Purpose: Collect a stack of optical sections in the z-axis and use constrained iterative deconvolution to rid the sections of blurred information, which in turn will enhance the actual data.

This type of application can be customized to fit an existing upright or inverted research grade microscope, or come complete as a turn-key imaging system. The system functions by using a motor to drive either the fine focus knob of the microscope or a piezoelectric focusing collar, which fits under the objective. Pictured here is a z motor with encoder (shown on bottom right corner of the image). This unit will read the exact position on the stage, regardless of any play in the fine focus or backlash of the z motor.

There are also other requirements for proper deconvolution. Two are hardware related, and the third is software. One hardware requirement is a light sensitive camera (pictured above is a Cooke SensiCam). Because of the number of z sections obtained, perhaps over 100, the sample will be prone to photobleaching if exposed for too long. Therefore the speed at which the camera can acquire and process the image becomes critical. Second, mercury light sources have an inherent flicker. Since CI relies on the pixel values of every plane within the stack to apply the proper deconvolution, any flicker from the light source will change those values and invalidate the information. While not specifically a requirement, it is highly advised to use a xenon power supply (pictured above is a Sutter DG-4). Lastly, for deconvolution to perform optimally, a specific point spread function must be performed within the software on a fluorescent bead stack to determine, and subsequently mathematically adjust for, the values by which the optical system spreads light.

Deconvolution is quickly becoming a standard practice in fluorescence imaging to provide the best overall image quality. Pictured here is an example of how the software eliminates the blurred information to provide a clear image of the specimen.