In spinning disk microscopy using a Yokogawa scan head, the camera exposure times are dependent upon the intensity of fluorescence emission gathered by the specimen and vary widely from one sample to another, and the disk rotation speed must be carefully adjusted to match the camera exposure time. As a result, uniform image capture can only be performed if the exposure time is an integral multiple of the time necessary to sweep one scan frame (30 degrees of disk rotation). In cases where the exposure time is not matched (in effect, is not an exact multiple of the rotation speed), scanning will continue into the next frame and can produce a striped pattern superimposed on the image (see Figure 1(c)). This interactive tutorial explores matching disk rotation speed with camera exposure times.

The tutorial initializes with a Yokogawa disk spinning at 1800 rpm and the camera frame rate set to 360 Hertz with an exposure time of 50 milliseconds. The disk speed is not synchronized to the camera exposure time, as indicated in the red box positioned beneath the disk and camera parameter values. In order to operate the tutorial, use the Disk Speed slider to increase the rotational speed of the spinning disk to transition through values that are synchronized to the camera exposure time. Once the camera exposure and disk speed are synchronized, the red box turns green to indicate a match. The CCD Exposure pull-down menu can be used to change the camera exposure time.

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Mismatches between disk rotation speed and camera integration periods are generally not a problem for long exposure times (greater than 100 milliseconds) where averaging occurs between frames, but can seriously hamper imaging success when exposure times are reduced to a few milliseconds. Fortunately, even though the Yokogawa disk is capable of producing 12 images from each 360-degree rotation, these individual segments do not have defined start and stop points so that any exposure corresponding to one twelfth of a disk rotation will capture a complete image irrespective of the point at which the exposure was started. Image banding can also occur when the frame shift of accumulated charge during EMCCD readout conflicts with the disk rotation speed (Figure 1(d)). This artifact can be addressed by pushing the vertical shift times to shorter values.

The Yokogawa CSU-X1 features an advanced disk drive motor that allows for fine-tuning the adjustment of rotation speed to match the camera exposure time. Furthermore, the unit incorporates a dynamic balancing mechanism for the rotating parts to prevent vibrations (as well as accompanying imaging artifacts) that might occur at the highest speeds.

Contributing Authors

Mismatches between disk rotation speed and camera integration periods are generally not a problem for long exposure times (greater than 100 milliseconds) where averaging occurs between frames, but can seriously hamper imaging success when exposure times are reduced to a few milliseconds.

Adam M. Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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