OHSU

Hi-Res Widefield

CoreDV_120

The Deltavision CoreDV Widefield Deconvolution System is a high resolution widefield microscope for acquiring images of live or fixed samples. The system is built on an Olympus IX71 inverted microscope and features an LED transmitted light source for differential interference contrast  and a 7-color solid state illumination unit for exciting fluorophores across the visible spectrum.  Polychroic beam splitters, optimized for imaging with Blue-Green-Red-FarRed fluorophore combinations in fixed cells or CFP/YFP and GFP/RFP combinations of expressed proteins in living cells, are combined with emission switching through fast filter wheels.  Images are captured on a Nikon CoolPix HQ cooled CCD camera. The motorized stage is controlled by XYZ nanomotors for accurate z-stack and point-visiting functions. 

The strengths of this system over laser-scanning confocals is the sensitivity to detect dimmer signal in thinner specimens.  For increasing apparent contrast and resolution in the standard widefield images acquired, 3D data sets are deconvolved based on the iterative constrained algorithm of Sedat and Agard.  Deconvolution is a mathematical process where an objective-specific standard, called a point spread function (PSF), is applied to each voxel (measurement in X, Y and Z) in each color to determine the degree of blur occurring. The PSF is determined a priori based on images of a sub-resolution bead to provide the algorithm information on how light behaves in the specific system.  The deconvolved image is more defined and resembles the narrow plane of a confocal image. There is a offline computer station available for this process.

Another advantage of this system over laser-scanning confocals is the gentle illumination and speed, which makes it a preferred setup for live-cell imaging.  While not as fast as a spinning disk, it combines multiple color and Z-plane abilities to acquire high resolution images at rates fast enough to observe many cellular functions of interest.  For live-cell imaging, temperature, humidity and CO2 environment are tightly controlled in the full stage enclosure. A continuous focus device maintains the sample in perfect registration with the objective.  The system has recently been upgraded to perform 3-line laser-based total internal reflection fluorescence microscopy (405 nm, 488 nm, and 561 nm) (TIRF). The same lasers can also be used for photomanipulations, such as photo-activation or photo-switching,  FRAP (fluorescence recovery after photo-bleaching), FLIP (fluorescence loss in photo-bleaching) and FRET (Forster resonance electron transfer).