An increasing number of investigations are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology laboratories, as well as a routine methodology that is practiced in the wide ranging fields of neurobiology, developmental biology, pharmacology, and many other related biomedical research disciplines. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated in the presence of synthetic fluorophores and/or fluorescent proteins.

Maintaining Live Cells on the Microscope Stage - Tight control of the culture environment is one of the most critical factors in successful live-cell imaging experiments. In particular, the conditions under which cells are maintained on the microscope stage, although widely variable in many requirements depending upon the organism, often dictate the success or failure of an experiment. Aspects of the environment that are readily manipulated include the physical parameters of the chamber in which the cells are grown and imaged, temperature control, atmospheric conditions (gas mixture and humidity), nutritional supplements, growth medium buffering (pH), and osmolarity of the culture medium.

Live-Cell Imaging Culture Chambers - Specimen chambers are an integral and critical branch in the history of live-cell imaging, and a wide spectrum of designs have been published over the years describing systems that offer excellent optical properties while allowing specimens to be maintained for varying amounts of time. Ranging in complexity from the simple preparation of a sealed coverslip on a microscope slide to sophisticated perfusion chambers that enable tight control of virtually all environmental variables, culture chambers are designed to to allow living specimens to be observed with minimal invasion at high resolution.

Optical System and Detector Requirements for Live-Cell Imaging - In designing an optical microscopy system for live-cell investigations, the primary considerations are detector sensitivity (signal-to-noise), the required speed of image acquisition, and specimen viability. The relatively high light intensities and long exposure times that are typically employed in recording images of fixed cells and tissues (where photobleaching is the major consideration) must be strictly avoided when working with living cells. In virtually all cases, live-cell microscopy represents a compromise between achieving the best possible image quality and preserving the health of the cells. Rather than unnecessarily oversampling time points and exposing the cells to excessive levels of illumination, the spatial and temporal resolutions set by the experiment should be limited to match the goals of the investigation.

The Automatic Microscope: Shutters, Filter Wheels, Focus, Stage Control, and Illumination Systems - Motorized microscope components and accessories enable the investigator to automate live-cell image acquisition and are particularly useful for time-lapse experiments that range in timescale intervals from milliseconds to tens or hundreds of minutes. Auxiliary components such as electromechanical shutters, microprocessor-controlled filter changers (filter wheels), motorized stages, and axial focus control mechanisms can be retrofitted to a research grade microscope and interactively controlled by a companion workstation computer using a variety of commercially available image acquisition software packages. However, it should be noted that assembling a fully automated and optimized multi-dimension optical imaging system is an extremely complex task.

Introduction to Fluorescent Proteins - The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology. When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.

Imaging Parameters for Fluorescent Proteins - The wide spectrum of fluorescent proteins and derivatives uncovered thus far are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These unique probes have proven extremely useful as reporters for gene expression studies in both cultured cells and entire animals. In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments, as well as a tracer of intracellular protein trafficking. Quantitative imaging of fluorescent proteins is readily accomplished with a variety of techniques, including widefield, confocal, and multiphoton microscopy, to provide a unique window for exposing the intricacies of cellular structure and function.

Cell Motility - In multicellular tissues, such as those found in animals and humans, individual cells employ a variety of locomotion mechanisms to maneuver through spaces in the extracellular matrix and over the surfaces of other cells. Examples are the rapid movement of cells in developing embryos, organ-to-organ spreading of malignant cancer cells, and the migration of neural axons to synaptic targets. Unlike single-celled swimming organisms, crawling cells in culture do not possess cilia or flagella, but tend to move by coordinated projection of the cytoplasm in repeating cycles of extension and retraction that deform the entire cell. The digital videos presented in this gallery investigate animal cell motility patterns in a wide variety of morphologically different specimens.

General Literature Sources - The technique of imaging living cells on the microscope stage has become an increasingly useful tool with the emergence of synthetic fluorophores and fluorescent proteins to serve as qualitative and quantitative reporters of intracellular structure and dynamics. Live-cell imaging now spans multiple modalities, including widefield (fluorescence, phase contrast, and differential interference contrast), laser scanning confocal, multiphoton, and spinning disk microscopy. This section features a bibliography of literature sources for books, review articles, and the original research reports on the wide spectrum of methodologies currently being implemented for studying living cells.

ZEISS Campus Live-Cell Imaging Reference Library - The introduction of genetically-encoded fluorescent protein fusions as a localization marker in living cells has revolutionized the field of cell biology, and the application of photostable quantum dots looms on the horizon. Live-cell imaging techniques now involved a wide spectrum of imaging modalities, including widefield fluorescence, confocal, multiphoton, total internal reflection, FRET, lifetime imaging, superresolution, and transmitted light microscopy. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of live-cell imaging.

Live-Cell Imaging Chambers - Specimen chambers have been an integral part of the history of microscopy and a number of designs have been published over the years describing systems that offer excellent optical properties while allowing specimens to be maintained for varying amounts of time. The basic requirements for live-cell imaging chamber configurations range from simple microscope slides with sealed coverslips to elaborate and complex designs that control a host of environmental variables. The literature references in this section describe simple chambers, perfusion systems, and enclosed microscope incubators that enable a significant degree of control over the imaging environment.

Motorized Microscope Accessories - Microscope configurations for live-cell imaging greatly benefit from the application of motorized components to automate rapid filter changes, focus, stage translation, and control of illumination shutters. Listed below are links to the websites for manufacturers and distributors of high-speed filter wheels, electromechanical shutters, axial focus systems, and translational motorized stages suitable for live-cell imaging.

Live-Cell Imaging and Perfusion Chambers - Although many investigators have fabricated customized live-cell imaging chambers designed to meet specific requirements over the years, a wide range of commercial perfusion and imaging chambers are now available. These chambers offer numerous designs, including glass bottom Petri dishes, multi-well chambers mounted on microscope slides, heating stages with a variety of interchangeable perfusion adapters, and specialized chambers with conductive coatings for tight control of temperature. Listed in this section are links to the manufacturers and distributors of specimen chambers for live-cell imaging.

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