Applications in Confocal Microscopy

The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, multiphoton microscopy, total internal reflection, DNA hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging. Many of these powerful techniques are described in the sections listed below.

Colocalization of Fluorophores in Confocal Microscopy - During the examination and digital recording of multiply labeled fluorescent specimens, two or more of the emission signals can often overlap in the final image due to their close proximity within the microscopic structure. This effect is known as colocalization and usually occurs when fluorescently labeled molecules bind to targets that lie in very close or identical spatial positions. The application of highly specific modern synthetic fluorophores and classical immunofluorescence techniques, coupled with the precision optical sections and digital image processing horsepower afforded by confocal and multiphoton microscopy, has dramatically improved the ability to detect colocalization in biological specimens.

Classification and Applications of Fluorescent Probes - In general, fluorescent probes are classified according to their excitation and emission characteristics, as well as their chemical and biological properties. The tabulation in this section reviews examples of probes in each of the important biological classes, including nucleic acids, polysaccharides, lipids, membranes, cytoplasm, ion concentration, and specific organelles. Immunological reagents are also of fundamental importance in fluorescence microscopy as are the fluorescent protein probes.

Fluorescent and Bioluminescent Proteins - Over the past several years, research with the green fluorescent protein (GFP) and its many genetic variants has become a major staple in the foundation of investigative cell biology. In addition, the bioluminescent proteins luciferase and aequorin are useful for studying fluctuations in intracellular ion concentrations and the detection of adenosine triphosphate (ATP). Spectral variants of these luminescent and fluorescent proteins are now commercially available, and open up the possibility of multiple labeling experiments in living cells.

Green Fluorescent Protein (GFP) - The green fluorescent protein (GFP) and its spectral variants (yellow, YFP); cyan, (CFP); blue, (BFP); and red (dsRFP)) are in rapidly becoming important investigational tools in the various disciplines associated with the life sciences including medicine and biology. A quick search of the Internet or the recent literature reveals dozens or more reports each month. The green fluorescent protein was originally isolated from the jellyfish and is a specialized protein that emits fluorescence when exposed to excitation light. Its primary importance for current research lies in the ability of the purified jellyfish GFP gene to express the fluorescent protein in other living organisms. When a non-specific fluorescent protein (or a variant chimera) gene is introduced into a tissue culture line, the entire cellular cytoplasm will emit a green fluorescence.

The Fluorescent Protein Color Palette - A broad range of fluorescent protein genetic variants have been developed over the past several years that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Extensive mutagenesis efforts in the original jellyfish protein have resulted in new fluorescent probes that range in color from blue to yellow and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone Discosoma striata and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, red, and far-red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.

Optical Highlighter Fluorescent Proteins - Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. These effects result in the direct and controlled highlighting of distinct molecular pools within the cell.

Embryonic Stem Cells - Embryonic stem cell lines, which were originally produced from the inner core of human blastocysts as well as those of other mammals, are now widely established in the research community using traditional in vitro culture. The cell lines preserve their undifferentiated state and normal nuclei during subculture, however, they remain capable of differentiation into virtually any type of tissue. Proliferated embryonic stem cells first become stem cells (such as neuronal stem cells, muscle stem cells, vascular endothelial stem cells, and hematopoietic stem cells) according to the specific culture conditions, and then differentiate into neurons, muscle cells, vascular endothelial cells and blood cells. However, unlike the fertilized egg, a cluster of embryonic stem cells cannot independently develop into a human being (or other animal).

Epitope Tagging - An epitope (also known as an antigenic determinant) is a biological structure or sequence, such as a protein or carbohydrate, which is recognized by an antibody as an antigen. Recognition of the antigen occurs when an appropriate structure is formed in an area of a protein or polysaccharide in which amino acids or sugars are arranged linearly. Most proteins usually have several kinds of epitopes. The antibody offers an important technique (termed an immunoassay) for identifying specific cellular components (proteins, lipids, carbohydrates, etc.) to track the function, distribution, and modification of the protein of interest within living and fixed cells.

Fluorescence Lifetime Imaging Microscopy (FLIM) - Multi-color staining with fluorescent dyes is actively used for observing the distribution of biological materials (such as proteins, lipids, nucleic acids, and ions) in the field of tissue and cell research. The detection technology for fluorescence observation has advanced to a level at which a single fluorescent dye molecule can be detected under the best of circumstances. This section reviews several of the important aspects of fluorescence lifetime imaging microscopy (FLIM), a new fluorescence microscopy technology. In addition to multi-color staining, fluorescence lifetime imaging can also be utilized to visualize the factors that affect the fluorescence lifetime properties of the dye molecule, that is, the state of the environment around the molecule.

Fluorescence Photobleaching Investigations - Both fluorescence loss in photobleaching (FLIP) and the related methodology of recovery after photobleaching (FRAP) are techniques for observing the movement of intracellular materials through photobleaching of fluorescence. A specific area of a floating fluorescent dye on a cell membrane, an organelle (endoplasmic reticulum and Golgi apparatus) membrane, or a floating fluorescence-labeled protein on these membranes is bleached, and the loss or recovery of fluorescence is observed to examine fluidity in the lateral direction. The techniques of FLIP and FRAP are also used to confirm the continuity of membranes.

Fluorescence Resonance Energy Transfer (FRET) - The precise location and nature of the interactions between specific molecular species in living cells is of major interest in many areas of biological research, but investigations are often hampered by the limited resolution of the instruments employed to examine these phenomena. Conventional widefield fluorescence microscopy enables localization of fluorescently labeled molecules within the optical spatial resolution limits defined by the Rayleigh criterion, approximately 200 nanometers (0.2 micrometer). However, in order to understand the physical interactions between protein partners involved in a typical biomolecular process, the relative proximity of the molecules must be determined more precisely than diffraction-limited traditional optical imaging methods permit. The technique of fluorescence resonance energy transfer (more commonly referred to by the acronym FRET), when applied to optical microscopy, permits determination of the approach between two molecules within several nanometers, a distance sufficiently close for molecular interactions to occur.

Multiphoton Microscopy - Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with highly specific fluorophores. The technique features attractive advantages over confocal microscopy for imaging living cells and tissues with three-dimensionally resolved fluorescence imaging. Multiphoton excitation, which occurs only at the focal point of the microscope, minimizes the photobleaching and photodamage that are the ultimate limiting factors in imaging live cells. This advantage allows investigations on thick living tissue specimens that would not otherwise be possible with conventional imaging techniques.

Total Internal Reflection Fluorescence Microscopy - Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. The technique is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology. The basic concept of TIRFM is simple, requiring only an excitation light beam traveling at a high incident angle through the solid glass coverslip or plastic tissue culture container, where the cells adhere. Refractive index differences between the glass and water phases regulate how light is refracted or reflected at the interface as a function of incident angle. At a specific critical angle, the beam of light is totally reflected from the glass/water interface, rather than passing through and refracting in accordance with Snell's Law. The reflection generates a very thin electromagnetic field (usually less than 200 nanometers) in the aqueous medium, which has an identical frequency to that of the incident light.

Fiber FISH (Fluorescence in situ Hybridization) - The term Fiber FISH refers to the common practice of fluorescence in situ (FISH) conducted on preparations of extended chromatin fibers. In mapping DNA fragments of interest by conducting FISH investigations on chromosomes, signals within a distance of several million base pairs are indistinguishable from each other because of the multifold structure of DNA strands in the metaphase chromosomes. The resolution of signals improves if the chromosomes are used before they progress to full condensation. What can we do when we want to map more adjacent DNA clones? The characterization of entire genome DNA sequences will resolve the problem of creating a map in scale of one base pair, but is extremely time-consuming.

Cameleons: Calcium Ion Probes - Cameleons are a new class of indicators for calcium ion concentrations in living cells, which operate through a conformational change that results in fluorescence resonance energy transfer (FRET) in the presence of calcium ions. In the past, fluorescent probes, such as Fura-2, Indo-1, and Fluo-3 were very popular for measuring fluctuations in calcium ion concentrations within living cells. In 1997, Dr. Atsushi Miyawaki (of the Riken Brain Science Institute in Wako, Japan) developed a novel probe for calcium ion measurement. This probe consists of an artificial protein modified from green fluorescent protein (GFP), and was named cameleon (after the chameleon reptile, but dropping the "h" in the name). The cameleon molecular structure is modeled as a fusion product between two fluorescent proteins (having differing excitation and emission characteristics), calmodulin (CaM), and the calmodulin-binding domain of myosin light chain kinase (M13). Calmodulin is capable of binding with free calcium ions and the M13 chain can bind with calmodulin after it has bound the calcium ions. The genes of these four proteins are joined linearly, and the fusion genes are expressed in a variety of cells.

Specimen Preparation Using Synthetic Fluorophores and Indirect Immunofluorescence - Confocal microscopy was becoming more than just a novelty in the early 1980s due to the upswing in applications of widefield fluorescence to investigate cellular architecture and function. As immunofluorescence techniques, as well as the staining of subcellular structures using synthetic fluorophores, became widely practiced in the late 1970s, microscopists grew increasingly frustrated with their inability to distinguish or record fine detail in widefield instruments due to interference by fluorescence emission occurring above and below the focal plane. Today, confocal microscopy, when coupled to the application of new advanced synthetic fluorophores, fluorescent proteins, and immunofluorescence reagents, is one of the most sophisticated methods available to probe sub-cellular structure. The protocols described in this section address the specimen preparation techniques using synthetic fluorophores coupled to immunofluorescence that are necessary to investigate fixed adherent cells and tissue cryosections using widefield and confocal fluorescence microscopy.

Fluorescent Protein Literature Sources - Dramatically expanding the ability to visualize molecular events with high temporal and spatial resolution in the optical microscope, fluorescent proteins represent perhaps the most important probes ever developed for investigations in cellular and plant biology. With the emergence of the Internet as a practical vehicle for the international dissemination of information, a majority of the scientific publications have begun to make their content available on-line in the form of abstracts, HTML documents, and downloadable portable document format (PDF) files that can be viewed and printed locally. The references listed in the following sections contain links to indexing services in order to provide investigators with a quick pathway to the original literature targeting fluorescent proteins.