Coherence-controlled holographic microscope (CCHM) combines off-axis holography and an achromatic grating interferometer allowing for the use of light sources of arbitrary degree of temporal and spatial coherence. This results in coherence gating and strong suppression of coherent noise and parasitic interferences enabling CCHM to reach high phase measurement accuracy and imaging quality. The achievable lateral resolution reaches performance of conventional widefield microscopes, which allows resolving up to twice smaller details when compared to typical off-axis setups. Imaging characteristics can be controlled arbitrarily by coherence between two extremes: fully coherent holography and confocal-like incoherent holography. The basic setup parameters are derived and described in detail and experimental validations of imaging characteristics are demonstrated.

Fresnel Incoherent Correlation Holography (FINCH) allows digital reconstruction of incoherently illuminated objects from intensity records acquired by a Spatial Light Modulator (SLM). The article presents wave optics model of FINCH, which allows analytical calculation of the Point Spread Function (PSF) for both the optical and digital part of imaging and takes into account Gaussian aperture for a spatial bounding of light waves. The 3D PSF is used to determine diffraction limits of the lateral and longitudinal size of a point image created in the FINCH set-up. Lateral and longitudinal resolution is investigated both theoretically and experimentally using quantitative measures introduced for two-point imaging. Dependence of the resolving power on the system parameters is studied and optimal geometry of the set-up is designed with regard to the best lateral and longitudinal resolution. Theoretical results are confirmed by experiments in which the light emitting diode (LED) is used as a spatially incoherent source to create object holograms using the SLM.

Transmitted-light coherence-controlled holographic microscope (CCHM) based on an off-axis achromatic interferometer allows us to use light sources of arbitrary degree of temporal and spatial coherence. Besides the conventional DHM modes such as quantitative phase contrast imaging and numerical 3D holographic reconstruction it provides high quality (speckle-free) imaging, improved lateral resolution and optical sectioning by coherence gating. Optical setup parameters and their limits for a technical realization are derived and described in detail. To demonstrate the optical sectioning property of the microscope a model sample uncovered and then covered with a diffuser was observed using a low-coherence light source.

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI− phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre – an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

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Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells’ activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells’ membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

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