POWRE: An Extended-Depth-of-Focus, Differential Interference Contrast (DIC) Microscope for Improved Observations of Living Cells

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

A new kind of differential interference contrast (DIC) microscope that will allow biologists to observe live-cell preparations with all the high-resolution advantages of conventional DIC, but with the added advantage that many more features throughout the specimen volume will appear in focus in a single image, will be developed. At present, high resolution DIC techniques are hampered by an inherent narrow depth-of-focus. Sometimes called "optical sectioning" (not to be confused with confocal microscopy), DIC tends to image the highest spatial frequencies (i.e. the finest detail) only from features that are nearest the plane of focus. Features above or below this plane rapidly lose contrast and appear as low spatial frequency blur. Any biologist who has used DIC for imaging thick preparations appreciates that by focusing up and down rapidly, one can get a sense of the 3D morphology of a specimen. Unfortunately, there is no current mechanism for acquiring this depth information all at one time in a single image. For many biological applications requiring tracking of moving cell components over time, the limited focal depth exhibited by DIC means that features may rapidly move out of focus, which severely limits the ability to obtain useful, interpretable images. The new microscope will be called an extended-depth-of-focus, differential interference contrast (EDF-DIC) system. It will be based on novel "wavefront coding" optical techniques that recently have been used to convert a standard fluorescence microscope into one that acquires sharp images from extended depths at much faster rates than are possible with confocal or widefield microscopes. Specifically, the creation of an EDF-DIC system requires simply inserting a custom-designed phase plate into the imaging path of a standard DIC microscope and then applying a very fast, single-iteration digital filter to the recorded image. This also has the advantage that it can be easily adapted to virtually any existing microscope design. It is anticipated that the new EDF-DIC microscope will supply biologists with a promising new tool for a wide range of investigations of the dynamical processes of living cells over time. To name a few examples, the new EDF-DIC system should provide biologists with the capability to perform the following: observe the 3D spatial distribution and dynamic instability of microtubules (e.g. in budding yeast) or view all the chromosomes in focus at once throughout the mitotic cycle in a broad range of applications throughout the animal and plant kingdoms.