Deconvolution

The approximate dimensions of a haploid yeast cell are 5 x 7 um. Since the objective lenses used for the examination of yeast have depths of focus that are less than 0.4 um, any acquired image is contaminated with out-of-focus light from above and below the focal plane. The observed image is a convolution of the true image with a smearing function. The smearing function is primarily a property of the objective, and is known as the point spread function, or PSF. As the name implies the PSF describes how a point object spreads out to take on an observed shape in the acquired image. Based on the PSF deconvolution attempts to reconstruct the true image by a mathematical process that removes the out-of-focus light.

Visualizing the yeast mitochondria gives a powerful example of the advantage of three-dimensional deconvolution. The morphology of the yeast mitochondria changes during the growth, but in general is tubular and associated with the periphery of the cell. In the live cell images below the mitochondria were labeled using TRX3-YFP fusion. The cells were visualized on the integrated DeltaVision system with a 60X (numerical aperture, 1.4) objective, fluorescence filter sets from Omega Optical, and a cooled, charge-coupled Photometrics' Quantix camera from Roper Scientific. (Since these images were captured we have switched cameras to a Coolsnap HQ, from Roper, and do all our acquisitions with a 100X objective, usually with 2 x 2 binning.) Forty optical sections were acquired in which the focus was progressively changed by 0.2um. The image on the left shows a 2-D projection of the stack of 40 sections taking the maximum pixel value at each position. On the right is a movie showing every other interval of the 40 sections.

Here on the left is a 2-D projection of the same stack after 15 cycles of constrained, iterative deconvolution. On the right is a movie showing every other interval of the deconvolved images.

 

Finally the Deltavision software can construct a volume view which incorporates all of the data into a 3-dimensional image. We present this 3-D image of the yeast mitochondria as a movie that rotates around the x-axis for 360°.

Here is another 3-D movie in which TRX3-GFP has been pseudocolored green and the image rotated around the Y-axis 180°. In the upper cell you can see the mitochondria as it moves into the bud.