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.

Chameleons: Calcium Ion Probes - Chameleons 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). 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.

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.

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.

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.

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.

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.

Colocalization of Fluorophores in Confocal Microscopy - Two or more fluorescence emission signals can often overlap in digital images recorded by confocal microscopy due to their close proximity within the specimen. 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. This interactive tutorial explores the quantitative analysis of colocalization in a wide spectrum of specimens that were specifically designed either to demonstrate the phenomenon, or to alternatively provide examples of fluorophore targets that lack any significant degree of colocalization.

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