Hyperion Image Analysis Depicts a Preliminary Landscape of Tumor Immune Microenvironment in OSCC with Lymph Node Metastasis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) constitutes the most common types of oral cancer. Because its prognosis varies significantly, identification of a tumor immune microenvironment could be a critical tool for treatment planning and predicting a more accurate prognosis. This study is aimed at utilizing the Hyperion imaging system to depict a preliminary landscape of the tumor immune microenvironment in OSCC with lymph node metastasis. METHODS: We collected neoplasm samples from OSCC patients. Their formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained and stained utilizing a panel of 26 clinically relevant metal-conjugated antibodies. Detection and analysis were performed for these stained cells with the Hyperion imaging system. RESULTS: Four patients met our inclusion criteria. We depicted a preliminary landscape of their tumor immune microenvironment and identified 25 distinct immune cell subsets from these OSCC patients based on phenotypic similarity. All these patients had decreased expression of CD8+ T cells in tumor specimens. Variety in cell subsets was seen, and more immune activated cells were found in patient A and patient B than those in patient C and patient D. Such differences in tumor immune microenvironments can contribute to forecasting of individual prognoses. CONCLUSION: The Hyperion imaging system helped to delineate a preliminary and multidimensional landscape of the tumor immune microenvironment in OSCC with lymph node metastasis and provided insights into the influence of the immune microenvironment in determination of prognoses. These results reveal possible contributory factors behind different prognoses of OSCC patients with lymph node metastasis and provide reference for individual treatment planning.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

[Experimental corneal imaging and corneal surgery with non-amplified femtosecond laser pulses]. / Experimentelle Hornhautbildgebung und Hornhautchirurgie mit nicht verstärkten Femtosekundenlaserpulsen.

BACKGROUND: Non-amplified femtosecond laser was used to induce multiphoton effects for corneal tissue imaging and for tissue ablation. MATERIAL AND METHODS: A non-amplified titanium-sapphire laser was coupled to a laser scanning microscope in order to examine human and porcine cornea. Tissue was subjected to imaging and lesions were created using identical optical pathways at pulse energies below 2 nJ. RESULTS: Cellular components and the extracellular matrix were selectively imaged by applying autofluorescence and second harmonic generation at submicron resolution. Intrastromal linear scanning at higher power resulted in luminescent plasma along the scanning line. Lesion width decreased with increasing tissue depth and increased with increasing laser power at the target. Light microscopy showed intact stromal tissue around the area of the lesion. CONCLUSIONS: High-resolution images as well as high precision tissue lesions were created in the cornea using low energy femtosecond laser pulses. Easy switching between tissue imaging and ablation seems to be suitable for diagnostic and therapeutic applications.

Differentiation and visualization of diverse cellular phenotypic responses in primary high-content screening.

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating "scaffold hopping."

Laser scanning cytometry and its applications: a pioneering technology in the field of quantitative imaging cytometry.

Imaging cytometry plays an increasingly important role in all fields of biological and medical sciences. It has evolved into a complex and powerful discipline amalgamating image acquisition technologies and quantitative digital image analysis. This chapter presents an overview of the complex and ever-developing landscape of imaging cytometry, highlighting the imaging and quantitative performance of a wide range of available instruments based on their methods of sample illumination and the detection technologies they employ. Each of these technologies has inherent advantages and shortcomings stemming from its design. It is therefore paramount to assess the appropriateness of all of the imaging cytometry options available to determine the optimal choice for specific types of studies. Laser scanning cytometry (LSC), the original imaging cytometry technology, is an attractive choice for analysis of both cellular and tissue specimens. Quantitative performance, flexibility, and the benefits of preserving native sample architecture and avoiding the introduction of artificial signals, particularly in cell-signaling studies and multicolor tissue analysis, are speeding the adoption of LSC and opening up new possibilities for developing sophisticated applications.

Multiple biomarker expression on circulating tumor cells in comparison to tumor tissues from primary and metastatic sites in patients with locally advanced/inflammatory, and stage IV breast cancer, using a novel detection technology.

Patients with locally advanced/inflammatory breast cancer (LABC/IBC) face a high likelyhood of recurrence and prognosis for relapsed, or de novo stage IV metastatic breast cancer (MBC) remains poor. Estrogen (ER) and HER2 receptor expression on primary or MBC allow targeted therapies, but an estimated 10-18% of tumors do not exhibit these biomarkers and survival in these cases is even poorer. Variations in discordance rates for the expression of ER and HER2 receptors have been observed between primary and metastatic tumors and such discordances may lead to suboptimal treatment. Circulating tumor cells (CTCs) are considered the seeds of residual disease and distant metastases and their characterization could help guide treatment selection. To explore this possibility, we used multiple biomarker assessment of CTCs in comparison to primary and metastatic tumor sites. Thirty-six patients with LABC/IBC, or stage IV MBC were evaluated. Blood samples were procured prior to initiating or changing therapy. CTCs were identified based on presence of cytokeratin and nucleus staining, and the absence of CD45. A multimarker assay was developed to simultaneously quantify expression of HER2, ER, and ERCC1, a DNA excision repair protein. Novel fiber-optic array scanning technology (FAST) was used for sensitive location of CTCs. CTCs were detected in 82% of MBC and 62% LABC/IBC cases. Multiplex marker expression was successfully carried out in samples from18 patients with MBC and in 8 patients with LABC/IBC that contained CTCs. In MBC, we detected actionable discordance rates of 40 and 23%, respectively for ER and HER2 where a biomarker was negative in the primary or metastatic tumor and positive in the CTCs. In LABC/IBC, actionable discordances were 60 and 20% for ER and HER2, respectively. Pilot trials evaluating the effectiveness of treatment selections based on actionable discordances between biomarker expression patterns on CTCs and primary or metastatic tumor sites may allow for a prospective assessment of CTC-based individualized targeted therapies.

An automated microfluidic sample preparation system for laser scanning cytometry.

Laser scanning cytometry (LSC) is emerging as a clinical tool. In one application a "Clatch" slide, named after the inventor, is used in conjunction with LSC for cell surface marker immunophenotyping of patient samples. The slide requires time consuming and laborious pipetting steps, making a test tedious and prone to handling errors. The Clatch slide also uses a significant number of cells, limiting the number of analyses on paucicellular samples. This paper presents an automated microfluidic system consisting of a control circuit, a microfluidic system, and an aluminum frame, capable of performing immunophenotyping procedures. This prototype system reduces 36 pipetting steps to 1, reduces the amount of cell sample from 180 µL to 56 µL, and shortens the time used by technicians.

Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.

Cell type-specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon-excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.

Flow cytometry and laser scanning cytometry, a comparison of techniques.

OBJECTIVE: Flow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications. METHODS: A Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting. RESULTS: Both techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer's disease. CONCLUSIONS: Understanding the differences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capabilities with the cellular characteristics they wish to measure delineating molecular and cellular events occurring in health and disease. Discovery of mechanisms in cells using FCM and LSCM provide evidence needed to guide future treatment and interventions.

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry.

The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting.

Visualization of microscopy-based spectral imaging data from multi-label tissue sections.

Combining images taken with light of specific wavelengths can dramatically enhance light-microscopic images. This technology is enabled by the availability of programmable filters that can be set to transmit light only of particular wavelengths. Spectral imaging technologies have become an important part of microscopy, and are particularly useful for analyzing samples that have been labeled with multiple (two or more) molecular markers. The most commonly used methodology for separating the markers from each other is linear unmixing, which results in a quantitative image of the location and amount of each marker present in the sample. The very complexity of these multilabel samples requires a high degree of sophistication in methods to visualize the results of unmixing. This article describes a wide range of useful visualization tools designed to better enable discrimination of different features in multilabeled tissue or cell samples. These commercially available tools can be attached to the standard laboratory light microscope to significantly enhance the power of light microscopy.

Methods and applications of laser-enabled analysis and processing (LEAP).

The LEAP (Laser-Enabled Analysis and Processing) platform combines in situ imaging with laser manipulation to efficiently identify, purify, and monitor expansion of high secreting clones. It also allows for rapid analysis of cell population heterogeneity. This unit describes the LEAP instrumentation as well as basic and alternate protocols of the major applications in recombinant human or humanized IgG expression. The protocols include fluorescent cell counting, secreted recombinant IgG capture and detection, and IgG-secreting clone selection by laser processing.

In vivo multispectral, multiparameter, photoacoustic lymph flow cytometry with natural cell focusing, label-free detection and multicolor nanoparticle probes.

Compared with blood tests, cell assessment in lymphatics is not well-established. The goal of this work was to develop in vivo lymph tests using the principles of flow cytometry. Cells in living animals were counted by laser (420-2,300 nm) generation of photoacoustic (PA) signals in individual cells hydrodynamically focused by lymph valves into a single file flow, and using endogenous absorption as intrinsic cell-specific markers, or gold nanorods, nanoshells, and carbon nanotubes as multicolor probes. PA data were verified by high-speed transmission, photothermal, and fluorescent imaging. Counting of melanoma and immune-related cells in normal, apoptotic, and necrotic states in lymphatics in vivo was demonstrated to have the unprecedented sensitivity as one metastatic cell among millions of white blood cells. The time-resolved PA spectral identification of flowing cells was achieved using multicolor labels and laser pulses of different wavelengths and time delays. Multiparameter, noninvasive, portable flow cytometer can be used for preclinical studies on animals with the potential of translation to humans for in vivo PA mapping of colorless lymph vessels and sentinel nodes with simultaneous single cell detection and metastasis assessment without labeling or use of contrast dyes and/or novel low-toxic multicolor probes with different absorption spectra.

High-content analysis in preclinical drug discovery.

High-Content Analysis (HCA) has developed into an established tool and is used in a wide range of academic laboratories and pharmaceutical research groups. HCA is now routinely proving to be effective in providing functionally relevant results. It is essential to select the appropriate HCA application with regard to the targeted compound's cellular function. The cellular impact and compound specificity as revealed by HCA analysis facilitates reaching definitive conclusions at an early stage in the drug discovery process. This technology therefore has the potential to substantially improve the efficiency of pharmaceutical research. Recent advances in fluorescent probes have significantly boosted the success of HCA. Auto-fluorescent proteins which minimally hinder the functioning of the living cell have been playing a decisive role in cell biology research. For companies the severely restricted license conditions regarding auto-fluorescent proteins hamper their general use in pharmaceutical research. This has opened the field for other solutions such as self-labeling protein technology, which could potentially replace the well established methods that utilize auto-fluorescent proteins. In addition, direct labeling techniques have improved considerably and may supersede many of the approaches based on fusion proteins. Following sample preparation, treated cells are imaged and the resulting multiple fluorescent signals are subjected to contextual and statistical analysis. The extraordinary advantage of HCA is that it enables the large-scale and simultaneous quantification and correlation of multiple phenotypic responses and physiological reactions using sophisticated software solutions that permit assay-specific image analysis. Hence, HCA once more has demonstrated its outstanding potential to significantly support establishing effective pharmaceutical research processes in order to both advance research projects and cut costs.

Laser surgery and optical trapping in a laser scanning microscope.

Optical manipulation opens up many new possibilities for experiments in the field of microbiology and is a very powerful tool for investigating cellular structure. In this emerging field imaging retains an important role, and systems that combine advanced imaging techniques with optical manipulation tools, such as laser scalpels or optical tweezers, are an important starting point for researchers. We present a flexible experimental platform that contains both a laser scalpel and optical tweezers, in combination with confocal and multiphoton microscopy. A simple manipulation of the external optics is used to retain the three-dimensional imaging capabilities of the microscopes. Two applications of the system are presented. In the first, the laser scalpel is used to initiate diffusion of a fluorescent dye through Escherichia coli mutants, which exhibit abnormal cell division, forming filaments, or chains of bacteria. The diffusion assay is used to assess the potential for the exchange of cytoplasmic material between neighboring cells. The second application investigates the binding of endoplasmic reticulum (ER) to chloroplasts in Pisum sativum (garden pea). Individual plant protoplasts are ruptured using the laser scalpel, allowing individual chloroplasts to be trapped and manipulated. Strands of the ER which are attached to the chloroplast are identified. The magnitude and nature of the binding between the chloroplast and the ER are investigated.

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.

Microtransponders, the miniature RFID electronic chips, as platforms for cell growth in cytotoxicity assays.

BACKGROUND: An electronic radio frequency (RF) microchip, the microtransponder (MTP), has been developed as a platform for assays in the fields of genomics and proteomics. Upon activation by light, each MTP provides a unique RF identification (ID) signal that matches a chip to the specific biological material attached to it. The MTP is powered by a photocell and has an antenna that transmits the signal. The aim of the present study was to explore utility of MTPs as a platform for cell growth in cytotoxicity assays. METHODS: The MCF-7, MCF-116, A549, or T-24 cells growing on MTPs placed in petri dishes or slide chambers were cultured untreated or exposed to antitumor drugs topotecan, mitoxantrone, or onconase for up to 4 days. Their attachment to- and growth on- MTPs was assessed by fluorescence microscopy and laser scanning cytometry (LSC) and compared with growth on the dish surface in the MTP neighborhood. The MTPs were fixed in ethanol, stained with propidium iodide (PI), and interrogated in flow in the instrument capable to rapidly (up to 103 MTPs/s) identify their ID signal and measure fluorescence. RESULTS: The cells plated on MTPs exhibited similar attachment properties to those plated in culture dishes. When measured by LSC, they had similar mitotic activity, growth rate, and cell cycle distributions as the cells adhering to the culture dish in the neighborhood of MTPs. The fluorescence intensity of MTPs provided information about the cell number per MTP, which made it possible to assess cell growth rate and monitor the cytostatic/cytotoxic effects of the tested drugs. CONCLUSIONS: The MTP-based system holds promise for the multiplexed cell assays in which numerous different cell lines can be screened for their growth rate or sensitivity while exposed to particular agents in the same vessel. Other advantages of the system are the rapidity of the screening and a very large number of ID codes. Because many cell lines/types can be assayed in a single dish, the system also offers cost savings on tissue culture reagents.

Method for assessment of viability and morphological changes of bacteria in the early stage of colony formation on a simulated natural environment.

A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

Sinking velocities of phytoplankton measured on a stable density gradient by laser scanning.

Two particular difficulties in measuring the sinking velocities of phytoplankton cells are preventing convection within the sedimenting medium and determining the changing depth of the cells. These problems are overcome by using a density-stabilized sedimentation column scanned by a laser. For freshwater species, a suspension of phytoplankton is layered over a vertical density gradient of Percoll solution; as the cells sink down the column their relative concentration is measured by the forward scattering of light from a laser beam that repeatedly scans up and down the column. The Percoll gradient stabilizes the column, preventing vertical mixing by convection, radiation or perturbation of density by the descending cells. Measurements were made on suspensions of 15 microm polystyrene microspheres with a density of 1050 kg m-3; the mean velocity was 6.28 microm s-1, within 1.5% of that calculated by the Stokes equation, 6.36 microms-1. Measurements made on the filamentous cyanobacterium Planktothrix rubescens gave mean velocities within the theoretical range of values based on the range of size, shape, orientation and density of the particles in a modified Stokes equation. Measurements on marine phytoplankton may require density gradients prepared with other substances.