Particle tracking and TILLvisTRAC package

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Optimising image collection for tracking applications

To achieve the best possible result, the tracking of fluorescence objects requires a high level of precision and accuracy at each stage of data acquisition. Errors can be minimised by attention to detail (some general points of consideration):

• Microscope must be free of stage drift
• Coverslips of correct and consistent thickness
• Focal positioning with sub-resolution accuracy (piezo driven, see our integrated Pifoc objective positioning unit)
• Always use an objective of the highest quality (fully corrected optics, excellent transmittance over a board range of wavelengths), highest numerical aperture
• Use of high quality dichroics and filters
• Tube length errors caused by a non-parafocal position of the CCD detector (this again increases spherical aberration)
• Use of a high quality scientific grade CCD camera with high quantum efficiency, large dynamic range, and low dark counts (refer to the Imago range of CCD cameras on this site)
• Magnification must be matched to the individual pixel size of the CCD detector according to the Nyquist criteria; in general: required Mag. = (2 x pixel size) / [(0.61 x lex) / NA]
• A uniform field of illumination (optimised over the area visible to the CCD detector) limited by a field stop iris encircling the CCD detector field of view
• Background correction of all images prior to processing for background, light source instability (minimised in the Agilent high stability Polychrome light source)
• Reduction of photo-bleaching (optimised in the Agilent imaging system due to precise control over wavelength, illumination and CCD exposure)