Note how much brighter and blurrier the open pinhole image is and how much extra light comes from out of focus planes when the pinhole isn’t blocking it. This is obvious based on number of oversaturated red pixels.

Compare it with the images that you have just collected. If you close the pinhole too much, light can’t reach the detector.

At a Pixel Dwell Time of 12 µseconds (compared to a dwell time of 2.0 µseconds), the time to collect photons is increased by a factor of 6x. This means the image will be much BRIGHTER and the image scan rate will be much SLOWER. Most common dwell times are around the 2–5 µsecond range.

The resulting image is a merge of both AlexaFluor568 and AlexaFluor647.
Save this image to compare it to a sequentially acquired image.

The resulting image is a merge of both AlexaFluor568 and AlexaFluor647 but scanned sequentially.

Simultaneous

Sequential

Channel 3 3D image projection and rotation.

Note that each image is acquired frame by frame to ensure the absence of the spectral bleedthrough of the 568 into Channel 4 that was present in the simultaneous image.