Confocal laser‐scanned microscopy: Analog signal processing

Biological samples labelled with fluorescent markers often result in specimens with a dynamic range that is less than ideal for imaging with the confocal laser‐scanned microscope. This range can be either too great or too small, and thus prevents the entire image from being processed and/or displayed at the same time. The effect of analog signal processing to overcome this limitation was tested using a modified GW Electronics Image Enhancer in the image pipeline of a Bio‐Rad MRC‐500 Confocal Laser Scanning Microscope. The analog preprocessing did not interfere with subsequent digital processing. The use of homomorphic filtering allowed the dynamic range to be compressed. Fine structures with low signal levels were imaged with improved contrast while structures with high signal levels, did not saturate the detection and display systems. When the black levels were properly manipulated, the processor could be used to enhance signals that were near the noise level, in effect expanding the dynamic range. Both applications resulted in improved image quality and signal to noise. Other analog functions, notably logarithmic and exponentiation were also evaluated. Analog preprocessing produces images with better signal to noise than standard images after contrast stretching and background subtraction by off‐line digital processing. Since the analog processor functions in real time, image collection could be optimized directly, and subsequent digital processing further improved the images.