This tutorial explores combinations of fluorescence microscopy with additional contrast-enhancement techniques using both phase contrast and differential interference contrast (DIC) methods. Combined techniques are popular to avoid photobleaching by locating specific areas of the specimen with an enhanced contrast method (phase contrast or DIC) prior to observation and photomicrography with fluorescence illumination.

To operate the tutorial, first choose a method to combine with fluorescence (either phase contrast or DIC) using the radio buttons above the microscope viewport. Next, use the radio buttons below the viewport to toggle between fluorescence alone, the combination method (phase or DIC) alone, and both methods together.

Photobleaching, or dye photolysis, rapidly degrades fluorescent probes used to stain specimens and should be minimized by exposing the preparation to the lowest illumination levels as is possible. This effect is caused primarily by the photodynamic interaction between the fluorochrome and oxygen, which involves promoting the dye molecule from the singlet ground state to the relatively long-lived triplet excited state by a process termed intersystem crossing. Once the chromophore has been elevated to an exited state, it becomes more chemically reactive and may then participate in chemically irreversible reactions including decomposition, polymerization, oxidation (primarily by singlet oxygen) or reaction with another molecule. Reaction with oxygen will usually cause bleaching of the chromophore, depending upon the intracellular singlet oxygen concentration and the proximity of the chromophore to other internal cellular components such as proteins, lipids, or small molecules. Calculations indicate that singlet oxygen can produce chromophore photolysis over a distance exceeding 500 angstroms.

To minimize the effects of photobleaching, fluorescence microscopy can be combined with other techniques that are non-destructive to fluorochromes, such as differential interference contrast (DIC), Hoffman modulation contrast (HMC), and phase contrast. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique then, without relocating the specimen, switch the microscope to fluorescence mode. The results of a typical experiment of this type are illustrated in the tutorial. This tutorial illustrates, among other things, 3T3 fibroblasts in monolayer tissue culture imaged using phase contrast optics. The cell line was established from a National Institutes of Health line of Swiss mouse embryo cells, which are highly contact inhibited and useful for studies involving sarcoma virus formation and leukemia virus propagation. The WU Filter Cube radio button loads a photomicrograph of 3T3 fibroblasts imaged using fluorescent illumination (a mercury arc lamp and an Olympus WU filter cube) with cells stained by the fluorochrome 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid specific dye with an emission maximum at 461 nanometers, which is used to selectively stain nuclei and chromatin. Selecting the Phase Contrast radio button shows the same viewfield with phase contrast illumination. The Fluorescence/Phase Contrast radio button illustrates the two techniques used in combination to produce a beautiful photomicrograph of fluorescent-stained 3T3 cellular nuclei superimposed on a phase contrast image of the fibroblast cell membranes and internal organelles.

Several additional examples of combined techniques, this time using differential interference contrast (DIC), are also presented in the tutorial. Use the Fluorescence/Nomarski DIC radio button to toggle between phase contrast and DIC illumination. When in DIC mode, the WU Filter Cube radio button displays a fluorescence image of rat retina optic ganglion cells stained with the chromophore Fast Blue. Clicking on the Nomarski/DIC radio button toggles to an image of the retina cells imaged with DIC, and clicking on the Fluorescence/Nomarski DIC radio button located beneath the microscope viewport displays a combination of the two techniques. The Choose A Sample pull-down menu allows the visitor to select between rat retina tissue and cat brain tissue infected with cryptococcus. When the cat brain tissue is selected, and the WIB Filter Cube radio button is checked, a photomicrograph of the tissue imaged with fluorescence illumination through an Olympus WIB filter cube is displayed. The cells were stained with a combination of fluorescein-5-isothiocyanate (FITC) and Congo red (emission wavelength maxima of 520 and 614 nanometers, respectively). Selecting the Nomarski/DIC radio button reveals an image of the cat brain tissue using DIC optics and a full-wave retardation plate. Note the pseudo three-dimensional appearance of the photomicrograph. The two techniques used in combination can be viewed by clicking on the Fluorescence/Nomarski DIC radio button located beneath the microscope viewport.

The rate of photobleaching is dependent upon several factors, including the chemical reactivity of the chromophore, the intracellular chemical environment, and the intensity and wavelength of the excitation light. Some fluorescent dyes are readily susceptible to photobleaching while other are relatively stable. In many instances, the specimen may recover from the effects of photobleaching, particularly if it is kept cool and in a dark environment. Photobleaching should be distinguished from another fluorescence artifact termed quenching, which occurs by reduction (or in some cases, enhancement) of fluorescence intensity by competing processes such as temperature, high oxygen concentrations, and molecular aggregation in the presence of salts or halogen compounds. Sometimes quenching results from the transfer of energy to other acceptor molecules residing physically close to the excited fluorochromes, a phenomenon known as resonance energy transfer. This particular phenomenon has become the basis for a newer technique of measuring distances far below the lateral resolution of the light microscope. Impurities in the chromophore may also reduce fluorescence intensity either by photobleaching or quenching.

Photobleaching can be minimized by reducing exposure times (as discussed above) or by lowering the excitation energy (lamp intensity), however these remedies are accompanied by the undesirable effect of reducing the chromophore fluorescence emission energy. Neutral density filters can be placed in the light path before the light reaches the excitation filter, thus diminishing the excitation light intensity. It is also helpful, where possible, to deoxygenate specimens (but not living cells or tissues) and to use specific antifade reagents such as n-propylgallate and other inhibitors, which are commercially available. Chemicals that are capable of quenching singlet oxygen can also be employed to reduce the effects of photobleaching. Examples are the amino acid histidine and diphenylisobenzofuran. Fading effects can also be reduced, in some instances, by changing the pH concentration of the mounting medium. Although chemical methods can reduce photobleaching, the most effective remedy is to increase detection sensitivity (coupled with reduced excitation energy) using a low-light level CCD digital camera designed specifically for fluorescence microscopy.

The occurrence of photobleaching has led to a technique known as FRAP, Fluorescence Recovery After Photobleaching. This technique is based upon bleaching by short laser bursts and subsequent observation of the recovery of fluorescence caused by the diffusion of fluorochromes into the bleached area.

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BACK TO DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY

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