Many biological researches require observation of the sample at a sub-micrometer scale. However, most biological
samples are transparent, and thus are barely visible in conventional transmission microscopy. Techniques like
interference contrast or oblique illumination permit to record an improved contrast, but are useful for morphological
studies only, because the interaction of incoherent, non-polarized and polychromatic illumination with matter is very
complex, so that the recorded contrast cannot be linked to local physical properties of the sample, as for example the
index of refraction. We have developed a diffractive tomographic microscope, which permits the observation of
unstained-, transparent samples in 3-D. This device is based on a combination of microholography, which records the
field diffracted by the specimen in both amplitude and phase using a high numerical aperture objective and a phase
stepping interferometer, with a variable illumination of the sample (tomography) using a high numerical aperture
condenser. The successive holograms are numerically recombined in the Fourier space, and the reconstruction of the
specimen index of refraction distribution is based on the first Born approximation for weakly diffractive samples.
Examples of biological specimens observed with this technique are given, and possible evolutions of the instrument are
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.
A prototype of a tomographic microscope has bene realized, which uses phase-shifting holography to sense the wave diffracted by an object. The observed object is successively illuminated by a series of about 1000 plane waves. Each diffracted wave yields a part of the frequency representation of the object, and superposition of these parts yields a 3D frequency representation. An inverse Fourier transform yields the 3D spatial representation, from which sections, projections or stereoscopic images can be extracted. This prototype has been used to image a variety of biological samples. Images show a resolution limit of about a quarter of a wavelength and a depth of field of about 40 microns.
Important factors for the understanding of image characteristics include horizontal and vertical resolution, ability to image horizontal and/or vertical surfaces, ability to distinguish variations of refractive index from variations of absorptivity. These and other imaging characteristics of the tomographic microscope are discussed on the basis of a set of images of biological samples. The connection between characteristics of a three-dimensional image and its frequency representation is explained. Influence of an object's characteristics (thickness, refractive index, ...) on image quality is described. Possible improvements and their impact on image quality is also discussed.