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3D Quantitative Imaging

This research activity aims to investigate an alternative approach for the morphological characterization of microscopic objects (biological or not biological) using a technology based on interferometric microscopy. This method (which does not use fluorescent probes, aspect of fundamental importance for biological applications) is based on holographic processing allowing improving the resolution and the contrast in order to obtain greater efficiency in the 3D reconstruction of the sample. A great advantage of the interferometric imaging is the possibility to three-dimensionally reconstruct the sample even if the acquired 2D image (hologram) is out of its focus plane, through a numerical re-focusing without any mechanical sample scanning. Furthermore, this technique allows to obtain quantitative information and to carry out several numerical analyzes (for example, estimation of an area, profiles along particular directions, estimating the volume of an object). This activity is based on the potential of this technique for morphological analysis of micrometric objects. Additionally, this technique allows to obtain information about the state of polarization of the sample (for examples: birefringence and dichroism).

Moreover, the interferometric imaging is very versatile, indeed it is possible to combine it with other characterization technique, such as Raman spectroscopy.

The ability to see detailed relationships between morphology and physiology in selected biological or not biological samples with sub-micrometric resolution in a non-destructive and non-invasive way and within a functional correlated context could be extremely important. With this aim, the second objective of this research activity is to improve the characterization technique without involving any harmful procedure that could alter the sample by developing an advanced optical technique able to provide simultaneously as many parameters as possible on sample under test. In particular, we aim to combine the Holographic imaging and the Raman spectroscopy to provide a simultaneously and complete characterization of living cells in term of morphology, motility and biochemical features. The development of the combined optical approach would give additional information, allowing accurate and non-destructive diagnosis of cell alterations. Our approach combines innovative physics to capture appropriate image/spectra with appropriate links to biomedical and clinical personnel for fast and accurate translation to clinical problems. Therefore, the potential scientific benefits include the generation of novel information through optical fingerprinting of cells and the application of that information as diagnostic tool.

Imaging by structured light and single pixel detection

On the other hand, the use of more or less complex light patterns and single pixel detection represents a very fast way to obtain images in comparison to all scan-based microscopies (confocal, two-photon, Raman microscopies etc.), where the desired resolution is directly proportional to the acquisition time. Since modern spatial light-modulators such as digital mirror devices (DMDs) can reach several tens of KHz in refresh rates, the imaging of living (i.e. moving) samples such as single cells can be obtained with very high resolution in relatively short acquisition times respect to pixel-by-pixel scanning. The use of orthogonal patterns as light probes can make the process even more efficient. We introduced new encoding methods of the phase of illuminating optical fields which allow to minimize, for a given resolution, the number of needed light probes.

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