The scientific literature contains abundant resources in the form of books, review articles, and original research reports that deal with numerous topics in optical microscopy. The Carl Zeiss MicroImaging Online Campus Reference Library contains links to selected reports that should be useful to investigators seeking introductory material on a variety of techniques, probes, light sources, and live-cell imaging applications.
Colocalization Analysis - Colocalization analysis is a powerful tool in confocal and deconvolution microscopy for the demonstration of spatial and temporal overlap in the distribution patterns of fluorescent probes. A number of commercial software packages contain colocalization algorithms and a number of techniques have been introduced to address specific applications. Many of the references listed below are review articles that thoroughly discuss a wide range of parameters for colocalization analysis and should be useful as a starting point for gathering information on this subject.
Deconvolution Microscopy - Over the past several decades, deconvolution microscopy has become a mainstream image processing tool for deciphering the substructure of living and fixed specimens in three dimensions. Routinely applied to widefield optical sections, as well as those obtained in confocal and structured illumination, the technique has benefited from the continued development of advanced algorithms and turnkey systems. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of deconvolution.
Fluorescence Microscopy - The application of fluorescence illumination and detection in optical microscopy has ushered in a wide range of advanced applications for live-cell imaging and in vivo observations. The articles tabulated in this section discuss the basic aspects of fluorescence, microscope configuration, fluorescent probes, software, light sources, detectors, objectives, filter sets, and a variety of other pertinent topics.
Fluorescent Proteins - The growing class of fluorescent proteins useful for detecting events in living cells and animals has almost single-handedly launched and fueled a new era in biology and medicine. These powerful research tools have provided investigators with a mechanism of fusing a genetically encoded optical probe to a practically unlimited variety of protein targets in order to examine living systems using fluorescence microscopy and related technology. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of fluorescent protein technology.
Fluorescence Recovery After Photobleaching (FRAP) - Taking advantage of the ability to track dynamic behavior in living cells using fluorescent protein fusions to intracellular targets, the technique of fluorescence recovery after photobleaching (FRAP) and associated methods (loss in photobleaching; FLIP, and inverse FRAP; iFRAP) are proving highly useful for studying the kinetic behavior of proteins. All of these experiments rely on selectively photobleaching the fluorescence within a region of interest with a high-intensity laser, followed by monitoring the diffusion of new fluorescent molecules into the bleached area over a period of time with low-intensity laser light. Photobleaching techniques are ideal for determining kinetic properties, including the diffusion coefficient, mobile fraction, and transport rate of proteins in live-cell imaging.
Förster Resonance Energy Transfer (FRET) - The dynamic interaction between proteins and other biomolecules in living cells plays a significant role in a wide spectrum of essential processes. Although classically investigated in fluorescence microscopy using co-localization techniques, these interactions can provide additional information when applied to fluorescent proteins in live-cell imaging microscopy using resonance energy transfer techniques. The references listed in this section point to review articles in the scientific literature that should provide an excellent starting point for investigators seeking information on FRET methodology.
Laser Scanning Confocal Microscopy - A majority of the literature pertaining to review articles on laser scanning confocal microscopy has been published in textbooks, edited article collections, and symposia, with only an intermittent sprinkling of papers in the scientific journals. The reviews listed in this section should be available to those students and investigators who have access to subscriptions through their host institutions.
Live-Cell Imaging - 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.
Multiphoton Microscopy - The application of nonlinear excitation techniques to the imaging of synthetic fluorophores and fluorescent proteins in biology and medicine has witnessed increasing attention over the past several years, primarily due to the introduction of turnkey pulsed laser systems coupled to advanced instrumentation. The references described in this section contain review articles and original research reports on multiphoton microscopy with emphasis on the theoretical background, microscope configuration, specimen preparation, deep tissue imaging, and numerous applications.
Photoactivation and Photoconversion - The ability to selectively initiate or alter fluorescence emission profiles in fluorescent proteins has resulted in the creation of a new class of probes for exploring protein behavior and dynamics in living cells. As the fluorescence intensity or spectral alterations of highlighters generally occur only after photon-mediated conversion, newly synthesized non-photoactivated protein pools remain unobserved and do not complicate experimental results. This section provides sources for selected review articles and original research reports on optical highlighter fluorescent proteins.
Spectral Imaging and Linear Unmixing - Spectral overlap in specimens labeled with synthetic fluorophores and fluorescent proteins can often lead to analysis artifacts when interpreting images. The technique of spectral imaging, which involves gathering incremental emission lambda stacks, coupled to linear unmixing can significantly aid in the interpretation of images and in FRET measurements. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spectral imaging.
FRET with Spectral Imaging and Linear Unmixing - In FRET applications, spectral imaging can be considered a variation of the sensitized emission technique that relies on excitation of the donor alone, followed by acquisition of the entire emission spectrum of both the donor and acceptor fluorescence instead of capturing data in two independent channels. Spectral imaging FRET assumes that gathering of the entire fluorescence spectrum will enable overlapping spectral profiles to be separated according to the distinct shapes of the spectra rather than simply monitoring emission intensity in a limited bandwidth region using a filter.
Spinning Disk Confocal Microscopy - Spinning disk confocal microscopy is rapidly emerging as the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems are capable of providing high acquisition speeds with acceptable contrast and minimal photobleaching at the low light levels available with this technique. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spinning disk confocal microscopy.
Structured Illumination - Often referred to as a "poor man's confocal microscope", structured illumination is emerging as a powerful technique for optical sectioning in widefield microscopy at high resolution. Although current implementations are limited in speed and multi-channel acquisition by the requirement of capturing multiple images, new technological innovations are occurring rapidly in this field. The references listed in this section point to original research reports and review articles that should provide the starting point for a thorough understanding of structured illumination.
Superresolution Microscopy - The traditional diffraction limit in fluorescence microscopy (approximately 200 nanometers) has limited applications to gross approximations of molecular positioning in cellular substructures. To address this challenge, several research groups have developed new techniques (superresolution microscopy) that manipulate laser and fluorophore physics to break the diffraction barrier and yield resolutions down to 50 nanometers or less. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of superresolution microscopy.
Total Internal Reflection Fluorescence Microscopy (TIRFM) - Often referred to in the literature as evanescent wave microscopy, total internal reflection fluorescence microscopy (TIRFM) is proving to be a powerful technique for examining phenomena occurring at the plasma membrane in living cells and for imaging single molecules. TIRFM has grown in utility and popularity as manufacturers have provided increasingly sophisticated turnkey instrumentation coupled to advanced software interfaces. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of TIRFM and related methodology.
Optical Highlighter Fluorescent Protein Original References - Optical highlighter fluorescent proteins, which include the photoactivatable GFP (PA-GFP), the green-to-red photoconverter Kaede, and the photoswitchable Dronpa, allow direct and controlled activation of distinct molecular pools of the fluorescent proteins within the cell. Listed in this section are key references to many of the original articles describing the discovery and properties of optical highlighters.