This PDF file contains the front matter associated with SPIE Proceedings Volume 7902, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

The online analysis of rapid cellular processes by morphological alterations strongly depends on the ability to rapidly visualize and to quantify cell shape and intracellular structures. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogenous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyzed the impact of specific intracellular refractive index manipulation by microinjection of refractive index changing agents on the DHM phase contrast. Glycerol was chosen as osmolyte, which combines high solubility in aqueous solutions and cellular compatibility. We present data showing that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle was proven by experiments inducing cell shrinkage and protein concentration. The integrity of cell membranes is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for DHM opens also prospects for application with other quantitative phase contrast imaging technologies.

High speed intravital microscopy has emerged as an essential tool for studying cellular dynamics in live tissue. A limitation of this technique, however, is that the timescale that a sample can be continuously imaged is limited by practical considerations to several hours. Long term observation of live tissue is of great interest for a variety of research areas. We present methods for observing long term cellular dynamics in live tissue based on three-dimensional registration of time-lapse intravital microscopy images. For these experiments we utilized a custom multimodal microscope that allows simultaneous and co-registered acquisition of optical coherence (OCM) and multiphoton (MPM) microscopy images. OCM allows the structure of a sample to be visualized based on backscattered light while MPM excited fluorescence allows individual cells and cell function to be visualized. The OCM images of tissue structure are used to register data sets taken at different time points. The transformations of the OCM images are applied to MPM images to determine the migration of cell populations. This method of image registration is applied to in vivo tracking of bone-marrow derived GFP-labeled stem cells in mouse skin following bone marrow transplants from GFP donors into species-matched wildtype hosts. The use of three-dimensional image registration of time-lapse microscopy images enables tracking these cells after local cutaneous injury, and for investigating the role of skin stem cells in wound healing.

In vivo multispectral reflectance imaging has been extensively used in the somatosensory cortex (SsC) in anesthetized rodents to collect intrinsic signal during activation and derive hemodynamics signals time courses. So far it has never been applied to the Olfactory Bulb (OB), although this structure is particularly well suited to the optical study of brain activation due to the its well defined organization, the ability to physiologically activate it with odorants, and the low depth of the activated layers. To obtain hemodynamics parameters from reflectance variations data, it is necessary to take into account a corrective factor called Differential Pathlength (DP). It is routinely estimated using Monte Carlo simulations, modeling photons propagation in simplified infinite geometry tissue models. The first goal of our study was to evaluate the influence of more realistic layered geometries and optical properties on the calculation of DP and ultimately on the estimation of the hemodynamics parameters. Since many valuable results have been obtained previously by others in the SSc, for the purpose of validation and comparison we performed Monte Carlo simulations in both the SSC and the OB. We verified the assumption of constant DP during activation by varying the hemoglobin oxygen saturation, total hemoglobin concentration and we also studied the effect of a superficial bone layer on DP estimation for OB. The simulations show the importance of defining a finite multilayer model instead of the coarse infinite monolayer model, especially for the SSc, and demonstrate the need to perform DP calculation for each structure taking into account their anatomofunctional properties. The second goal of the study was to validate in vivo multispectral imaging for the study of hemodynamics in the OB during activation. First results are presented and discussed.

Abnormal microvascular function and angiogenesis are key components of various diseases that can contribute to the perpetuation of the disease. Several skin diseases and ophthalmic pathologies are characterized by hypervascularity, and in cancer the microvasculature of tumors is structurally and functionally abnormal. Thus, the microvasculature can be an important target for treatment of diseases characterized by abnormal microvasculature. Motivated largely by cancer research, significant effort has been devoted to research on drugs that target the microvasculature. Several vascular targeting drugs for cancer therapy are in clinical trials and approved for clinical use, and several off-label uses of these drugs have been reported for non-cancer diseases. The ability to image and measure parameters related to microvessel function preclinically in laboratory animals can be useful for development and comparison of vascular targeting drugs. For example, blood supply time measurements give information related to microvessel morphology and can be measured with first-pass fluorescence imaging. Hemoglobin saturation measurements give an indication of microvessel oxygen transport and can be measured with spectral imaging. While each measurement individually gives some information regarding microvessel function, the measurements together may yield even more information since theoretically microvessel morphology can influence microvessel oxygenation, especially in metabolically active tissue like tumors. However, these measurements have not yet been combined. In this study, we report the combination of blood supply time imaging and hemoglobin saturation imaging of microvessel networks in tumors using widefield fluorescence and spectral imaging, respectively. The correlation between the measurements in a mouse mammary tumor is analyzed.

Light-emitting-diode induced chlorophyll fluorescence analysis is employed to investigate the effect of water and salt stress upon the growth process of physicnut(jatropha curcas) grain oil plants for biofuel. Red(Fr) and far-red (FFr) chlorophyll fluorescence emission signals around 685 nm and 735 nm, respectively, were observed and examined as a function of the stress intensity(salt concentration and water deficit) for a period of time of 30 days. The chlorophyll fluorescence(ChlF) ratio Fr/FFr which is a valuable nondestructive and nonintrusive indicator of the chlorophyll content of leaves was exploited to monitor the level of stress experienced by the jatropha plants. The ChlF technique data indicated that salinity plays a minor role in the chlorophyll concentration of leaves tissues for NaCl concentrations in the 25 to 200 mM range, and results agreed quite well with those obtained using conventional destructive spectrophotometric methods. Nevertheless, for higher NaCl concentrations a noticeable decrease in the Chl content was observed. The Chl fluorescence ratio analysis also permitted detection of damage caused by water deficit in the early stages of the plants growing process. A significant variation of the Fr/FFr ratio was observed sample in the first 10 days of the experiment when one compared control and nonwatered samples. The results suggest that the technique may potentially be applied as an early-warning indicator of stress caused by water deficit.

Laser tissue welding is a potential medical treatment method especially on closing cuts implemented during any kind of surgery. Photothermal effects of laser on tissue should be quantified in order to determine optimal dosimetry parameters. Polarized light and phase contrast techniques reveal information about extend of thermal change over tissue occurred during laser welding application. Change in collagen structure in skin tissue stained with hematoxilen and eosin samples can be detected. In this study, three different near infrared laser wavelengths (809 nm, 980 nm and 1070 nm) were compared for skin welding efficiency. 1 cm long cuts were treated spot by spot laser application on Wistar rats' dorsal skin, in vivo. In all laser applications, 0.5 W of optical power was delivered to the tissue, 5 s continuously, resulting in 79.61 J/cm² energy density (15.92 W/cm² power density) for each spot. The 1st, 4th, 7th, 14th, and 21st days of recovery period were determined as control days, and skin samples needed for histology were removed on these particular days. The stained samples were examined under a light microscope. Images were taken with a CCD camera and examined with imaging software. 809 Nm laser was found to be capable of creating strong full-thickness closure, but thermal damage was evident. The thermal damage from 980 nm laser welding was found to be more tolerable. The results showed that 1070 nm laser welding produced noticeably stronger bonds with minimal scar formation.

Autofluorescence and Raman spectra, images and decay kinetics of U251-MG glioblastoma cells prior and after activation of tumor suppressor genes are compared. While phase contrast images and fluorescence patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy. Slight differences are also observed in Raman spectra of these cell lines, in particular at wave numbers around 970 cm^-1. While larger numbers of fluorescence and Raman spectra are evaluated by the method of multivariate data analysis, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

Recently autofluorescence imaging (AFI) endoscopy, visualizing tissue fluorescence in combination with reflected light, has been adopted as a technique for detecting neoplasms in the colon and other organs. However, autofluorescence colonoscopy is not infallible, and improvement of the detection method can be expected to enhance the performance. Colonic mucosa contains metabolism-related fluorophores, such as reduced nicotinamide adenine dinucleotide, which may be useful for visualizing neoplasia in autofluorescence endoscopy. We examined sliced cross-sections of endoscopically resected tubular adenomas under a microscope. Fluorescence images acquired at 365-nm excitation (F_365ex) and 405-nm excitation (F_405ex), and reflectance images acquired at 550 nm (R₅₅₀) were obtained. Fluorescence ratio (F_365ex/F_405ex) images and reflectance/fluorescence ratio (R₅₅₀/F_405ex) images were calculated from the acquired images. The fluorescence ratio images could distinguish adenomatous mucosa from normal mucosa more clearly than the reflectance/fluorescence ratio images. The results showed that the autofluorescence ratio imaging is a potential technique for increasing the diagnostic power of autofluorescence endoscopy.

We report the feasibility of tumor detection and delineation in vivo using multimode optical imaging of targeted gallium corrole (HerGa). HerGa is highly effective for targeted HER2+ tumor elimination in vivo, and it emits intense fluorescence. These unique characteristics of HerGa prompted us to investigate the potential of HerGa for tumor detection and delineation, by performing multimode optical imaging ex vivo and in vivo; the imaging modes included fluorescence intensity, spectral (including ratiometric), lifetime, and two-photon excited fluorescence, using our custombuilt imaging system. While fluorescence intensity imaging provided information about tumor targeting capacity and tumor retention of HerGa, ratiometric spectral imaging offered more quantitative and specific information about HerGa location and accumulation. Most importantly, the fluorescence lifetime imaging of HerGa allowed us to discriminate between tumor and non-tumor regions by fluorescence lifetime differences. Finally, two-photon excited fluorescence images provided highly resolved and thus topologically detailed information around the tumor regions where HerGa accumulates. Taken together, the results shown in this report suggest the feasibility of tumor detection and delineation by multimode optical imaging of HerGa, and fluorescent chemotherapy agents in general. Specifically, the multimode optical imaging can offer complementary and even synergetic information simultaneously in the tumor detection and delineation by HerGa, thus enhancing contrast.

Optical imaging and MRI have both been used extensively to study tumor microenvironment. The two imaging modalities are complementary and can be used to cross-validate one another for specific measurements. We have developed a modular platform that is capable of doing optical microscopy inside an MRI instrument. To do this, an optical relay system transfers the image to outside of the MR bore to a commercial grade CCD camera. This enables simultaneous optical and MR imaging of the same tissue and thus creates the ideal situation for comparative or complementary studies using both modalities. Initial experiments have been done using GFP labeled prostate cancer cells implanted in mouse dorsal skin fold window chamber. Vascular hemodynamics and vascular permeability were studied using our imaging system. Towards this goal, we developed a dual MR-Optical contrast agent by labeling BSA with both Gd-DTPA and Alexa Fluor. Overall system design and results of these preliminary vascular studies are presented.

We present a novel line confocal microscope for investigating biological samples that simultaneously acquires images from three fluorescent channels. This instrument provides the majority of the benefits of confocal microscopes, such as depth sectioning and improved contrast, while allowing more of the excitation illumination to reach the sample area, resulting in reduced exposure times. One configuration of the line confocal fluorescent microscope is designed to work with a four laser illuminator, and three discrete cameras, while an alternate configuration allows the instrument to act as a multispectral microscope that captures the fluorescent emission over the visible and NIR wavelength range.

Until now, the cancer was examined by diagnosing the pathological changes of tumor. If the examination of cancer can diagnose the tumor before the cell occur the pathological changes, the cure rate of cancer will increase. This research develops a human-machine interface for hyper-spectral microscope. The hyper-spectral microscope can scan the specific area of cell and records the data of spectrum and intensity. These data is helpful to diagnose tumor. This research aims to develop a new system and a human-machine interface to control the hyper-spectral microscope. The interface can control the moving speed of motor, the exposure-time of hyper-spectrum, real-time focus, image of fluorescence, and record the data of spectral intensity and position.

We have taken a three-pronged approach to improving the current standard of melanoma detection: (a) we are developing a new hyperspectral imaging-based medical device aimed at noninvasively detecting melanoma (b) we used a commercially available hand-held microscope with polarization control as a dermoscope, to begin establishing an inexpensive, portable imaging capability that could help assess the risk of a particular lesion (pigmented nevus) harboring melanoma (c) we created an updated ABCD algorithm and user interface software that more accurately generates a single risk number (Total Dermoscopy Score), for allowing a trained clinician to better assess the need for seeing the patient whose internet-uploaded nevus images they are evaluating. The hyperspectral instrument (a) is discussed elsewhere, and we focus here on (b) and (c), in the hope of increasing melanoma awareness and early detection.

Ultrafast laser microbeam is finding growing usage in causing highly localized damage to cellular structures. This has specifically enhanced efficiency of optoporation-injection of exogenous impermeable substances into the cell by transient pore formation. However, kinetics of laser microbeam induced pore formation and sealing of membrane has not been visualized at nanoscale resolution. Here, we report realization of live atomic force microscopy (AFM) imaging of ultrafast tunable Ti: Sapphire laser microbeam assisted cellular microsurgery. AFM imaging was carried out using Nanonics Multiview system in parallel to exposure of the laser beam. Red blood cells (RBCs) were chosen as cellular model for micro-surgery due to their smooth surface topography. The transparent nature of the Nanonics fiber-optic AFM cantilever allowed simultaneous bright field/phase contrast imaging of the RBC. Measurement of pore size by AFM revealed true pore size as a function of laser exposure duration in contrast to phase contrast imaging. Further, AFM imaging of live cells showed fine topography of sealed pores that could not be comprehended from conventional microscopy.

F_oF₁-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in F_oF₁-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We evaluate two different methods to trap the enzyme inside the confocal volume in order to extend the observation times. Monte Carlo simulations show that optical tweezers with low laser power are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen (Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian electrokinetic trap (ABELtrap) which is capable to apparently immobilize single molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent particles is achieved by applying a real time, position-dependent feedback to four electrodes in a microfluidic device. The standard deviation from a given target position in the ABELtrap is smaller than 200 nm. We develop a combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution.

A three dimensional finite element method is used to model the forces acting on red blood cells trapped on an optical waveguide surface. The parameters are chosen to correspond to strip waveguides made of tantalum pentoxide (Ta₂O₅). A wavelength of 1070 nm is used and the cells are taken to be spherical. Gradient and scattering forces experienced by the cells are studied and found to be highly dependent on the refractive index of the cells. Gradient forces are found to be one order of magnitude larger than scattering forces. Only the lower part of the cells is in contact with the evanescent field of the waveguide. For low refractive indices, we find that the lower 0.5-1 μm of the cells is sufficient to determine the optical forces. For the cell sizes considered, all forces increase with the size.

In this study, an objective-based two-dimensional (2D) surface plasmon (SP)-enhanced optical trapping system with a spatial light modulator (SLM) has been developed to trap dielectric particles in freeform pattern. Through a gold film with a thickness of 45 nm in the near infrared region, a 40-fold electric field enhancement is reached and hence a strong 2D trapping force distribution with SP excitation has been demonstrated. Furthermore, the algorithm called weighted Gerchberg-Saxton can provide a freeform pattern which is used to control the trapping force distributions in the image space based on SLM. Unlike the patterns formed by finite gold areas fabricated on a glass surface, the freeform plasmonic trapping is a more convenient and efficient method to manipulate nanoparticles and biomolecules arbitrarily.

Cancer studies require a thorough understanding of how human gene expressions and DNA modifications are translated at the proteome level. In order to unravel the large and complex interactions between proteins, we have developed a compact lifetime-based flow cytometer, utilising a commercial microfluidic chip, to screen large non-adherent cell populations. Fluorescent signals from cells are detected using time correlated single photon counting (TCSPC) in the burst integrated fluorescence lifetime (BIFL) mode and used to determine the lifetime of each cell. Initially, the system was tested using 10 μm highly fluorescent beads to determine optical throughput and detection efficiency. The system was validated with 293T monkey kidney adenocarcinoma cell line transiently transfected with a FRET standard, consisting of eGPF and mRFP1 fluorescent proteins linked by a19 amino-acid chain. Analysis software was developed to process detected signals in BIFL mode and chronologically save the transient burst data for each cell in a multi-dimensional image file.

Filamentous fungi include serious plant and animal pathogens that explore their environment efficiently in order to penetrate the host. This environment is physically and chemically heterogeneous and the fungi rely on specific physical and chemical signals to find the optimal point/s of attack. This study presents a methodology to introduce distinct structures with dimensions similar to the hyphal diameter and specific chemical surface groups into a controllable environment in order to study the fungal response. We introduced 3.3 μm polystyrene beads covered with Epoxy surface groups into microfluidic channels made from PDMS by rapid replica molding. The experimental setup resulted in different areas with low and high densities of beads as well as densely packed patches. The observations of the fungus exploring the areas long-term showed that the growth parameters were altered significantly, compared with the values measured on agar. The fungus responded to both, the physical and chemical parameters of the beads, including temporary directional changes, increased branching angles, decreased branching distances, decreased apical extension velocities and occasional cell wall lysis. The wealth and magnitude of the observed responses indicates that the microfluidic structures provide a powerful platform for the investigation of micron-sized features on filamentous fungi.

While optical tweezers have been widely used for the manipulation and organization of microscopic objects in three dimensions, observing the manipulated objects along axial direction has been quite challenging. In order to visualize organization and orientation of objects along axial direction, we report development of a Digital holographic microscopy combined with optical tweezers. Digital holography is achieved by use of a modified Mach-Zehnder interferometer with digital recording of interference pattern of the reference and sample laser beams by use of a single CCD camera. In this method, quantitative phase information is retrieved dynamically with high temporal resolution, only limited by frame rate of the CCD. Digital focusing, phase-unwrapping as well as online analysis and display of the quantitative phase images was performed on a software developed on LabView platform. Since phase changes observed in DHOT is very sensitive to optical thickness of trapped volume, estimation of number of particles trapped in the axial direction as well as orientation of non-spherical objects could be achieved with high precision. Since in diseases such as malaria and diabetics, change in refractive index of red blood cells occurs, this system can be employed to map such disease-specific changes in biological samples upon immobilization with optical tweezers.

We report red blood cell (RBC) stretching using a Zeiss Axioplan microscope, modified for phase contrast and optical trapping using a 808 nm diode laser bar, as a tool to characterize RBC dynamics along a linear optical trap. Phase contrast offers a convenient method of converting small variations of refractive index into corresponding amplitude changes, differentially enhancing the contrast near cell edges. We have investigated the behavior of RBCs within both static and dynamic microfluidic environments with a linear optical stretcher. Studies within static systems allow characterization of cell interactions with the line optical force field without the complicating forces associated with hydrodynamics. In flowing, dynamic systems, cells stretch along the optical trap down microfluidic channels and are eventually released to recover their original shape. We record the dynamic cell response with a CMOS camera at 250 fps and extract cell contours with sub-pixel accuracy using derivative operators. To quantify cell deformability, we measure the major and minor axes of individual cells both within and outside of the trap, which also allows measurement of cell relaxation. In these studies, we observe that cell rotation, stretching, and bending along the linear optical trap, are tightly coupled to the modulation of optical power and cell speed inside our microfluidic systems.

Laser-based drug delivery is attractive for the targeting capability due to high spatial controllability of laser energy. Recently, we found that photomechanical waves (PMWs) can transiently increase the permeability of blood vessels in skin, muscle and brain of rats. In this study, we examined the use of annular-shaped PMWs to increase pressure at target depths due to superposition effect of pressure waves. This can increase the permeability of blood vessels located in the specific depth regions, enabling depth-targeted transvascular drug delivery. Annular PMWs were produced by irradiating a laser-absorbing material with annular-shaped pulsed laser beams that were produced by using an axicon lens. We first examined propagation and pressure characteristics of annular PMWs in tissue phantoms and confirmed an increased pressure at a target depth, which can be controlled by changing laser parameters. We injected Evans blue (EB) into a rat tail vein, and annular PMWs (inner diameter, 3 mm; outer diameter, 5 mm) were applied from the myofascial surface of the anterior tibialis muscle. After perfusion fixation, we observed fluorescence originating from EB in the tissue. We observed intense fluorescence at a target depth region of around 5 mm. These results demonstrate the capability of annular PMWs for depth-targeted transvascular drug delivery.

We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and excessive photobleaching during sequential experiments.

Bioluminescence Imaging (BLI) is an increasingly useful and applicable technique that allows for the non-invasive observation of biological events in intact living organisms, ranging from single cells to small rodents. Though the photon production occurs within the host, significant exposure times can be necessary due to the low photon flux compared to fluorescence imaging. The optical absorption spectrum of haemoglobin strongly overlaps most bioluminescent emission spectra, greatly attenuating the total detectable photons in animal models. We have developed and validated a technique that is able to red-shift the bioluminescent photons to the more desirable optical region of > 650 nm, a region of minimal absorbance by hemoglobin. This red-shift occurs by using bioluminescence as an internal light source capable of exciting a fluorophore, such as a fluorescent protein or a quantum dot, that emits in the red. Interestingly, in the absence of an absorber, this excitation can occur over substantial distances (microns to centimeters), far exceeding distances associated to, and thereby precluding, resonance energy transfer phenomena. We show this novel technique yields a substantial increase in the number of red photons for in vitro and ex vivo conditions, suggesting eventually utility for in vivo studies on, for example, intact living mice.

The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

Impedance-based Coulter counters and its derivatives are widely used cell analysis tools in many laboratories and use normally DC or low frequency AC to perform these electrical analyses. The emergence of micro-fabrication technologies in the last decade, however, provides a new means of measuring electrical properties of cells. Microfluidic approaches combined with impedance spectroscopy measurements in the radio frequency (RF) range increase sensitivity and information content and thus push single cell analyses beyond simple cell counting and sizing applications towards multiparametric cell characterization. Promising results have been shown already in the fields of cell differentiation and blood analysis. Here we emphasize the potential of this technology by presenting new data obtained from viability studies on microorganisms. Impedance measurements of several yeast and bacteria strains performed at frequencies around 10 MHz enable an easy discrimination between dead and viable cells. Moreover, cytotoxic effects of antibiotics and other reagents, as well as cell starvation can also be monitored easily. Control analyses performed with conventional flow cytometers using various fluorescent dyes (propidium iodide, oxonol) indicate a good correlation and further highlight the capability of this device. The label-free approach makes on the one hand the use of usually expensive fluorochromes obsolete, on the other hand practically eliminates laborious sample preparation procedures. Until now, online cell monitoring was limited to the determination of viable biomass, which provides rather poor information of a cell culture. Impedance microflow cytometry, besides other aspects, proposes a simple solution to these limitations and might become an important tool for bioprocess monitoring applications in the biotech industry.

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAP^TM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

Antimicrobial peptides (AMPs) are an essential part of the innate immune system that serves as a first line of defense against invading pathogens. Recently, immunomodulatory activities of AMPs have begun to be appreciated, implying the usefulness of AMPs in the treatment of infectious disease. The aim of this strategy is the modulation of host immune responses to enhance clearance of infectious agents and reduce tissue damage due to inflammation. Although AMPs could be used as therapeutic agents, a more detailed understanding of how they affect host cells is needed. Hence, several AMPs have been investigated for their potential as a new class of antimicrobial drugs in this study. Synthetic AMPs and AMPs of natural origin were tested on human leukocytes by flow cytometry. Dose- and time-dependent cytotoxic effects could be observed by propidium iodide staining. Different leukocyte subtypes seem to be susceptible to AMP treatment while others were not affected, even in high concentrations. In conclusion, AMPs have an impact on host immune cells. However, their role in stimulation of chemokine production and enhanced leukocyte recruitment remains a crucial aspect and further studies are needed.

With a diffraction imaging flow cytometer, we have acquired and analyzed the diffraction imaging data from 5 types of cultured cells. A gray level co-occurrence matrix (GLCM) algorithm was applied to extract the interference fringe related textures from the diffraction image data. Six GLCM parameters were chosen and imported into a support vector machine algorithm for automated classification of about 20 cells for each of the 5 cell types. We found that the GLCM based algorithm has the capacity for rapid processing of diffraction images and yield feature parameters for subsequent cell classification except the T- and B-lymphocytes.

Digital excited state lifetime measurements in cytometry were performed on multi-tagged Chinese Hamster Ovary (CHO) cells in order to discriminate between spectrally overlapping fluorescent species. Fluorescence lifetime was determined through digital Fourier analysis with a specialized data acquisition system subsequent to multi-frequency intensity modulation by a solid-state laser excitation source. This work demonstrates that square wave modulation coupled with digital lifetime signal processing can lead to separation of ethidium bromide (EB) and propidium iodide (PI), in cells stained with both dyes. By driving the square wave modulation of the laser at 2 MHz, we were able to access the multiple harmonics present within that wave. In an offline analysis, the phase differences of scatter and fluorescence channels were examined at each harmonic of the primary frequency. The phase difference revealed approximate fluorescence lifetimes of 27.1-ns and 13.0-ns for the EB and PI, respectively. Although the absolute lifetime of each species was not resolved to high accuracy, this work shows a clear separation of the lifetime value calculated at each harmonic. The calculated values that most closely corresponded to the single-dye and multiple-dye average lifetimes were found at the fundamental harmonic frequency (2 MHz) as well as the 4th harmonic (14MHz) frequency. At 2 and 14MHz the average lifetime was 27.1ns and 13.0ns, respectively.

CytometryML is an XML schema based translation, extension and amalgamation of the DICOM and ISAC standards. CytometryML consists of 4 major XML schemas: Series, Instance, Instrument, and Specimen; it also includes Image and List-Mode schemas. Series metadata, which is specific for an entire collection of images and/or list-mode files produced by a single instrument and derived from a single specimen, is stored together with associated metadata files in a container (ZIP) file. Each Instance container file includes binary image and/or list-mode files together with associated metadata files that are specific for a single or closely related group of instrument runs from a single specimen. The Archival Cytometry Standard (ACS) proposed Table of Contents schema including its Resource Description Framework (RDF) capabilities has been extended and modified for use in the Instance schema.

Implementation of AO in high performance microscopes is very dependent on the type of microscopy and the nature of the studied specimen. In this communication, we present the comparison between different implementations of AO sectioning microscopes. The analysis of the benefits and drawbacks of the correction strategies is also presented. Finally, we presented some results obtained by using the discussed strategies of correction.

Infrared (IR) microscopy has shown itself to be an important diagnostic tool for tissue analysis. To date, the main tool for performing IR microscopy has been the Fourier transform infrared (FTIR) microscope. FTIR microscopes utilize incandescent bulbs for light sources, and require cryogenically cooled detectors for the weak, optically poor probe signals. Image acquisition times can be tens of minutes even for sophisticated instruments, and the size and cost of FTIR microscopes precludes their broader clinical use. The development of broadly tunable, external cavity quantum cascade lasers (ECqcL^TM) has created an ideal light source for IR microscopy. Spectrally brilliant probe beams that are diffraction limited, with intensities many orders of magnitude higher than incandescent sources, can be generated from compact, room temperature ECqcL^TM devices. Moreover, the increase in intensity allows the use of room temperature microbolometer focal plane arrays (FPAs) for detection. The combination of ECqcLs^TM and microbolometer FPAs opens the possibility of producing low cost, compact, room temperature IR microscopes with acquisition speeds thirty times that of state-of-the-art FTIR microscopes. The present study explores the challenges of creating this new generation of IR microscopes, and demonstrates the capabilities of the technology.

Spectroscopic and light scattering methods were used to gain insight into the existence and characterization of the cancer stem cell. Fundamental technical description of devices used have been reported elsewhere. We included alterations and implementation of these biophotonic instruments as applied to our objectives. We disassociated human tumor and submitted the cells to optical characterization to support our working hypothesis of stem cell origins to cancer and mechanisms. Single cell combined with population based analysis within the Pancreatic cancer system led us to information regarding the polarization state of cells possessing anchor proteins and drug influx pumps. Multispectral imaging combined with flow cytometry enabled us to target rare cells that appear to retain template DNA. rendering them resistant to anti-cancer drug therapy. In this study we describe an optical method that combines high-throughput population pattern and correlates each cell with an individual fluorescent and bright-field image.

In spite of the adverse ageing effects of glycation in vivo, in vitro this process is widely employed to increase stiffness and strength of tissues' and artificial scaffolds'. In-situ optical characterization methods that report on the structures within these materials could clarify the effects of glycation. We employed one-photon fluorescence and multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals to characterize collagen hydrogels modified with glyceraldehyde, ribose and glucose. We observed an increase in the in situ fluorescence as well as structural alterations within the materials during the course of glycation.

Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in humans following intradermal injections of microdose amounts of indocyanine green. However computational methods for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck, we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms calculating the apparent velocity and period must be validated to verify that they produce results consistent with the proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were compared. Paired Student's t-tests indicate that the differences between their average results are not significant. With the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent lymphatic imaging.

We have quantitatively and systemically measured the morphologies and dynamics of fluctuations in human RBC membranes using a full-field laser interferometry technique that accurately measures dynamic membrane fluctuations. We present conclusive evidence that the presence of adenosine 5'-triphosphate (ATP) facilitates nonequilibrium dynamic fluctuations in the RBC membrane and that these fluctuations are highly correlated with specific regions in the biconcave shape of RBCs. Spatial analysis reveals that these nonequilibrium membrane fluctuations are enhanced at the scale of the spectrin mesh size. Our results indicate the presence of dynamic remodeling in the RBC membrane cortex powered by ATP, which results in nonequilibrium membrane fluctuations.

Introduction: Methylprednisolone (MP) is frequently preoperatively administered in children undergoing open heart surgery. The aim of this medication is to inhibit overshooting immune responses. Earlier studies demonstrated cellular and humoral immunological changes in pediatric patients undergoing heart surgeries with and without MP administration. Here in a retrospective study we investigated the modulation of the cellular immune response by MP. The aim was to identify suitable parameters characterizing MP effects by cluster analysis. Methods: Blood samples were analysed from two aged matched groups with surgical correction of septum defects. Group without MP treatment consisted of 10 patients; MP was administered on 21 patients (median dose: 11mg/kg) before cardiopulmonary bypass (CPB). EDTA anticoagulated blood was obtained 24 h preoperatively, after anesthesia, at CPB begin and end (CPB2), 4h, 24h, 48h after surgery, at discharge and at out-patient followup (8.2; 3.3-12.2 month after surgery; median and IQR). Flow cytometry showed the biggest MP relevant changes at CPB2 and 4h postoperatively. They were used for clustering analysis. Classification was made by discriminant analysis and cluster analysis by means of Genes@work software. Results & conclusion: 146 parameters were obtained from analysis. Cross-validation revealed several parameters being able to discriminate between MP groups and to identify immune modulation. MP administration resulted in a delayed activation of monocytes, increased ratio of neutrophils, reduced T-lymphocytes counts. Cluster analysis demonstrated that classification of patients is possible based on the identified cytomics parameters. Further investigation of these parameters might help to understand the MP effects in pediatric open heart surgery.

This paper proposes a new segmentation technique developed for the segmentation of cell nuclei in both 2D and 3D fluorescent micrographs. The proposed method can deal with both blurred edges as with touching nuclei. Using a dual scan line algorithm its both memory as computational efficient, making it interesting for the analysis of images coming from high throughput systems or the analysis of 3D microscopic images. Experiments show good results, i.e. recall of over 0.98.

Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

Leaf cells of living plants exhibit strong fluorescence from chloroplasts, the reaction centers of photosynthesis. Mutations in the photosystems change their structure and can, thus, be monitored by recording the fluorescence spectra of the emitted chlorophyll light. These measurements have, up to now, mostly been carried out at low temperatures (77 K), as these conditions enable the differentiation between the fluorescence of Photosystem I (PSI) and Photosystem II (PSII). In contrast, at room temperature, energy transfer processes between the various photosynthetic complexes result in very similar fluorescence emissions, which mainly consist of fluorescence photons emitted by PSII hindering a discrimination based on spectral ROIs (regions of interest). However, by statistical analysis of high resolution fluorescence spectra recorded at room temperature, it is possible to draw conclusions about the relative PSI/PSII ratio. Here, the possibility of determining the relative PSI/PSII ratio by fluorescence spectroscopy is demonstrated in living maize plants. Bundle-sheath chloroplasts of mature maize plants have a special morphologic characteristic; they are agranal, or exhibit only rudimentary grana, respectively. These chloroplasts are depleted in PSII activity and it could be shown that PSII is progressively reduced during leaf differentiation. A direct comparison of PSII activity in isolated chloroplasts is nearly impossible, since the activity of PSII in both mesophyll- and bundle-sheath chloroplasts decays with time after isolation and it takes significantly longer to isolate bundle-sheath chloroplasts. Considering this fact the measurement of PSI/PSII ratios with the 77K method, which includes taking fluorescence spectra from a diluted suspension of isolated chloroplasts at 77K, is questionable. These spectra are then used to analyze the distribution of energy between PSI and PSII. After rapid cooling to 77K secondary biochemical influences, which attenuate the fluorescence emanated from PSI, are frozen out. Due to their characteristic morphology, maize chloroplasts of mesophyll and bundle-sheath cells are an appropriate system for demonstrating the applicability of our in vivo method which, unlike the common 77K method, does not require the isolation of chloroplasts. In mesophyll chloroplasts of higher land plants, the thylakoids have a heterogenic morphology of appressed and non-appressed membrane domains, called the grana and the stroma lamellae. PSII is enriched in the grana, whereas PSI is enriched in the stroma lamellae. Changes in chloroplast membrane structure and composition, according to changes in the PSI/ PSII ratio, can be triggered by light quality and carbon source deficiency. Here, we demonstrate the applicability of statistical analysis of fluorescence spectra to detect changes in the PSI/PSII ratio resulting from structure changes in the thylakoid membrane.

A heating device is presented, which can be attached on top of a standard microtiter plate, permitting individual and fast heating of the specimen by resistor elements without direct contact. All wells of the microtiter plate remain accessible from above and beyond for sensory (e.g. optical) measurements, independently from the bottom geometry of the wells. By various circuit configurations it is possible to realize either homogenous heating all over the plate or heating of individual arrays or clusters of wells, e.g. for maintaining defined temperature gradients. Main applications include quantitative measurements of temperature dependent parameters in the area of diagnostics, serology, cell culture- and immunological research. The heating device is validated by various referencing temperature kinetics and infrared images, as well as by selective investigations of cell membranes upon total internal reflection of a laser beam. For this purpose, particularly with regard to potential pharmaceutical applications, fluorescence decay kinetics of cancer cells incubated with temperature-dependent dyes, e.g. 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), is determined.

We present the study of the correlation properties of RBC flickering using double-path interferometric quantitative phase microscopy (QPM) using detrended fluctuation analysis (DFA). For DFA of RBC membrane fluctuations, we have measured time series thickness variations of a normal RBC for 20 seconds. The amplitude of membrane fluctuations in RBC have showed significantly larger than the background noise level without a RBC. We have demonstrated a practical DFA application for QPM by studying the correlation property of RBC membrane fluctuations in a noninvasive manner. By measuring the fractal scaling exponents of the time series RBC thickness variations obtained from QPM, we have analyzed the correlation properties of RBC membrane fluctuations and the background noise without a sample. The exponents for a normal RBC revealed the long-range correlation property in time series during 20 seconds. However, the averaged exponent for background noise outside a cell was close to the exponent of white noise.

Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for in vivo optical imaging of the urinary tract. Particularly for bladder cancer, real time optical biopsy of suspected lesions will likely lead to improved management of bladder cancer. With pCLE, micron scale resolution is achieved with sterilizable imaging probes (1.4 or 2.6 mm diameter), which are compatible with standard cystoscopes and resectoscopes. Based on our initial experience to date (n = 66 patients), we have demonstrated the safety profile of intravesical fluorescein administration and established objective diagnostic criteria to differentiate between normal, benign, and neoplastic urothelium. Confocal images of normal bladder showed organized layers of umbrella cells, intermediate cells, and lamina propria. Low grade bladder cancer is characterized by densely packed monomorphic cells with central fibrovascular cores, whereas high grade cancer consists of highly disorganized microarchitecture and pleomorphic cells with indistinct cell borders. Currently, we are conducting a diagnostic accuracy study of pCLE for bladder cancer diagnosis. Patients scheduled to undergo transurethral resection of bladder tumor are recruited. Patients undergo first white light cystocopy (WLC), followed by pCLE, and finally histologic confirmation of the resected tissues. The diagnostic accuracy is determined both in real time by the operative surgeon and offline after additional image processing. Using histology as the standard, the sensitivity, specificity, positive and negative predictive value of WLC and WLC + pCLE are calculated. With additional validation, pCLE may prove to be a valuable adjunct to WLC for real time diagnosis of bladder cancer.

Analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging (FLIM). Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of lifetime to be measured. We have found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. We have also proposed dual channel waveform measurement scheme that may suppress these errors. It is shown that there may exist more than 2 ns drift in a measured temporal waveform during the period of the first 12 minutes after electronics components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as large as 51 ps without a proposed scheme. We have shown that this error can be reduced to 9 ps with our dual-channel waveform measurement method.

Hyperspectral imaging (HSI) is a chemical imaging modality with spectroscopic information. This technique was often used in agricultural or pharmaceutical industries. But there have been a few reports for clinical medical applications. In near-infrared (NIR) wavelength region, the significant absorption peaks are often observed by the overtone of midinfrared molecular vibration. In addition, NIR light has a high penetration because of low scattering and less absorption by water or protein. In this study, we constructed the NIR-HSI system and the high-contrast subcutaneous adipose tissue imaging was conducted in-vitro. In the absorption spectra which are obtained by our NIR-HSI system, the characteristic absorption bands were observed around 1200 nm and 1700 nm. In the processed images using these wavelength bands, subcutaneous adipose tissue was observed through a skin. In a hyperspectral image by another processing using all wavelengths, a high-contrast image of subcutaneous adipose tissue is also obtained. NIR-HSI system is a powerful diagnostic technique for adipose tissues distribution and their morphological change on/inside a tissue.

Optical tweezers is a technique to trap and to manipulate micron sized objects under a microscope by radiation pressure force exerted by a laser beam. Optical tweezers has been utilized for single-molecular measurements of force exerted by molecular interactions and for cell palpation. To extend applications of optical tweezers we have developed a novel optical tweezers system combined with a pulse laser. We utilize a pulsed laser (Q-switched Nd: YAG laser, wavelength of 1064 nm) to assist manipulations by conventional optical tweezers achieved by a continuous wave (CW) laser. The pulsed laser beam is introduced into the same optics for conventional optical tweezers. In principle, instantaneous radiation force is proportional to instantaneous power of laser beam. As a result, pulsed laser beam generates strong instantaneous force on an object to be manipulated. If the radiation force becomes strong enough to get over an obstacle structure and/or to be released from adhesion, the object will be free from these difficulties. We have named this technique as Pulse Laser beam Assisted optical Tweezers (PLAT). We have successfully demonstrated to manipulate objects surface on a living cell for "in vivo manipulation."

Wavelet Transform based multi-resolution analysis has been used to characterize the intrinsic fluorescence of both dysplastic and normal human cervical tissues. The fluorescence spectra corresponding to 325nm and 370nm excitation from cervical dysplastic tissues of 48 patients from diverse age groups are studied in detail using Morlet wavelet. The wavelet modulus maxima lines for 325nm excitation indicated a distinct shift for dysplastic tissues towards the lower wavelengths with respect to normal ones. For 370nm excitation however, the shift for dysplastic tissues were towards the higher wavelengths. Sensitivities of 72% and 81% for spectral shifts of 325 and 370nm excitations were observed in the wavelength band of 425-500nm of the intensity spectra.

Infrared spectroscopic imaging of cancerous kidney tissue was performed by means of FTIR microscopy. The spectra of thin tissue cryosections were collected with 64x64 MCT FPA detector and imaging area was increased up to 5.4×5.4 mm by mapping by means of PC controlled x,y stage. Chemical images of the samples were constructed using statistical treatment of the raw spectra. Several unsupervised and supervised statistical methods were used. The imaging results are compared with results of the standard histopathological analysis. It was concluded that application of method of cluster analysis ensures the best contrast of the images. It was found that border between cancerous and normal tissues visible in the infrared spectroscopic image corresponds with the border visible in histopathological image. Closer examination of the infrared spectroscopic image reveals that small domains of cancerous cells are found beyond the border in areas distant from the border up to 3 mm. Such domains are not visible in the histopathological images. The smallest domains found in the infrared images are approx. 60 μm.

An automated cell detection and sorting system was developed, combining both the optofluidic intracavity spectroscopy (OFIS) technique and dielectrophoresis (DEP). The OFIS method utilizes a microfluidic channel as a Fabry-Perot cavity to produce characteristic transmission spectra of individual cells. The concept behind optical detection is that a decrease in spectral intensity beyond a threshold indicates that a cell is present. Upon detection, an RF voltage is automatically applied to electrodes, trapping the cell with DEP forces. The system then sorts the cell into one of two microfluidic channels based on resulting optical analysis. A further advantage is that RF joule heating can be measured from known dn/dT values of the medium, which is useful for investigating cell viability issues.

Fiber based functional near infra-red (fNIR) spectroscopy has been considered as a cost effective imaging modality. To achieve a better spatial resolution and greater accuracy in extraction of the optical parameters (i.e., μa and μ's), broadband frequency modulated systems covering multi-octave frequencies of 10-1000MHz is considered. A helmet mounted broadband free space fNIR system is considered as significant improvement over bulky commercial fiber fNIR realizations that are inherently uncomfortable and dispersive for broadband operation. Accurate measurements of amplitude and phase of the frequency modulated NIR signals (670nm, 795nm, and 850nm) is reported here using free space optical transmitters and receivers realized in a small size and low cost modules. The tri-wavelength optical transmitter is based on vertical cavity semiconductor lasers (VCSEL), whereas the sensitive optical receiver is based on either PIN or APD photodiodes combined with transimpedance amplifiers. This paper also has considered brain phantoms to perform optical parameter extraction experiments using broadband modulated light for separations of up to 5cm. Analytical models for predicting forward (transmittance) and backward (reflectance) scattering of modulated photons in diffused media has been modeled using Diffusion Equation (DE). The robustness of the DE modeling and parameter extraction algorithm was studied by experimental verification of multi-layer diffused media phantoms. In particular, comparison between analytical and experimental models for narrow band and broadband has been performed to analyze the advantages of our broadband fNIR system.

Novel beam-based optical tweezers, employed in microfluidic devices, has enabled efficient and non-contact actuation of microscale samples. We report development of an optical isolator based on pincushion distortion introduced into astigmatic optical tweezers. While objects in the range of 1 to 5 microns (polystyrene particles and bacteria) were transported away along the curvilinear trajectories of the pincushion profile, objects in 10-20 micron range (e.g. cells) could easily be trapped in the center of the pincushion profile. This enabled efficient isolation of cell(s) from its surrounding with high spatial and temporal precision and thus opens up new possibility to control and study interaction of cells (and other microscopic objects) with surrounding objects without requiring presence or actuation of physical valves. The trapped and isolated cell(s) could be transported by maneuvering the sample stage or beam. Further, optical clearing of wide microscopic area was achieved using the distorted profile without requiring beam scanning or sample movement. The distorted tweezers was used to clear floating microscopic particles near axonal networks as well as from the top surface of retina explant. Theoretical simulation of force exerted by such beam profiles and experimental demonstration of its potential in microfluidic isolation and manipulation is discussed.

Imaging mass spectrometry based on matrix-assisted laser desorption/ionization gives distributions of multiple biomolecules and identification of analytes in a tissue section. We are developing a stigmatic imaging mass spectrometer using a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. A mesh pattern image was obtained with spatial resolution of 3 μm. Separation of stigmatic ion images according to the mass-to-charge ratio was also successfully demonstrated with the micro-dot pattern made with crystal violet and methylene blue. An ion image of a micro-dot pattern sample was observed after ten cycles in the multi-turn circuit of MULTUM-IMG, and the pattern of the sample was maintained. Then, stigmatic ion images of the ions of crystal violet and methylene blue produced from the stained section of a brain were observed. An ion image of whole part of a hippocampus (3.25 mm × 1.5 mm) in the brain section can be obtained within 13 minutes.

Detection and quantification of circulating cells in live animals is a challenging and important problem in many areas of biomedical research. Current methods involve extraction of blood samples and counting of cells ex-vivo. Since only small blood volumes are analyzed at specific time points, monitoring of changes in cell populations over time is difficult and rare cells often escape detection. The goal of this research is to develop a method for enumerating very rare circulating cells in the bloodstream non-invasively. This would have many applications in biomedical research, including monitoring of cancer metastasis and tracking of hematopoietic stem cells. In this work we describe the optical configuration of our instrument which allows fluorescence detection of single cells in diffusive media at the mesoscopic scale. Our instrument design consists of two continuous wave laser diode sources and an 8-channel fiber coupled multi-anode photon counting PMT. Fluorescence detector fibers were arranged circularly around the target in a miniaturized ring configuration. Cell-simulating fluorescent microspheres and fluorescently-labeled cells were passed through a limb mimicking phantom with similar optical properties and background fluorescence as a limb of a mouse. Our data shows that we are able to successfully detect and count these with high quantitative accuracy. Future work includes characterization of our instrument using fluorescently labeled cells in-vivo. If successful, this technique would allow several orders of magnitude in vivo detection sensitivity improvement versus current approaches.