3-D optical fluorescent microscopy becomes now an efficient tool for volume investigation of living biological samples.
Developments in instrumentation have permit to beat off the conventional Abbe limit, in any case the recorded image
can be described by the convolution equation between the original object and the Point Spread Function (PSF) of the
acquisition system. If the goal is 3-D quantitative analysis, whether you improve the instrument capabilities, or (and)
you restore the data. These last is until now the main task in our laboratory. Based on the knowledge of the optical
Transfer Function of the microscope, deconvolution algorithms were adapted to automatic determine the regularisation
threshold in order to give less subjective and more reproducible results. The PSF represents the properties of the image
acquisition system; we have proposed the use of statistical tools and Zernike moments to describe a 3-D system PSF and
to quantify the variation of the PSF. This first step toward standardization is helpful to define an acquisition protocol
optimizing exploitation of the microscope depending on the studied biological sample.
We have pointed out that automating the choice of the regularization level; if it facilitates the use, it also greatly
improves the reliability of the measurements. Furthermore, to increase the quality and the repeatability of quantitative
measurements a pre-filtering of images improves the stability of deconvolution process. In the same way, the PSF pre-filtering
stabilizes the deconvolution process. We have shown that Zernike polynomials can be used to reconstruct
experimental PSF, preserving system characteristics and removing the noise contained in the PSF.
Fluorescent microscopes suffer from limitations; photobleaching and phototoxicity effects, or influence of the sample
optical properties to 3-D observation. Amplitude and phase of the object can be reached with optical tomography based
on a combination of microholography with a tomographic illumination. So indices cartography of the specimen can be
obtained, and combined with fluorescence information it will open new possibilities in 3-D optical microscopy.