Coherent anti-stokes Raman scattering microscopy: a biological review.

Microscopic imaging of cells and tissues are generated by the interaction of light with either the sample itself or contrast agents that label the sample. Most contrast agents, however, alter the cell in order to introduce molecular labels, complicating live cell imaging. The interaction of light from multiple laser sources has given rise to microscopy, based on Raman scattering or vibrational resonance, which demonstrates selectivity to specific chemical bonds while imaging unmodified live cells. Here, we discuss the nonlinear optical technique of coherent anti-Stokes Raman scattering (CARS) microscopy, its instrumentation, and its status in live cell imaging.     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

Cell viability and probe-cell membrane interactions of XR1 glial cells imaged by atomic force microscopy.

As atomic force microscopy (AFM) imaging of live specimens becomes more commonplace, at least two important questions arise: 1) do live specimens remain viable during and after AFM, and 2) is there transfer of membrane components from the cell to the AFM probe during probe-membrane interactions? We imaged live XR1 glial cells in culture by single- or dual-pass contact or tapping-mode AFM, examined cell viability at various postimaging times, and report that AFM-imaged live XR1 cells remained viable up to 48 h postimaging and that cell death rates did not increase. To determine if nonlethal, transient interactions between the AFM probe and cell membrane led to transfer of XR1 cell membrane phospholipid components on the probe, we treated the scanned probes with the lipid-binding fluorophore FM 1-43. Confocal microscopy revealed that phospholipid membrane components did accumulate on the probe, and to a generally greater extent during contact-mode imaging than during tapping-mode imaging. Moreover, membrane accumulations on the probe were greater when live XR1 cells were damaged or perturbed, yet membrane did not accumulate in fluorescently detectable quantities during repeated "force curves" during control experiments. Taken together, our data indicate that although AFM imaging of live cells in culture does not affect long-term cell viability, there are substantial probe-membrane interactions that lead to transfer of membrane components to the probe.     

DNA Electroporation,Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.     

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.     

Inducing local DNA damage by visible light to study chromatin repair

Dynamics of DNA repair and recruitment of repair factors to damaged DNA can be studied by live cell microscopy. DNA damage is usually inflicted by a laser beam illuminating a DNA-interacting photosensitizer in a small area of the nucleus. We demonstrate that a focused beam of visible low intensity light alone can inflict local DNA damage and permit studies of DNA repair, thus avoiding potential artifacts caused by exogenous photosensitizers.     

Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp

To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.     

Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission

Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.     

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.     

Self-protection and survival of arbovirus-infected mosquito cells

Arboviruses are serious pathogens for men but cause little damage to their arthropod vectors. We have studied how a mosquito cell line derived from one of the relevant vectors for arboviruses responds to Bunyamwera virus, a well-characterized arbovirus. Confocal, live cell microscopy and electron microscopy showed that Bunyamwera virus induces deep changes in mosquito cells. Early in infection these cells develop long projections and create new intercellular connections where cell organelles and viral proteins are detected. Live cell microscopy shows that these connections are developed before viral protein can be detected by immunofluorescence. Interestingly, their proliferation is accompanied by a progressive trapping of the nucleocapsid and RNA polymerase viral proteins into large cytoplasmic aggregates. A significant drop in the release of infectious virions then follows. Before that, numerous viruses assemble in peripheral Golgi stacks and they apparently exit the cells immediately since they do not accumulate intracellularly. This mechanism of assembly seems to cause little damage to the integrity of cell endomembranes. The characterization of the antiviral mechanisms operating in mosquito cells can be of great help in the fight against pathogenic arboviruses.     

Calyculin A-induced neurite retraction is critically dependent on actomyosin activation but not on polymerization state of microtubules

Calyculin A (CL-A), a toxin isolated from the marine sponge Discodermia calyx, is a strong inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A). Although CL-A is known to induce rapid neurite retraction in developing neurons, the cytoskeletal dynamics of this retraction have remained unclear. Here, we investigated the cytoskeletal dynamics during CL-A-induced neurite retraction in cultured rat hippocampal neurons, using fluorescence microscopy as well as polarized light microscopy, which can visualize the polymerization state of the cytoskeleton in living cells. We observed that MTs were bent while maintaining their polymerization state during the neurite retraction. In addition, we also found that CL-A still induced neurite retraction when MTs were depolymerized by nocodazole or stabilized by paclitaxel. These results imply a mechanism other than depolymerization of MTs for CL-A-induced neurite retraction. Our pharmacological studies showed that blebbistatin and cytochalasin D, an inhibitor of myosin II and a depolymerizer of actin, strongly inhibited CL-A-induced neurite retraction. Based on all these findings, we propose that CL-A generates strong contractile forces by actomyosin to induce rapid neurite retraction independently from MT depolymerization.     

Cell cycle dependent TN-C promoter activity determined by live cell imaging

The extracellular matrix protein tenascin-C plays a critical role in development, wound healing, and cancer progression, but how it is controlled and how it exerts its physiological responses remain unclear. By quantifying the behavior of live cells with phase contrast and fluorescence microscopy, the dynamic regulation of TN-C promoter activity is examined. We employ an NIH 3T3 cell line stably transfected with the TN-C promoter ligated to the gene sequence for destabilized green fluorescent protein (GFP). Fully automated image analysis routines, validated by comparison with data derived from manual segmentation and tracking of single cells, are used to quantify changes in the cellular GFP in hundreds of individual cells throughout their cell cycle during live cell imaging experiments lasting 62 h. We find that individual cells vary substantially in their expression patterns over the cell cycle, but that on average TN-C promoter activity increases during the last 40% of the cell cycle. We also find that the increase in promoter activity is proportional to the activity earlier in the cell cycle. This work illustrates the application of live cell microscopy and automated image analysis of a promoter-driven GFP reporter cell line to identify subtle gene regulatory mechanisms that are difficult to uncover using population averaged measurements.     

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.     

Golgi inheritance under a block of anterograde and retrograde traffic

In mitosis, the Golgi complex is inherited following its dispersion, equal partitioning and reformation in each daughter cell. The state of Golgi membranes during mitosis is controversial, and the role of Golgi-intersecting traffic in Golgi inheritance is unclear. We have used brefeldin A (BFA) to perturb Golgi-intersecting membrane traffic at different stages of the cell cycle and followed by live cell imaging the fate of Golgi membranes in those conditions. We observed that addition of the drug on cells in prometaphase prevents mitotic Golgi dispersion. Under continuous treatment, Golgi fragments persist throughout mitosis and accumulate in a Golgi-like structure at the end of mitosis. This structure localizes at microtubule minus ends and contains all classes of Golgi markers, but is not accessible to cargo from the endoplasmic reticulum or the plasma membrane because of the continuous BFA traffic block. However, it contains preaccumulated cargo, and intermixes with the reforming Golgi upon BFA washout. This structure also forms when BFA is added during metaphase, when the Golgi is not discernible by light microscopy. Together the data indicate that independent Golgi fragments that contain all classes of Golgi markers (and that can be isolated from other organelles by blocking anterograde and retrograde Golgi-intersecting traffic) persist throughout mitosis.     

Thickness profiling of formaldehyde-fixed cells by transmission-through-dye microscopy

Conventional light microscopy techniques are poorly suited for imaging the vertical cell dimension. This can be accomplished using transmission-through-dye (TTD) imaging, in which cell thickness is directly converted into image intensity in the presence of extracellular dye with strong absorption. We have previously described applications of TTD to living cells using the dye Acid Blue 9 (AB9) to generate contrast. In this work, we investigated the possibility of extending TTD to chemically fixed cells. This would depend on preservation of cell impermeability to the dye; by using a method based on fluorescence quenching, we found that formaldehyde-fixed cells remain impermeable to AB9. Fixation enables imaging of cell surfaces in the presence of high concentrations of AB9, bringing the vertical resolution to several nanometers per pixel; that is at least an order of magnitude better than resolution achievable with live cells. TTD images collected with high-NA objectives are often contaminated by Becke lines resulting from intracellular organelles, and we show how to distinguish them from features on the cell surface. Quantification of cell thickness and volume on fixed cells is also possible during the early stages of fixation; this can be useful, for example, for measuring volume kinetics following rapid introduction of a stimulus.     

The vaccinia virus F11L gene product facilitates cell detachment and promotes migration

We previously showed that infection with vaccinia virus (VV) induces cell motility, characterized by contractility and directed migration. Motility is temporally regulated because cells are motile immediately after infection, whereas late in infection motility ceases and cells resettle. Motility and its cessation are accompanied by temporal rearrangements of both the microtubule and the actin networks. Because the F11L gene has previously been implicated in VV-induced migration, we now explore the role of F11L in contractility, migration, the cessation of motility and the cytoskeletal rearrangements. By live cell imaging using a VV that lacks an intact F11L gene, we show that F11L facilitates cell detachment and is required for migration but not for contractility. By light microscopy, F11L expression induces a remodeling of the actin, but not the microtubule, network. The lack of migration correlates with smaller plaques, indicating that this process facilitates cell-to-cell spreading of VV. Late in infection, when motility ceases, cells re-establish cell-to-cell contacts in an F11L-independent manner. We finally show that VV-induced motility and its cessation correlate with a temporal regulation of the guanosine triphosphatase RhoA as well as the expression levels of F11L during the infectious cycle.     

Importance of various types of metabolic inhibition for cell damage caused by direct membrane damage

Cell damage is caused by energy depletion or by direct membrane damage, or a combination when a direct membrane damage affects energy depleted cells. In this report it was investigated whether the extent of direct membrane damage induced by lysophosphatidyl choline (LPC) or phospholipase C (PhC) on quiescent fibroblasts depended on the metabolic state of the cells. When glycolysis was inhibited cell damage was always extensively increased, whereas cell damage was also increased to a minor degree when exposed to PhC during sole inhibition of oxidative phosphorylation. Acceleration of glycolysis in cells with a low rate of glycolysis resulted in a dramatic improvement of the membrane susceptibility within a few minutes. Thus, susceptibility of the cell membrane to direct membrane damage depends on the metabolic state. The results also emphasize previous findings that glycolysis has a special role in maintaining membrane function and integrity.     

Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light ("dark conditions") or by supplementing the medium with 10 micrograms/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under "lighted conditions." No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 degrees C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 micrograms/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 micrograms/ml catalase does not protect cells from hypothermic (5 and 10 degrees C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.