Advanced Light Microscopy and Image Analysis Core

Timeline of imaging technology

The Core facility is designed to image both living and fixed material

Imaging living cells or tissues:
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 ºC. 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.

A multi-wavelength, three dimensional image stack of fixed Hela cells. The cells were prepared by indirect immuno-fluorescence and imaged with wide-field microscopy. Microtubules labeled for tubulin are shown in green, F-actin is labeled in red, and DNA is labeled in blue. The image stack on the left is the "raw" (unprocessed) data. The image stack on the right is deconvolved, using a generated point spread function. There is substantially higher resolution in both the lateral and axial planes of the deconvolved image stack.

(Click on Image for QuickTime movie - Size: 9.85 MB)

(QuickTime -- Download the free player)

Fluorescence imaging:
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.

Image analysis:
Once the images are collected there is an extensive array of image processing & analysis software to assist in data analysis/interpretation. These “tools” 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.