There are three major types of light microscopes: fluorescence, two-photon and confocal. All three are fluorescent microscopy techniques. Also widely used are other, older, light microscopy techniques, such as non-fluorescent bright field microscopy, and various contrast-enhancing techniques (such as phase contrast, Nomarski, and others).
All three fluorescent light microscopes are used to study molecules in living cells. They provide insights into how the molecules and the cells they compose behave normally, and how they are altered by disease, injury, treatment, or other experiences. Each type of microscope has its strengths and limitations.
Fluorescence microscopes are used with fluorescent probes that emit light of short wavelength to reveal biochemical activities within a cell in human and animal tissue cultures. Fluorescence microscopes have the highest resolution of all cellular imaging devices. This enables them to be used to identify a single fluorescently labeled molecule or differentiate activities of several differently colored fluorescent molecules in the same cell. This process is referred to as “subcellular” resolution of molecular activities within a cell.
Fluorescently labeled cells are excited by laser or incandescent light. The fluorescent label (probe), which is designed to go to a specific molecule or region of a cell, absorbs a “photon” of energy supplied by the laser or incandescent light. Then the photon is excited to emit light at a particular wavelength, depending upon the specific probe used. The emitted light is recorded as a photographic image, video, fluorescence decay trace, or as “photo multiplier tube” signals from serial points that are displayed and analyzed by computer to provide flexible images.
Most fluorescent light microscope probes emit light of short wavelengths, which is visible with the naked eye, and useful for imaging molecules or cells close to the surface. Visible light does not pass through tissue well, however, and these techniques are suitable only in cases where the distances traveled in tissue by the probe are small (micrometers in length). As a result, the tissue grown in laboratory cultures needs to be very thin, from 1 to 20 microns thick, to be viewed with a fluorescence light microscope. The tissue grown for visualization with the other two main types of light microscopes, confocal and multi-photon, can be much thicker.
Confocal laser scanning microscopy provides the ability to simultaneously collect multiple images in digital form from serial sections of thick tissue specimens, and flexibly display and analyze them via computer. Confocal imaging is undertaken in thick tissue cultures and in small laboratory animals. The blur-free images are taken point by point using “photo multiplier tubes” that provide sensitive and fast registration of the intensity of emitted light. The points are then reconstructed by computer, rather than projected through a microscope’s eyepiece.
Confocal laser scanning imaging relies extensively on fluorescent probes of longer wavelength to monitor dynamic processes such as: cellular integrity (differentiating live cells from those that are dying to make way for new cells—called “apoptosis”—and cell death); membrane fluidity, transduction of cellular signals, activities of enzymes, and movements of proteins; and, the migration of cells in the developing animal embryo. Additionally, confocal laser screening microscopes facilitate study of brain synapses (communication junctions between two nerve cells) and cell circuitry (formation of neural networks), as do multi-photon techniques.
Multi-photon laser microscopy relies on the simultaneous absorption of two or more photons by a molecule to image fluorescent probes with longer wavelengths that penetrate deeper into tissues. It is used in thick tissue cultures and small laboratory animals, often to study cellular actions over time in the brain. As an example, multi-photon imaging visualizes actions of immune cells residing in the brain (microglia) and of antibodies summoned to the brain to fight infections and cancers. Scientists follow the actions of innate immune cells, called “dendritic” cells, to see how they pass information on how to recognize a specific invader to certain immune T-cells so that they can attack the intruder.
In addition to studies in human and animal tissues and small laboratory animals, hand-held fiberoptic confocal and multi-photon devices have been developed that can be used for endoscopic imaging in humans. This process entails using a combination of imaging techniques. Fluorescence resonance energy transfer (FRET) is undertaken with microscopic imaging to reveal the interaction between two or more fluorescent probes in tissue cultures. This interaction of adjacent probes is used to monitor the assembly or fragmentation of molecules, such as occurs in the fusion of two cell membranes or the binding of a molecule to its receptor. This imaging with light microscopes is limited to tissue cultures because the signal from fluorescent-and FRET-generated signals is relatively weak and is poorly transmitted through living tissue. To undertake such studies in small laboratory animals requires the use of more sophisticated imaging devices, called macroscopic optical imaging scanners.