Focus maintenance is a challenging problem in multidimensional wide-field microscopy. Most automated microscopes use software algorithms, which are applied to z-sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, autofocus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine autofocus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic-based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.
[Show abstract][Hide abstract] ABSTRACT: Digital-holographic metrology enables quantitative phase contrast microscopy of reflective and (partially) transparent samples. In this way, new application fields are opened up for nondestructive investigations of technical samples as well as for marker-free and time-resolved analysis of cell biological processes. Studies on long-term biological processes require permanent focus position readjustment to maintain an optimum image quality. Digital holographic microscopy permits subsequent numerical focusing by variation of the propagation distance. Here, the determination of the optimal propagation distance for a sharply focused image is of particular importance. At the Laboratory of Biophysics image definition quantification algorithms were adapted to the requirements of digital holographic microscopy. In order to obtain robust and reliable algorithms, the object-dependent optical absorption properties were taken into consideration. Automatic focus tracking is demonstrated on investigations with digital holographic microscopy on both technical amplitude objects and cytological pure phase objects.
Proceedings of SPIE - The International Society for Optical Engineering 06/2007; 6633. DOI:10.1117/12.727784 · 0.20 Impact Factor
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