How does fluorescent microscopy differ from scanning electron microscopy, and how do you know which technique to use for a specific application? Here is some basic information that can help you make a more informed decision.
Fluorescent Microscopy
Fluorescent microscopy is an imaging technique used to visualize specific features of biological specimens (i.e., microbes, cells, cellular components, etc.) at small concentrations with a good signal-to-background ratio.
In fluorescent microscopy, the sample is stained with a fluorescent dye (fluorophore) prior to imaging. As the test sample is illuminated through the lens of the microscope using a higher energy source (xenon lamp or mercury arc lamp), the fluorophores absorb the energy and emit it as a weak light with a longer wavelength as they return to their normal energy level. A magnified view of the target sample can then be observed as the emission filter separates the background fluorescence from the reflected light.
Fluorescence microscopy is mostly used for imaging cellular structures and genetic materials within the cell, as well as for viewing specific cells in a larger population (with techniques such as FISH) and checking cell viability studies on cell populations.
Despite its advantages, this technique has several limitations.
- Due to its large size, the fluorophore tag might interfere with the metabolic pathway under investigation.
- The excitation light may adversely affect the viability of live tissues.
- In its excited state, the fluorophore may react with oxygen and produce free radicals.
- The fluorophore may fail to fluoresce when it undergoes covalent modification while in the excited state (photobleaching).
Scanning Electron Microscopy
Prior to imaging, the samples are stained (either through shadow casting or negative staining) and sectioned by microtome, if necessary. Special preparations (i.e., coating non-conducting specimen with electrically conducting materials, impregnating biological samples with osmium to increase bulk conductivity, embedding samples in a resin, chemical fixation or cryofixation of biological samples, and freeze-fracturing of lipid membranes and their proteins) may also be needed to increase their stability and electrical conductivity.
Instead of using visible light for illumination, a scanning electron microscope (SEM) scans the surface of the sample with a focused beam of electrons in a raster pattern to produce extremely detailed three-dimensional images of the sample.
The interaction between these electrons and the atoms in the sample produce signals (e.g., transmitted electrons, elastically and inelastically scattered electrons, back-scattered electrons, secondary electrons, x-rays, and visible light) that contain information about the composition, morphology, and surface topography of the sample.
In conventional SEM, samples are observed in high vacuum while in variable pressure or environmental SEM, imaging can be done in low vacuum or wet conditions and at a wide range of temperatures.
SEM offers several advantages, which include the following:
- SEM samples require minimal preparations prior to imaging.
- It provides better resolution (as low as 10 nanometers) and can magnify the image of the sample up to two million times its original size.
- It allows for faster imaging and can complete SEI, BSE, and EDS analyses in under five minutes.
- SEM has a wide range of applications and can be used for studying subcellular structures, cell metabolism, and intracellular pathogens and viruses.
- With the proper training and technology, SEMs are easy to operate.
As for its limitations, SEMs can be expensive and can require specialized storage and housing conditions. Ideally, these instruments must be kept in an area with no electric, magnetic, or vibration interference and should be well-maintained at all times. Moreover, SEMs cannot detect very light elements, especially those with atomic numbers less than 11.
Additionally, special training is needed in preparing the samples, operating the equipment, and interpreting the resulting data. Operators and researchers should also observe the safety protocols at all times since there is a small risk of radiation exposure from the scattering of the electrons beneath the sample surface.
