New York State Dept. of Health
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Last Updated: 04/21/2021
Adherent cells or tissue fragments can be imaged in a variety of methods depending on the question being asked. If drug delivery is required, a perfusion style chamber is used; if gas exchange for the growth media is needed glass bottom Petri dishes can be used; if long term (> 3 days) sterility is needed, there are sealed Rose chambers for imaging. Regardless of what the experimental design, the specimen can be maintained from RT to 50 ÂºC. Non-destructive image modes i.e. phase contrast, differential interference contrast or polarized light coupled with shutter and filtered illumination provide as close to homeostatic conditions as possible.
Once the images are collected there is an extensive array of image processing & analysis software to assist in data analysis/interpretation. These â€œtoolsâ€ range from the simple, contrast manipulation to complex tracing and counting algorithms. There are also different forms of deconvolution available for "removing" the blur from fluorescent specimens, or transmitted light images. If multiple molecules are labeled, co-localization can be determined for either a static image or an image sequence. There are both automatic and semi-automatic 2 and 3-D tracking/montaging algorithms available.
Multiple labeled biomolecules such as GFP etc. or fluorophore can be imaged in four-dimensions. This can be done over time (5th dimension) for live specimens as well as statically for fixed specimens. Specimens can be as thin as a single cell or as thick as a whole organism (i.e. drosphilia) or anything in between. Conventional two-dimensional imaging can be done at high sensitivity, i.e. low bleaching rates. Three-dimensional imaging can be done in wide-field for relatively thin specimens or by confocal microscopy for thick specimens.
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