both feed-forward modules, in particular the program embedded in the species' DNA, and feed-back processes, in particular the response to external environmental conditions. Living organisms have to solve a perennial problem: for an environment with limited resources (finite and spatial distribution of nutrients, geometrical limitations, competition from other species, etc.), finite growth rate and penalties applied to nonoptimal behaviour, what is the best strategy that will maximise growth and survival of the individual and the species? Identifying how organisms resolve this problem is very difficult due to the high complexity involved. However, filamentous fungi and other simple organisms, provide a means to address these issues due to the availability of microscopic imaging and microfabricated geometries in which the behaviour can be tested. Semiconductor fabrication (photolithography, RIE etching, and soft lithography techniques) was used here to produce artificial micro-networks, which are physically and chemically structured 3D microenvironments. Continuous imaging of fungal behaviour in microfluidics networks required the use of PDMS, which is transparent and O₂-permeable. Using these structures, the dynamic growth behaviour of two fungal species Neurospora crassa and Armillaria mellea was observed microscopically in real-time. Growth parameters such as the tip extension velocities, branching angles and branching distances on plain surfaces differed vastly between the species. Features in the micronetworks of a size similar to the hyphal diameter induced the largest change in growth parameters such as, the largest decrease in branching distances in N. crassa and a near complete suppression for A. mellea. Despite these fundamental differences, both species negotiated the structures successfully. These results open the way to asking more fundamental questions, such as which species use algorithms that are most efficient for solving a particular type of mathematical problem coded in a network? Can these natural algorithms be used for either better control of fungal growth and metabolism for both medical and biotechnology applications; or even for new computation paradigms?

Although high voltage direct current (DC) shock is a standard technique to terminate ventricular fibrillation (VF), it can cause severe pain and tissue damage. The exact effect of the DC electric field, which can depolarize the heart during VF is still unknown. We hypothesized that low-energy DC field in combination with pacing (pacing+DC) could terminate VF by affecting the ventricular propagation pattern. In six Langendorff-perfused isolated rabbit hearts with the ablated sinoatrial (SA) node, the DC field was delivered to the left ventricle (cathode) and right ventricle (anode). We designed a timed protocol using LabVIEW programming that delivers pacing, DC and pacing+DC stimuli for two seconds time intervals each. The pacing pulse (with varying pacing cycle length: 300ms-30ms) was delivered to the apex. Transmembrane voltage was recorded with optical mapping technique for 16 seconds at a sampling rate of 2ms/frame. We crushed the sinoatrial node to reduce the heart rate. The baseline activation appeared to have endocardial origins with a mean escape ventricular rate of 60 ± 5bpm at baseline. The DC field (30mA-60mA) alone increased the mean heart rate to 120±5bpm. Although DC alone terminated VF in a few cases, the rate of termination was very low (6.2%). However, when pacing+DC was applied, it was possible to terminate VF in 34 of 130 episodes in six rabbits. The rate of successful defibrillation of VF with pacing+DC was significantly higher than that with DC alone (20% vs 6.2%, p<0.01). Pacing alone never terminated the VF. In conclusion, DC field may affect the conduction velocity in normal condition. Pacing+DC intervention could lead to regularization of VF propagation and eventually to termination. Further improvement of this approach may offer a higher success rate of defibrillation with lower energy requirements.

Angular Domain Imaging (ADI) employs an angular filter array to accept photons within a small acceptance angle along the axis of an aligned laser light source and preferentially reject scattered light. Simulations show that the accepted photons travel the shortest paths between source and detector and are therefore the earliest to arrive. We fabricated angular filter arrays using silicon bulk micromachining and found that an array of 60 μm square shape microtunnels 1 cm in length accepted photons within 0.48 degree of axis of the micro-tunnels. This small acceptance angle rejected most of the scattered light and sub-millimeter resolution targets could be resolved in a few centimeters of turbid medium with at least six times reduced mean free path. ADI through media with higher scattering coefficients was not achievable due to unwanted acceptance of late arriving scattered photons. To reject the late arriving photons, we added time-domain filtration and linear polarization to ADI. The implementation of a time-gated camera, a 780 nm femtosecond pulsed laser, and linear polarization to our ADI system resulted in improved image contrast. The use of ADI with time-gating (gate width 250 ps) and linear polarization enabled visualization of sub-millimeter absorbing objects with approximately eight times higher image contrast compared to ADI in a scattering medium equivalent to six times reduced mean free path.

Photomultiplier tubes (PMTs) are often used in scanning imaging systems requiring high sensitivity, due to their low noise and high gain. Solid-state photomultipliers (SSPMs), an array of independent Geiger-mode avalanche photodiodes, each with an integrated quenching resistor, have shown potential to outperform PMTs in terms of signal to noise ratio (SNR) because of higher achievable photon detection efficiency and lower excessive noise factor. Here, the factors affecting SNR of commercially available PMTs and SSPMs will be compared under different wavelengths (simulating dye emissions: 500-700 nm) in order to quantify the potential performance gain when PMTs are replaced by SSPMs.

We present a novel method for high-speed measurements of fluorescence lifetime, in which fluorescence signal for precise lifetime determination is acquired in a short time on the order of microseconds. Our method is based on analog signal that contains a number of fluorescence photons in a pulse, on the contrary to the conventional time-correlated single-photon counting in which only a single photon is permitted for a fluorescence pulse. Because this method does not have any problem of photon counting pile-up, the measurement speed is not limited by the single-photon constraint and can increase up to the excitation repetition rate. In order to extract the accurate lifetime information from the analog signal contaminated by the slow instrumental response function (IRF), we have developed a new signal processing method, in which the lifetime is determined by difference between mean arrival time of the analog photo-electronic pulse of fluorescence signal and one of IRF signal. By both experimental and theoretical studies, we have verified that the measurement accuracy and precision are nearly independent of the width of the IRF so that inexpensive narrowbandwidth photo-detectors and low-speed electronics can be used for this method. Excellent accuracy and precision have been obtained experimentally for high-speed measurements completed in a few microseconds. These results suggest that our method can be well applied to measurement of fast dynamic phenomena and real time fluorescence lifetime imaging microscope with low cost.

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. Thanks to a decoupling procedure cell thickness and intracellular refractive index can be measured separately. Consequently, Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), two highly relevant clinical parameters, have been measured non-invasively at a single cell level. The DHM nanometric axial and microsecond temporal sensitivities have permitted to measure the red blood cell membrane fluctuations (CMF) on the whole cell surface.

Time-angular domain imaging (TADI) employs an angular filter array, which functions to accept quasi-ballistic photons with trajectories near the axis of a collimated light source. At high scattering coefficients, image contrast declines due to background signals from scattered photons that have trajectories compatible with the angular filter array. We attempted to correct for the background signal using a temporal discrimination technique and image subtraction. During TADI through turbid media, photons at early arrival times represent a mixture of quasi-ballistic and scattered photons, while late arriving photons represent scattered photons. We captured two TADI images of a resolution target suspended midway through a 2 cm thick cuvette filled with 0.30% Intralipid^TM. A 780 nm, 100 ps pulsed laser (PicoTA, PicoQuant) was used to trans-illuminate the cuvette. Detection was performed after the angular filter array (500 elements with 60 μm × 60 μm square-shaped cross section and 1 cm length) with a gated camera (Picostar HR, LaVision). The first TADI image was collected at a short gate delay with respect to the minimum transit time, and resulted in a projection of the target. A long gate delay was used to collect the second TADI image and the projection of the target was not apparent. A corrected image (two - one) was digitally computed. Analysis of the first image compared to the corrected image revealed a 2.1-fold increase in contrast-to-noise ratio for the corrected image. Therefore, images collected with TADI were improved by processing successive images at different gate delays.

We report our recent introduction of a dynamic spectral imaging technique into a microscope system for specimen examination and classification. It uses a dynamic spectral filter and a diffraction grating on an intermediate image plane for fast spectral image acquisition that only captures interested spectral image frames. The reconfigurable capability enables the optimal spectral band selection for a specific application without hardware component modification. It can achieve 8 nm fine spectral resolution in the whole visible spectral band suitable for fluorescent image evaluation. With a fast spectral imaging update, it can support large quantify specimen screening and classification.

By taking advantage of the continuous spectrum collected for each image pixel by the Hyperspectral Microscopy Imaging (HMI) system, the data spectra can be de-convolved into a set of standard spectra coefficients for each pixel. The coefficients are calculated by the analysis program and indicate the relative amounts of each standard necessary to reproduce the data spectra for each image pixel. Images of a single color or composite images of two or more colors can be produced for visual analysis. The HMI system is composed of an Olympus inverted microscope, imaging spectrograph, CCD camera, motorized X-Y stage and illumination sources. This system has been used to scan and analyze pathology tissue samples which have been stained with 4 standard fluorochromes (not including the dynamic background spectrum) attached to specific antibodies. The typical wavelength range is 420nm to 785nm with the longest wavelength markers emitting in the spectral region where the human eye is not sensitive. Quantitative values are recorded and can be compared to the visual interpretations.

Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

Arterial remodeling, i.e. changes in size and/or structure of arteries, plays an important role in vascular disease. Conflicting findings have been reported as to whether an abundance of collagen causes inward or outward remodeling, phenomena that result in either a smaller or larger lumen, respectively. We hypothesize that the amount, type and quality of collagen influence the remodeling response. Here, we create mechanical injury to the rat carotid artery using a balloon catheter, and this leads to inward remodeling. Treatment of the artery with Connective Tissue Growth Factor (CTGF) causes outward remodeling. We investigated the arterial composition in injured CTGF-treated and non-CTGF-treated and sham CTGF-treated and non-CTGF treated arteries 14 days post-injury (n = 7-8 per group) using infrared imaging. A Perkin Elmer Spotlight Spectrum 300 FT-IR microscope was used for data collection. Cross-sections of paraffinembedded arteries were scanned at 2 cm^-1 spectral resolution with spatial resolution of 6.25 μm/pixel, and data analyzed using Malvern Instruments ISys 5.0. Post-injury, we found a nearly 50% reduction in the average 1338/AM2 area ratio (correlated to collagen helical integrity). The most dramatic change was a 600% increase in the 1660/1690 peak height ratio, which has previously been related to collagen crosslink maturity. In all cases, CTGF treatment resulted in the observed changes in peak parameters normalized back to control values. Overall, these preliminary studies demonstrate that infrared imaging can provide insight into the underlying molecular changes that contribute to arterial disease.

A Total Quality Management (TQM) approach can be used to analyze data from biomedical optical and imaging platforms of tissues. A shift from individuals to teams, partnerships, and total participation are necessary from health care groups for improved prognostics using measurement analysis. Proprietary measurement analysis software is available for calibrated, pixel-to-pixel measurements of angles and distances in digital images. Feature size, count, and color are determinable on an absolute and comparative basis. Although changes in images of histomics are based on complex and numerous factors, the variation of changes in imaging analysis to correlations of time, extent, and progression of illness can be derived. Statistical methods are preferred. Applications of the proprietary measurement software are available for any imaging platform. Quantification of results provides improved categorization of illness towards better health. As health care practitioners try to use quantified measurement data for patient diagnosis, the techniques reported can be used to track and isolate causes better. Comparisons, norms, and trends are available from processing of measurement data which is obtained easily and quickly from Scientific Software and methods. Example results for the class actions of Preventative and Corrective Care in Ophthalmology and Dermatology, respectively, are provided. Improved and quantified diagnosis can lead to better health and lower costs associated with health care. Systems support improvements towards Lean and Six Sigma affecting all branches of biology and medicine. As an example for use of statistics, the major types of variation involving a study of Bone Mineral Density (BMD) are examined. Typically, special causes in medicine relate to illness and activities; whereas, common causes are known to be associated with gender, race, size, and genetic make-up. Such a strategy of Continuous Process Improvement (CPI) involves comparison of patient results to baseline data using F-statistics. Self-parings over time are also useful. Special and common causes are identified apart from aging in applying the statistical methods. In the future, implementation of imaging measurement methods by research staff, doctors, and concerned patient partners result in improved health diagnosis, reporting, and cause determination. The long-term prospects for quantified measurements are better quality in imaging analysis with applications of higher utility for heath care providers.

In order to enhance cell culture growth in biosensors such as those for glucose detection must be developed that are capable of monitoring cell culture processes continuously and accurate. Fourier domain optical coherence tomography (FD-OCT) is used to obtain cell images with nanometer level resolution by analyzing the interference pattern by the mixing of reference and objective light to determine glucose concentration in doped double distilled water and create a glucose signature spectrum in salt-sugar solution. We demonstrate ultrahigh-resolution optical coherence tomography (OCT) imaging of in vitro biological cells and an improved deflection angle measurements formal and back projection method is used to reconstruct the two-dimensional glucose concentration performs refractive index distribution. Slopes of OCT signals decreased substantially and almost linearly with the increase of glucose concentration from 2.5 to 15 mg/dl. Phantom studies demonstrated 1% accuracy of scattering- coefficient measurement. Our theoretical and experimental studies suggest that glucose concentration can potentially be measured non-invasively with high sensitivity and accuracy with OCT systems.

Cell Transformation Assays (CTA) rely on the detection of phenotypic changes, namely foci, induced by chemicals (e.g., xenobiotics or candidate drugs) in mammalian cells such as C3H10T1/2 mouse fibroblasts. A focus is a cell colony and as such is made visible by standardized techniques of light microscopy. Foci exhibit a variety of morphological features, by which three "Types" have been defined. Types II and III consist of cells having undergone neoplastic transformation. The assignment of a focus to a Type is based on the evaluation of phenotypic features by a trained human expert. An automated, two-stage morphological classifier of foci is described herewith. Morphological descriptors are extracted from light microscope images by the "spectrum enhancement" algorithm, which separates structure from texture. Said descriptors are submitted to a classifier, the first stage of which is trained to discriminate transformed cells from normal ones and the 2nd stage to discriminate Type III from Type II. The classifier operating in recognition mode (on images not used for training) is satisfactory in terms of confusion matrix entries. The whole procedure is aimed at removing subjectivity from the scoring and classification of foci and thus make CTA a more powerful tool in carcinogenesis studies.

Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular parameters, such as vascular density, perfusion rate, and permeability, using spatiotemporal profiles of indocyanine green. Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal tissues with high spatial resolution. The multimodality of this method allowed us to find a negative spatial correlation between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic drug; these findings were validated using two-photon microscopy. We suggest that DyFI could be a useful tool for the preclinical development of anti-angiogenic drugs.

We present the measurement of red blood cell (RBC) volume change induced by Ca²⁺ for a live cell imaging with full field quantitative phase microscopy (FFQPM). FFQPM is based on the Mach-Zehnder interferometer combined with an inverted microscopy system. We present the effective method to obtain a clear image and an accurate volume of the cells. An edge detection technique is used to accurately resolve the boundary between the cell line and the suspension medium. The measurement of the polystyrene bead diameter and volume has been demonstrated the validity of our proposed method. The measured phase profile can be easily converted into thickness profile. The measured polystyrene bead volume and the simulated result are about 14.74 μm³ and 14.14 μm³, respectively. The experimental results of our proposed method agree well with the simulated results within less than 4 %. We have also measured the volume variation of a single RBC on a millisecond time scale. Its mean volume is 54.02 μm³ and its standard deviation is 0.52 μm³. With the proposed system, the shape and volume changes of RBC induced by the increased intracellular Ca²⁺ are measured after adding ionophore A23187. A discocyte RBC is deformed to a spherocyte due to the increased intracellular Ca²⁺ in RBC. The volume of the spherocyte is 47.88 μm³ and its standard deviation is 0.19 μm³. We have demonstrated that the volume measurement technique is easy, accurate, and robust method with high volume sensitivity (<0.0000452 μm³) and this provides the ability to study a biological phenomenon in Hematology.

Introduction: The highest priority for the Advanced Cytometry Standard (ACS) is the interpretation of list-mode cytometry measurements. Other priorities of lesser importance are the capacity to reproduce a cytometry measurement and the implementation of a digital microscopy image standard. The sequential nature of these requirements is being accommodated by a flexible, modular design. A major feature of this modular design is the creation of a design for an Advanced Cytometry Standard Container (ACSC) that includes a Table of Contents (ToC) XML file, one or more binary data containing files and files that contain the meta-data that describes the binary data. Methods: The design and partial implementation of the CytometryML schemas have been based on the techniques of modularity (each schema describing one object), iterative (spiral) development, inheritance, and reuse. Data-types including their definitions have been reused from DICOM, FCS, and other standards. Results: A prototype ToC schema together with prototypes of many of the schemas that describe the contents of the ACSC have been created together with their supporting schemas. These schemas have been validated with two tools and XML pages were generated from the main element(s) of the highest level schemas. These elements describe the table of contents of the zipped container file and a flow-cytometry instrument. The zipped container file (ACSC) describes and contains the meta and binary data.

Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The novel slide-based imaging system is suitable for detection of fluorescence differences over a broad range of concentrations. This approach may lead to novel assays for measuring concentration differences in cell free solutions and cell cultures e.g. in secretion assays.

Slide-based cytometry (SBC) leads to breakthrough in cytometry of cells in tissues, culture and suspension. Carl Zeiss Imaging Solutions' new automated SFM combines imaging with cytometry. A critical step in image analysis is selection of appropriate triggering signal to detect all objects. Without correct target cell definition analysis is hampered. DNA-staining is among the most common triggering signals. However, the majority of DNA-dyes yield massive spillover into other fluorescence channels limiting their application. By microscopy objects of >5μm diameter can be easily detected by phase-contrast signal (PCS) without any staining. Aim was to establish PCS - triggering for cell identification. Axio Imager.Z1 motorized SFM was used (high-resolution digital camera, AxioCam MRm; AxioVision software: automatic multi-channel scanning, analysis). Leukocytes were stained with FITC (CD4, CD8) and APC (CD3) labelled antibodies in combinations using whole blood method. Samples were scanned in three channels (PCS/FITC/APC). Exposition-times for PCS were set as low as possible; the detection efficiency was verified by fluorescence. CD45-stained leukocytes were counted and compared to the number of PCS detected events. Leukocyte subtyping was compared with other cytometers. In focus the PCS of cells showed ring-form that was not optimal for cell definition. Out of focus PCS allows more effective qualitative and quantitative cell analyses. PCS was an accurate triggering signal for leukocytes enabling cell counting and discrimination of leukocytes from platelets. Leukocyte subpopulation frequencies were comparable to those obtained by other cytometers. In conclusion PCS is a suitable trigger-signal not interfering with fluorescence detection.

We describe the concept of laser rastering flow cytometry, where a rapidly scanning laser beam allows counting and classification of cells at much higher rates than currently possible. Modifications to existing flow cytometers to implement the concept include an acousto-optic deflector, fast analog-to-digital conversion, and a two-step digital-signal-processing scheme that handles the high data rates and provides key assay information. Results are shown that prove the concept, demonstrating the ability to resolve closely spaced cells and to measure cells at rates more than an order of magnitude faster than on conventional flow-cytometer-based hematology analyzers.

A novel multi-modality optic nerve head image fusion approach has been successfully designed. The new approach has been applied on three ophthalmologic modalities: angiogram, fundus, and oxygen saturation retinal optic nerve head images. It has achieved an excellent result by giving the visualization of fundus or oxygen saturation images with a complete angiogram overlay. During this study, two contributions have been made in terms of novelty, efficiency, and accuracy. The first contribution is the automated control point detection algorithm for multi-sensor images. The new method employs retina vasculature and bifurcation features by identifying the initial good-guess of control points using the Adaptive Exploratory Algorithm. The second contribution is the heuristic optimization fusion algorithm. In order to maximize the objective function (Mutual-Pixel-Count), the iteration algorithm adjusts the initial guess of the control points at the sub-pixel level. A refinement of the parameter set is obtained at the end of each loop, and finally an optimal fused image is generated at the end of the iteration. It is the first time that Mutual-Pixel-Count concept has been introduced into biomedical image fusion area. By locking the images in one place, the fused image allows ophthalmologists to match the same eye over time and get a sense of disease progress and pinpoint surgical tools. The new algorithm can be easily expanded to human or animals' 3D eye, brain, or body image registration and fusion.

We proposed and demonstrated an optical system for high-speed and high-resolution imaging of surface profiles. The system is a fiber-based composite interferometer in which a Michelson interferometer is used for the measurement of the surface profile of the sample and a Mach-Zehnder interferometer is used for compensation of phase deviation due to the systematic errors and environmental perturbations. In the system, a laser diode with wavelength of 531 nm was used as the light source. A two-axis translation stage was used for lateral scanning of the sample. In the reference arm, a reflection mirror was arranged on a translation stage driven by a piezoelectric transducer as the axial scanning component. The phase difference between the interferograms of both interferometers was acquired to obtain the surface height of the sample. The axial accuracy of ±5 nm was achieved where the imaging was acquired within one minute for a frame. The lateral resolution was at the diffraction limit of light. The system possesses the advantages of low cost, portability and flexibility. Furthermore, it can perform high-resolution imaging in large area without special shielding of the system as well as any special preparation of the sample.

We present a novel imaging mass spectrometry technique that uses femtosecond laser pulses to directly ionize the sample. The method offers significant advantages over current techniques by eliminating the need of a laser-absorbing sample matrix, being suitable for atmospheric pressure sampling, and by providing 10μm resolution, as demonstrated here with a chemical image of vegetable cell walls.

We present a novel method to determine the effective elastic constant (EEC) and effective restoring force (ERF) by using volumetric analysis of Red blood cell (RBC)s with Full field quantitative phase microscopy (FFQPM). We use the simple harmonic oscillator model to determine EEC and ERF. We investigate the EECs and ERFs of different shape of RBCs (discocyte, acanocyte, stomatocyte, and spherocyte) and we investigate the effective temporal coherence of RBCs by analyzing temporal volumetric behavior of the RBCs.

A novel and simplest polarization microscope by single polarization modulation method to visualize cytoskeletal structure in bio-cells have been developed. The polarization microscope visualizes cytoskeletal structure based on birefringence characteristics of cells. In the polarization microscope, a series of intensity images according to single modulation of rotating linear polarizer were obtained to visualize cytoskeletal structure. Phase retardation and principal axis orientation were calculated by analytic data processing from the acquired intensity images. Phase retardation and principal axis orientation of breast cancer cells, MCF 7 and MDA MB 231 were measured and visualized as their birefringence characteristics by the polarization microscope. Phase retardation sensitivity of the polarization microscope system also was measured in this paper.