Imaging in Volocity: Setting Exposure, Gain and Offset

Three successive images of the same section. Image 1 is overexposed, with a longer Exposure and lower Offset. Exposure was corrected in Image 2 by using navigating to the brightest part of the image and using AutoExposure. Contrast was improved by increasing Offset from 138 to 230. The effect of Gain was examined in Image 3. Holding Offset constant, increasing the Gain from 1.7 to 5X decreases Exposure from 101 to 36 ms, with no noticeable increase in noise or loss of detail. Therefore, at least within a certain range, Gain can be used to optimize Exposure time to minimize bleaching and keep acquisition times reasonable. (All images: Bilateral composite image of MCH (Zamir's rabbit anti-MCH, 1:1k) in the LHA. 10X, 7 X 5 tiles)

Image 1:

Exposure: 157ms

Gain: 1.7x
Offset: 138 levels

Image 2:

Exposure: 101ms
Gain: 1.7x
Offset: 230 levels

Image 3:

Exposure: 36ms
Gain: 5.0x
Offset: 230 levels