Confocal Microscopy

We are about to purchase a confocal microscope to help us with our research in dry eyes, corneal infections, pterygium etiologies, and goblet cell densities and health.

This paper may help in asking the right questions besides cost & time to assess images for the best confocal microscope.

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JBT Index Association Homepage
. 2015 Jul; 26(2): 54–65.
Published online 2015 Mar 19. doi:  10.7171/jbt.15-2602-003
PMCID: PMC4365987
PMID: 25802490

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques


Wide-field fluorescence microscopes achieve excellent resolution, contrast, sensitivity, and acquisition speeds. However, for thicker specimens, the out-of-focus features add a blurry haze to each image plane, reducing contrast. Many 3-dimensional (3D) imaging technologies have been developed to remove this out-of-focus haze and improve contrast, and thereby resolution, in the fluorescence microscope. The confocal laser-scanning microscope (CLSM) was first conceptualized and patented in 1957 by Marvin Minsky.1 However, it was the implementation and commercialization of the CLSM in the late 1980s—specifically for fluorescent biologic samples— that really moved the CLSM to the mainstream of biologic research.2 The development of a robust and reliable instrument, the use of rapidly scanning mirrors instead of slowly moving the sample, and the ability to remove out-of-focus blur to produce a series of thin optical slices of the specimen in 3D revolutionized fluorescence imaging. Images of a single image plane in the middle of a very thin sample (<5 μm), a sample of intermediate thickness (∼20 μm), and a thick sample (∼50 μm), imaged in wide-field or CLSM modes, demonstrate the need for confocality for thick samples (Fig. 1). For thin samples, there is only a slight advantage using confocal imaging (compare Fig. 1a and b), but for thicker samples, the removal of out-of-focus blur from each image section is crucial for visualizing and measuring features of interest in the specimen (compare Fig. 1c and d and 1e and f).
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