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 Microscopy 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.
Aperture Correlation Microscopy - Aperture correlation microscopy is a structured illumination technique that employs a specialized spinning disk having a grid pattern to acquire images in both transmitted and reflected light modes. The resulting images are calculated using a series of algorithms after acquisition. The references listed in this section point to original research reports and review articles on aperture correlation microscopy.
Basic Concepts in Microscopy - References that describe basic concepts necessary for a complete understanding of microscopy, including objectives, eyepieces, condensers, magnification, numerical aperture, resolution, contrast, and optical aberrations, along with a wide spectrum of additional considerations.
Textbooks on Basic Principles in Optical Microscopy - A list of the best textbooks that provide a general knowledge of the principles and practice of optical microscopy, as well as an introduction to contrast-enhancing techniques. The volumes listed here are useful in both the classroom and the research laboratory.
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.
Correlative Light and Electron Microscopy (CLEM) - The combination of electron microscopy with light microscopy (termed correlative light and electron microscopy; CLEM) has witnessed recent technological advances that have enabled the study of biological specimens at high resolution through the introduction of fluorescent probes that are capable of generating sufficient contrast for high-resolution electron microscopy.
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.
Differential Interference Contrast (DIC) Microscopy - Differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. Images produced in DIC microscopy have a distinctive shadow-cast appearance
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.
Fluorescence Correlation Spectroscopy (FCS) - Fluorescence Correlation Spectroscopy (FCS) is a tool that provides quantitative localized measurements of important physical parameters including mechanisms of transport, molecular mobilities, and densities of fluorescently labeled species. In the most basic configuration, FCS examines the inherent correlations exhibited by the fluctuating fluorescent signal from labeled molecules as they transition into and out of a specified excitation volume.
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.
Light Sheet Microscopy - Light sheet microscopy, often referred to as single plane illumination microscopy (SPIM), is a rapidly emerging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with impressive resolution. The light sheet technique illuminates on the region surrounding the focal plane of the detection objective in a twin objective configuration (where the objectives are juxtaposed at 45-degrees).
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.
Microscope Optical Systems - The microscope optical train typically consists of an illuminator (including the light source and collector lens), a condenser, specimen, objective, eyepiece, and detector, which is either some form of camera or the observer's eye. These components are often supplemented with contrast-enhancing optical elements.
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.
Optical Sectioning - The ability to image thin sections without having to mechanically slice a thick specimen is afforded by optical sectioning microscopy. Sections are achieved by eliminating the excitation and detection of fluorescence that originates in regions removed from the focal plane. The reviews listed in this section should be available to students and investigators who have access to subscriptions through their host institutions.
Phase Contrast Microscopy - Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by the Carl Zeiss laboratories into a commercial microscope for the first time several years later. The technique provides an excellent method of improving contrast in unstained biological specimens.
Polarized Light Microscopy - The polarized light microscope is designed to observe specimens that are visible due to their birefringent character. Polarizing microscopes must be equipped with a polarizer, positioned in the light path before the specimen, and an analyzer placed in the optical pathway between the objective rear aperture and the observation tubes or camera port.
Specimen Contrast - Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector.
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 for Optical Sectioning - 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.
Microscope Ergonomics - Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Microscope operators often must assume an unusual and challenging position.