Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.     

A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our “photooxidizer” consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy.     

New optics sheds light on biology. Advances in microscopy, genetics and biochemistry are together increasingly enabling scientists to watch molecules in action

Advances in science and technology enable scientists to peek at cellular and molecular events in vivo. The same technologies also provide new tools for cancer diagnostics and monitoring treatmentAdvances in light microscopy are about to transform molecular biology by enabling biologists to observe molecular processes in vivo. Although biochemical analysis or genomic sequencing, along with imaging techniques such as X-ray diffraction and electron microscopy, can provide detailed information about cellular structures and the products of particular processes, it is light microscopy that allows scientists to capture dynamic events in the cell. Basic research is not the only enterprise that benefits from improvements in microscopy; new optical techniques also have an increasing role in diagnosis and surgery—in particular for some cancers—to analyse tissue in real time.…it is light microscopy that allows scientists to capture dynamic events in the cellFor decades, light microscopy was regarded as the poor relative of electron microscopy and X-ray crystallography, both of which enable much greater resolutions, down to atomic scales around 0.1 nm. Yet both techniques come at the expense of the object because they can only analyse fixed samples—crystals in the case of X-ray crystallography and thin sections for electron microscopy. This has always been the advantage of light microscopy: to capture events in living systems. Now, various developments over the past two decades have come together to introduce real-time imaging. “Revolutionary improvements in laser light sources, detectors, or cameras, and the advent of computerized digital light and fluorescence microscopy have been essential for enabling real-time imaging and micro-endoscopy in living systems,” said Frank Chuang, associate director of research and education at the Centre for Biophotonics and Research at the University of California, Davis in Sacramento, USA.The first major step for real-time light microscopy was the development of fluorescence methods for labelling proteins in vivo. These methods exploit the fact that fluorescent compounds absorb electromagnetic radiation at one wavelength and emit light at a different wavelength. A sample can be illuminated by electromagnetic radiation at the prescribed wavelength, and only the fluorescent components of the sample, as well as areas very close to them, are observed. Using fluorophores as markers for specific proteins, scientists can obtain functional information as this allows them to track their movement.The opening move was the discovery of the green fluorescence protein (GFP) in the jellyfish Aequorea victoria in the late 1970s (Tsien, 1998). GFP is composed of 238 amino-acid residues and gives off bright green fluorescence on exposure to blue light. Since its discovery, GFP has been further improved by protein design and has been applied to studies of proteins that are marked with GFP in bacteria, yeast and fish, as well as plant and mammalian cells. For these discoveries, Martin Chalfie, Osamu Shimomura and Roger Tsien received the Nobel Prize in Chemistry in 2008.The first major step for real-time light microscopy was the development of fluorescence methods for labelling proteins in vivoGFP can be spliced into the genome next to the gene of the protein of interest, which is then expressed as a fluorescent fusion protein. GFP can also be introduced into target cells to analyse, for example, brain circuitry or the entry of viruses into cells during infection. “That was definitely a turning point […] and people can now capture not only stills but also dynamic scenarios of these proteins, to observe how they are moving, and where they are localized in living samples ranging from bacteria to entire living animals,” commented Michael Knop, professor of molecular biology at Heidelberg University, Germany, who applies live-cell microscopic imaging to studying protein function in meiosis.One problem though was that in traditional so-called wide-field light microscopy—in which the light source illuminates the whole sample—background light interferes with the area of interest. This was addressed by the confocal microscope, which eliminates the background by shining light through a pinhole so that it can be focused on a small target zone. This improves optical resolution immensely, but at the expense of signal intensity, because most of the light is blocked at the pinhole. Images have to be exposed for longer periods or at increased intensity, which causes greater photo damage to the object.This problem was eventually addressed by another important advance for light microscopy, the development of fast, ultra-sensitive charged-coupled-device (CCD) chips for photodetection. “This really started 10–12 years ago when a new generation of CCDs allowed the construction of cameras that could be used for very low light detection,” said Knop. CCD cameras help to reduce photo damage to samples and capture images more quickly, which increases the scope for making time-lapsed movies.These improvements led the way for other developments, notably the light-sheet microscope in 2003. It was pioneered at the European Molecular Biology Laboratory in Heidelberg, Germany, by a group headed by Ernst Stelzer, now Professor in Advanced Light Microscopy at the University of Frankfurt, Germany. The microscope works on the basis of illuminating the specimen from the side rather than from above; a sheet of light exposes only the narrow slice on which the microscope is focused at the time. Light-sheet microscopy involves two independently operated lenses for illumination and detection. By focusing the light only on the narrow optical section within the focal plane of observation, it further reduces photo damage or photo-bleaching.The other goal of light-microscopy development is improving resolution, which is needed to observe individual proteins and detail at the molecular levelLight-sheet microscopy has since been combined with other techniques for improving contrast and manipulating specimens to create even better three-dimensional images. It enabled the dramatic in vivo recording of embryonic development in a zebrafish, from the early 32-cell stage to the neurulation period, when cells start to differentiate to form organs. Individual cells were visualized with 60–90 seconds between samples, and four-megapixel image frames were recorded at a rate of five per second. This recording speed was important because cell movement could then be tracked with sufficient accuracy to study migration. The work showed that light-sheet microscopy exposed the embryo to 200 times less energy than a conventional microscope and 5,000–6,000 times less than a confocal fluorescence microscope (Keller et al, 2008).The other major goal of light-microscopy development is improving resolution, which is needed to observe individual proteins and detail at the molecular level. It requires overcoming the diffraction-limit rule, which states that the resolution cannot be better than half the wavelength of light, around 200 nm, which is insufficient even to observe organelles within cells. This limit occurs because the wavelength defines the minimum spot size that can be resolved from a single ray of light. Any smaller details will be blurred by interference from light emitted from neighbouring points in the sample.Even when a sample is being observed in vivo, the preparation—involving excitation with light—is not normalHowever, ways have been found to extract information from samples at higher resolution. One such technique is called STED (stimulated emission depletion) fluorescence microscopy, described in 1994 by Stefan Hell at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany (Hell & Wichmann, 1994). STED is based on the idea of blocking light that is emitted from around the point of interest, to prevent interference. It combines the normal pulse of light that stimulates fluorescence with a second pulse tuned to the frequency of the excited radiation, which cancels it out. This second pulse is directed close to the focal point from a doughnut-shaped emitter. By transmitting the second cancelling pulse from the rim of the doughnut only, the light is focused onto a ring around the focal point so that only a small area in the centre receives the pulse that generates fluorescence. This creates a coherent spot of emitted light corresponding to the molecules in the centre of the ring only, which yields a sharp image at a resolution beyond the diffraction limit. Furthermore, by increasing the intensity of the cancelling light that is emitted from the rim of the doughnut, the diameter of the coherent spot in the middle can be reduced to further increase resolution, in principle, down to the size of a single atom. In practice, one of the highest resolutions obtained so far is 2.6 nm, which is in the range of the size of individual proteins (Rittweger et al, 2009).Although STED and light-sheet microscopy—combined with other techniques and tools—promise to revolutionize cell biology by providing image sequences at higher resolutions and shorter timescales, they also bring new challenges or accentuate existing ones. The two main issues concern disturbing the system being observed and introducing visual artefacts that cannot be immediately identified, because the images are constructed, at least partly, by computers. This boils down to one fundamental problem: how to tell whether the image reflects the system being observed under normal environmental conditions. Even when a sample is being observed in vivo, the preparation—involving excitation with light—is not normal. “This is a major issue, because when you basically set up conditions to detect a protein of interest, you also interfere with the system,” commented Knop. “One of the typical problems in researching mammalian cells for example is that many proteins are over expressed when people label them.”In the case of the zebrafish embryo, it might seem as though normal development has occurred, but even when a healthy animal is produced it is impossible to be sure that some details of the mechanisms involved have not been changed by the observational process. This can be addressed by validating findings obtained by high-resolution optical microscopy in other ways. “Depending on the organism there are different options,” said Knop. “In yeast we can use genetics.”…the increasing power and resolution of light microscopy also increases the sensitivity of the observations to small fluctuations in the environmentFurthermore, the increasing power and resolution of light microscopy also increases the sensitivity of the observations to small fluctuations in the environment. This was an issue for Peter Sudbery at the University of Sheffield in the UK, who uses a microscope called Deltavision to study the growth of the fungus Candida. “Candida only forms hyphae at 37 °C, and even a small drop in temperature changes its behaviour,” he said. “Also small fluctuations in temperature of less than 0.1 °C cause the image to lose focus. So while the Deltavision can be programmed to make time-lapse movies with the exposures taken automatically at preset intervals, for videos longer than a few minutes the microscope has to be manually monitored constantly to check the focus and temperature.”Although artefacts and environmental control are important issues at the cutting edge of research near the limits of resolution, they are fortunately less critical for medical applications. Here, resolution at the cell level is usually not necessary; the main issue lies in developing sufficiently small systems that can accurately and conveniently observe the region of interest during diagnosis and surgery.The most prevalent application in medical imaging is for tumour detection—both during diagnosis and surgery—either to replace traditional biopsy, which is invasive and time-consuming, or to replace direct visual inspection, which can be inaccurate. One method is near-infra-red (NIR) microscopy, in which the patient ingests compounds that cause tumour cells to emit radiation in the NIR spectrum in response to excitation. This can either be used to identify specific tumours or general cancers, according to Stijn Keereweer, research fellow at the Erasmus Medical Centre in Rotterdam, the Netherlands. “Agents can target specific epitopes which differ between various tumours, but can also be targeted against general characteristics, such as MMP [matrix metalloproteinase] activity or increased glucose uptake,” said Keereweer. He added that by mounting cameras on laparoscopic probes, the technique could be extended to internal abdominal-cancer surgery, as well as for neck and head cancers when the observation can be performed by external systems.The advantage of these optical systems is that the surgical margin required to be sure that all malignant tissue has been removed can be reduced, thereby minimizing risk and injury to the patient. One of the hurdles for this technique is regulatory approval of the tumour-specific agents that are needed to elicit the fluorescence.However, there are emerging techniques that do not require such agents, by using conventional confocal laser microscopy without fluorescence. A study at the Garvan Institute of Medical Research in Sydney, Australia, showed that in vivo analysis of surface and near-surface tissue could be performed successfully in real time by using a contrast agent administered intravenously to show structural features (Nguyen et al, 2009).Techniques that avoid the need for patients to ingest an agent—for contrast or to generate fluorescence—would be ideal as they would reduce preparation time. This can be achieved by exploiting auto-fluorescence instead of adding fluorescent compounds, and has been shown to successfully identify oesophageal cancer, according to Bevin Lin at the Department of Biomedical Engineering at the University of California Davis. “Regarding oesophageal carcinoma, current screening methods can take 20–30 minutes,” he said. “There is a need for a rapid method of imaging that is less complicated and faster than topical or intravenous contrast agents. Therefore, significant advantages of auto-fluorescence microscopy include the elimination of contrast agents, preparation time, and complex instrumentation, which could directly translate to reduced cost and improved patient care.” As Lin pointed out, regulatory approval from the US Food and Drug Administration is still required for the endoscopy system, but he believes that there is great potential for optical systems. “Hopefully, optical methods will become as clinically mainstream as traditional methods,” he said.The most prevalent application in medical imaging is for tumour detection…There is similar expectation that real-time optical imaging will become as widely used in research once teething troubles associated with recognition of artefacts and disturbances to the systems under observation are overcome. “Effective, affordable and usable super-resolution imaging that could be used on living, non-fixed cells with GFP and variants would transform the whole of cell biology,” said Sudbery.     

Multicellular Computing Using Conjugation for Wiring

Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal “re-programming” and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a “computation” is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the “wiring” between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids. We design a multi-cellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing system under different conditions, and provide baseline information for future laboratory implementations.     

Molecular dynamics imaging in micropatterned living cells

Micropatterning approaches using self-assembled monolayers of alkyl thiols on gold are not optimal for important imaging modalities in cell biology because of absorption of light and scattering of electrons by the gold layer. We report here an anisotropic solid microetching (ASOMIC) procedure that overcomes these limitations. The method allows molecular dynamics imaging by wide-field and total internal reflection fluorescence (TIRF) microscopy of living mammalian cells and correlative platinum replica electron microscopy.     

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.     

A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.     

Fast Three-Dimensional Single-Particle Tracking in Natural Brain Tissue

Observation of molecular dynamics is often biased by the optical very heterogeneous environment of cells and complex tissue. Here, we have designed an algorithm that facilitates molecular dynamic analyses within brain slices. We adjust fast astigmatism-based three-dimensional single-particle tracking techniques to depth-dependent optical aberrations induced by the refractive index mismatch so that they are applicable to complex samples. In contrast to existing techniques, our online calibration method determines the aberration directly from the acquired two-dimensional image stream by exploiting the inherent particle movement and the redundancy introduced by the astigmatism. The method improves the positioning by reducing the systematic errors introduced by the aberrations, and allows correct derivation of the cellular morphology and molecular diffusion parameters in three dimensions independently of the imaging depth. No additional experimental effort for the user is required. Our method will be useful for many imaging configurations, which allow imaging in deep cellular structures.     

Imaging of single molecules in live cells

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.     

Strategies for High-Resolution Imaging of Epithelial Ovarian Cancer by Laparoscopic Nonlinear Microscopy

Ovarian cancer remains the most frequently lethal of the gynecologic cancers owing to the late detection of this disease. Here, by using human specimens and three mouse models of ovarian cancer, we tested the feasibility of nonlinear imaging approaches, the multiphoton microscopy (MPM) and second harmonic generation (SHG) to serve as complementary tools for ovarian cancer diagnosis. We demonstrate that MPM/SHG of intrinsic tissue emissions allows visualization of unfixed, unsectioned, and unstained tissues at a resolution comparable to that of routinely processed histologic sections. In addition to permitting discrimination between normal and neoplastic tissues according to pathological criteria, the method facilitates morphometric assessment of specimens and detection of very early cellular changes in the ovarian surface epithelium. A red shift in cellular intrinsic fluorescence and collagen structural alterations have been identified as additional cancer-associated changes that are indiscernible by conventional pathologic techniques. Importantly, the feasibility of in vivo laparoscopic MPM/SHG is demonstrated by using a “stick” objective lens. Intravital detection of neoplastic lesions has been further facilitated by low-magnification identification of an indicator for cathepsin activity followed by MPM laparoscopic imaging. Taken together, these results demonstrate that MPM may be translatable to clinical settings as an endoscopic approach suitable for high-resolution optical biopsies as well as a pathology tool for rapid initial assessment of ovarian cancer samples.     

Characterizing Organelles in Live Stem Cells Using Label-Free Optical Diffraction Tomography

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells—namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell—based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.     

Inhibition of Photosynthesis in Certain Algae by Extreme Red Light

This paper shows that in Porphyridium cruentum and in Chlorella pyrenoidosa (but apparently not in Anacystis nidulans) “extreme red” light (> 720 mμ) can inhibit photosynthesis produced by “far red” light (up to 720 mμ). From the action spectrum of this phenomenon, it appears that an unknown pigment with an absorption band around 745 mμ must be responsible for it.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption

Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic “scratch” assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the “wound” is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell–substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.     

Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI− phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.     

Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.     

Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction

Total internal reflection fluorescence microscopy (TIRFM) is becoming an increasingly common methodology to narrow the illumination excitation thickness to study cellular process such as exocytosis, endocytosis, and membrane dynamics. It is also frequently used as a method to improve signal/noise in other techniques such as in vitro single-molecule imaging, stochastic optical reconstruction microscopy/photoactivated localization microscopy imaging, and fluorescence resonance energy transfer imaging. The unique illumination geometry of TIRFM also enables a distinct method to create an excitation field for selectively exciting fluorophores that are aligned either parallel or perpendicular to the optical axis. This selectivity has been used to study orientation of cell membranes and cellular proteins. Unfortunately, the coherent nature of laser light, the typical excitation source in TIRFM, often creates spatial interference fringes across the illuminated area. These fringes are particularly problematic when imaging large cellular areas or when accurate quantification is necessary. Methods have been developed to minimize these fringes by modulating the TIRFM field during a frame capture period; however, these approaches eliminate the possibility to simultaneously excite with a specific polarization. A new, to our knowledge, technique is presented, which compensates for spatial fringes while simultaneously permitting rapid image acquisition of both parallel and perpendicular excitation directions in ∼25 ms. In addition, a back reflection detection scheme was developed that enables quick and accurate alignment of the excitation laser. The detector also facilitates focus drift compensation, a common problem in TIRFM due to the narrow excitation depth, particularly when imaging over long time courses or when using a perfusion flow chamber. The capabilities of this instrument were demonstrated by imaging membrane orientation using DiO on live cells and on lipid bilayers that were supported on a glass slide (supported lipid bilayer). The use of the approach to biological problems was illustrated by examining the temporal and spatial dynamics of exocytic vesicles.     

Alternate histories of cytokinesis: lessons from the trypanosomatids

Popular culture has recently produced several “alternate histories” that describe worlds where key historical events had different outcomes. Beyond entertainment, asking “could this have happened a different way?” and “what would the consequences be?” are valuable approaches for exploring molecular mechanisms in many areas of research, including cell biology. Analogous to alternate histories, studying how the evolutionary trajectories of related organisms have been selected to provide a range of outcomes can tell us about the plasticity and potential contained within the genome of the ancestral cell. Among eukaryotes, a group of model organisms has been employed with great success to identify a core, conserved framework of proteins that segregate the duplicated cellular organelles into two daughter cells during cell division, a process known as cytokinesis. However, these organisms provide relatively sparse sampling across the broad evolutionary distances that exist, which has limited our understanding of the true potential of the ancestral eukaryotic toolkit. Recent work on the trypanosomatids, a group of eukaryotic parasites, exemplifies alternate historical routes for cytokinesis that illustrate the range of eukaryotic diversity, especially among unicellular organisms.