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Chapter 14:
Speculation on Future Directions for Confocal and Multiphoton Excitation Microscopy
14.1 Correlative Microscopy From its beginnings, the field of microscopy was plagued by artifacts. Artifacts may be thought of as observed properties in the image of microscopic objects that are not inherent to the object in its natural state. Open any histology book and there is an important section on artifacts in microscopy. Artifacts can be placed into two classes: those associated with sample preparation which includes staining and genetically expressed fluorescent probes, and those associated with the optical system and the physics of photon detection. Examples of artifacts associated with the preparation of the sample include the following: improper sampling of a heterogeneous sample; fixation, mechanical sectioning, staining, heating and drying. Also to be included in this category are light damage to the sample, mechanical damage, thermal damage, and changes in living cells and tissues once cell death occurs. The latter category includes optical aberrations and insufficient spatial and or temporal resolution to adequately observe structure and physiological function. For example, with inadequate transverse resolution, high spatial frequency structures will not be apparent. When monitoring physiological events, if the temporal resolution is insufficient then the true time course of these events will not be correctly measured. Even when all due care is maintained in sample preparation and when the optical elements are selected to minimize many of the optical aberrations, the possibility of incorrect interpretation of the images still exists. That is when the power of correlative microscopy becomes apparent. As mentioned in Chapter 5, correlative microscopy is the use of two or more types of microscopy on the same sample. For example, a sample could be observed with both reflected light confocal microscopy and fluorescence light multiphoton excitation microscopy. Alternatively, a specimen can be observed with confocal and interference microscopy (DIC). Images obtained with optical low coherence tomography (OCT) can be compared with confocal microscopy and multiphoton excitation microscopy. When several different microscope techniques show similar structures on the same sample, then the likelihood that the images correspond to the structure of the specimen is increased. We will see increased use of correlative microscopy and this will increase the accuracy of our observations and measurements.
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