<title>Autofocus algorithms for fluorescence microscopy</title>

Abstract

A reliable autofocus is necessary for any combined image processing and automated microscope system analyze scan areas larger than a single field. Autofocus functions for brightfield microscopy have been reported in the literature. Autofocus implementations for fluorescence microscopy have to deal with some technical difficulties mainly due to the incoherence and faintness of the fluorescent light. In this presentation autofocus procedures are introduced for fluorescence microscopy based on image content information. A Leitz MPV II fluorescence microscope with x, y, z stepping motors was used as basic equipment. The microscope images were captured with an intensified target camera, digitized with a frame grabber and analyzed with a PC. We have constructed a digital autofocus system as well as an analogue autofocus detector. In the case of digital autofocus, the frame grabber images were analyzed while changing the z-position of the microscope slide. Three different focus functions were investigated: 1) edge-finding algorithms such as LoG and Canny operators; 2) different autocorrelation algorithms. In contrast to the digital focus criteria evaluation we constructed an electronic board with several differentiators and integrators. This module is designed for high speed autofocusing and directly coupled to the output of the camera. It makes it possible to obtain a focus value for the current image within one video cycle. All methods and functions were tested for different situations and the results compared.