Holographic Tomography (HT) is currently the most common tool in biomedical applications, which provides 3D distribution of refractive index (RI) and dry mass (dm) in biological cells and tissues. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis, which is now considered as important biophysical feature to be applied for future applications in digital phase histopathology or liquid biopsy, e.g., supported by deep learning procedures. RI investigations at cellular level may be performed based on living or chemically fixed cells. In this work we had focused on investigations of the influence of PFA fixation process on RI in cellular organelles: nucleus, nucleolus, nucleoplasm and cytoplasm. The research was carried out on the NRK-52E rat kidney epithelial cell line. Epithelial cells were chosen for the experiment due to the fact that they belong to the basic building cells of the human body and are often used for normal and extended toxicity experiments with nano particles. A commercial Tomocube HT-1S device was used for the research. RI of live and fixed cells was measured. Changes in RI after fixation are observed at the order of 10-3. The RI values decrease, and the mass density is found reduced in the fixated cells. In conclusion, our results demonstrate the need of standard procedures for the preparation of biological samples for phase tomographic measurements in the case of chemically fixed cells. Moreover the requirement for conversion factors to retrieve accurate RI and dm values that are comparable to living cells is discussed.
Limited angle holographic tomography (LAHT) is currently the most common tool in biomedical applications of 3D quantitative phase imaging. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis and provides highly accurate RI values in the full measurement volume. Recently several new systems have been built in laboratories and new devices have been released into the market. All of them apply algorithms and processing paths which significantly influence correctness of the results. In our work we perform study of the selected LAHT systems and compare their 3D metrological features and other functional parameters.
In holographic tomography (HT), the 3D refractive index distribution within weakly-scattering, phase-only biological object is retrieved. This key property of the technique is one of its most significant strengths compared to labelling-based methods of cell analysis such as fluorescence microscopy. As a consequence, however, it is required to acquire a set of holograms at several viewing directions, which hinders the measurement speed. In this paper we explore the prospect of multiplexing projections in order to decrease the number of scanning positions required for the full measurements. The presented analysis is based on experimental data acquired in a limited-angle holographic tomography system and emulates the performance of a spatial-light-modulator-based system in which multiple projections may be acquired simultaneously by generating a distribution of multiple point sources in the Fourier plane of the condenser lens. For this reason, the increase in acquisition speed strictly depends on the number of multiplexed holograms and results in decreased reconstruction quality. The performance of the system is demonstrated and analyzed with biological objects - human keratinocyte cells.
In this paper we propose and implement the systematic approach to investigation of skin tissue based on sequential determination of refractive index and dry mass density for representative skin cells (3T3 fibroblasts and HaCaT keratinocytes) during their growing in a culture from single cells up to formation of a single layer. The main measurement tool is digital holographic microscopy supported by reference measurements performed by holographic tomography. The results of the measurement are presented and discussed.