A vast numbers of investigators are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. As such, live-cell imaging has become an important analytical tool in most cell biology laboratories, as well as a routine methodology that is practiced in the wide ranging fields of neurobiology, developmental biology, pharmacology, and many of the other related biomedical research disciplines. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated in the presence of synthetic fluorophores and/or fluorescent protei A comparison between Confocal and Widefield Fluorescence Microscopy - Confocal microscopy offers several distinct advantages over traditional Widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane, that leads to image degradation, and the capability to collect serial optical sections from thick specimens.

The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope. Fluorescence Resonance Energy Transfer with Fluorescent Proteins - Fluorescent proteins are increasingly being applied as non-invasive probes in living cells due to their ability to be genetically fused to proteins of interest for investigations of localization, transport, and dynamics. In addition, the spectral properties of fluorescent proteins are ideal for measuring the potential for intracellular molecular interactions using the technique of Förster (or fluorescence) resonance energy transfer, FRET rmicroscopy. Because energy transfer is limited to distances of less than 10 nanometers, the detection of FRET provides valuable information about the spatial relationships of fusion proteins on a sub-resolution scale. This interactive tutorial explores various combinations of fluorescent proteins as potential FRET partners and provides information about critical resonance energy transfer parameters, as well as suggestions for microscope optical filter and light source configuration. 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. Fluorescent Protein Fluorophore Maturation Mechanisms - Autocatalytic formation of the fluorophore (also referred to as a chromophore, within the shielded environment of the polypeptide backbone during fluorescent protein maturation follows a surprisingly unified mechanism, especially considering the diverse natural origins of these useful biological probes. Shortly after synthesis, most fluorescent proteins slowly mature through a multi-step process that consists of folding, initial fluorophore ring cyclization, and subsequent modifications of the fluorophore. The spectral properties of fluorescent proteins are dependent upon the structure of the fluorophore as well as the localized interactions of amino acid residues in the immediate vicinity, and in some cases, residues far removed from the fluorophore. The interactive tutorials in this section explore fluorophore formation in a wide variety of spectrally diverse fluorescent proteins deduced from crystallographic studies. The Rat - The humble rat has had an outsized impact on human history. In the Middle Ages, the black rat Rattus rattus was blamed for spreading the Black Plague through its fleas, a pandemic that killed a third of Europe’s population, an estimated 34 million people. In modern times, however, a larger cousin, the brown rat, Rattus norvegicus has become an important model organism in biological research. Selective breeding of the Brown Rat has produced the albino laboratory rat. Rats grow quickly to sexual maturity and are easy to keep and breed in captivity. Scientists have bred many strains or “lines” of rats specifically for experimentation. Generally, these lines are not transgenic because the easy techniques of genetic transformation that work in mice do not work as well for rats. This has been a problem for investigators who view rat behavior and physiology as more relevant to humans and easier to observe than in mice. In October 2003, researchers succeeded in cloning two laboratory rats by the problematic technique of nuclear transfer. As cloning techniques are perfected, rats likely will become an important subject of genetic research