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.
Imaging the division process in living tissue culture cells. Methods 38: 2-16 (2006). A must-read paper for investigators contemplating launching into live-cell imaging applications. This review discusses phototoxicity, issues with maintaining living cells on the microscope stage, vibration artifacts during time-lapse imaging, temperature control, digital camera systems, and culture chambers.
Live Cell Imaging: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York, 631 pages (2005). Featuring over 30 chapters written by noted experts, this reference manual is perhaps the most important single source for information related to a wide spectrum of topics in live-cell imaging. The chapters address advanced fluorescence techniques, imaging applications, and specific examples of model systems.
Imaging the live plant cell. The Plant Journal 45: 573-598 (2006). Although centered on the many important issues surrounding the imaging of living plant tissue, most of the topics discussed in this review also apply to mammalian cells as well. Included are sections on specimen chambers, perfusion, digital imaging, camera systems, microscopy techniques, and data analysis.
Light microscopy techniques for live cell imaging. Science 300: 82-86 (2003). Briefly discussing the environmental considerations of maintaining cells in culture for observation under the microscope, this review highlights important aspects of choosing the optimum instrument settings, photobleaching artifacts, and phototoxicity. Also detailed are transmitted light, multiphoton, confocal, TIRF, FRET, FLIM, and widefield fluorescence imaging modalities.
Cell Motility Under the Microscope: Vorsprung durch Technik. Nature Reviews Molecular Cell Biology 5: 667-672 (2004). A nice historical account of live-cell imaging dating back to the invention of the microscope. This review focuses on the observation of cell motility and the techniques that have been applied to image a wide variety of specimens. Also discussed are the evolution of instrumentation utilized for time-lapse imaging, the increased use of computers in microscopy, and recent advances in fluorescent protein applications in live-cell imaging.
Quantitative motion analysis and visualization of cellular structures. Methods 29: 3-13 (2003). The authors present a comprehensive review of data analysis for dynamic live-cell imaging applications. Among the topics discussed are object detection, motion estimation, single-particle tracking, segmentation, thresholding, volume rendering, visualization, automated analysis, and related computational methods. The paper also reviews commercial software packages designed for image analysis of live cells.
Live cell imaging using wide-field microscopy and deconvolution. Cell Structure and Function 27: 335-341 (2002). The authors review the advantages and disadvantages of using various fluorescence labeling strategies for live-cell imaging. In addition to discussing basic considerations for maintaining cells and tissues on the microscope stage, data collection and analysis techniques are also addressed.
Using long-term time-lapse imaging of mammalian cell cycle progression for laboratory instruction and analysis. Cell Biology Education 4: 284-290 (2005). Focusing on long term time-lapse techniques, this review article approaches live-cell imaging from an educational perspective. Among the topics discussed are choosing the appropriate cell lines, simple imaging chambers, and the technology necessary for digital microscopy.
Use of quantum dots for live cell imaging. Nature Methods 1: 73-78 (2004). A protocol-based review of the application of quantum dots for live-cell imaging applications. The paper discusses cell lines, conjugation techniques, reagents, culture conditions, labeling restrictions, and imaging considerations using these unique fluorophores.
The beauty of the yeast: Live cell microscopy at the limits of optical resolution. Microscopy Research and Technique 51: 511-529 (2000). An excellent primer on live-cell imaging applications using yeast. The author discusses fluorescent protein fusions in yeasts, immunofluorescence, culture conditions, imaging chambers, cell immobilization, staining with synthetic dyes, and a wide variety of microscopy techniques.