| • | Introduction |
| • | The point spread function |
| • | Deconvolution by adaptive inverse modelling - the TILLvisDECO approach |
| • | Requirements for optimal processing of image data in deconvolution experiments |
| • | Equipment needed for high quality deconvolution results |
| • | References |
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Deconvolution and TILLvisDECO package
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Requirements for optimal processing of image data in deconvolution experiments
To achieve the best possible result, the deconvolution of fluorescence images requires a high level of precision and accuracy at each stage of data acquisition and processing. 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)
- Use of water immersion objective of highest NA (1.3) and quality. Oil immersion objectives are not recommended. This is because spherical aberrations are dependent on the nature of the optical path, which of course changes with focal position (as the objective front lens moves relative to the coverslip the thickness of the immersion fluid layer changes as well as the positional depth of the lens focus within the specimen). The change in spherical aberration (and hence PSF) is greatest when the mismatch in refractive index is maximised by using oil as the immersion fluid
- 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 TILL Photonics high stability Polychrome light source)
- Reduction of photobleaching (optimised in the TILL Photonics imaging system due to precise control over wavelength, illumination and CCD exposure)