Experimentální biofotonika
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- ItemTomographic microscope for low-coherent quantitative phase imaging(SPIE, 2025-02-17) Špaček, Matěj; Dvořák, Vladislav; Pachl, Přemysl; Buček, Jozef; Neumanová, Anna; Dostál, ZbyněkA novel tomographic microscope setup utilizing the principle of Holographic Incoherent-light-source Quantitative Phase Imaging is introduced. This setup combines the advantages of achromatic off-axis holography with the ultrafast operation of a digital micromirror device-based tomographic illuminator. The imaging theory is explained, and the optical design of the microscope is described. The functionality of the microscope modules is demonstrated experimentally using a light source of limited spatial coherence.
- ItemOn quantitativeness of diffraction-limited quantitative phase imaging(AIP Publishing, 2024-12-01) Bouchal, Zdeněk; Bouchal, Petr; Chmelíková, Tereza; Fiurásek, JaromírQuantitative phase imaging (QPI) has advanced by accurately quantifying phase shifts caused by weakly absorbing biological and artificial structures. Despite extensive research, the diffraction limits of QPI have not been established and examined. Hence, it remains unclear whether diffraction-affected QPI provides reliable quantification or merely visualizes phase objects, similar to phase contrast methods. Here, we develop a general diffraction phase imaging theory and show that it is intrinsically connected with Rayleigh's resolution theory. Our approach reveals the entanglement of phases under restoration, imposing diffraction bounds on spatial phase resolution and, unexpectedly, on phase accuracy. We prove that the phase accuracy depends on the size, shape, and absorption of objects forming the sample and significantly declines if the object size approaches the Rayleigh limit (a relative phase error of -16% for an Airy disk-sized object with low phase shift). We show that the phase accuracy limits can be enhanced at the cost of deteriorated phase resolution by attenuating the sample background light. The QPI diffraction limits are thoroughly examined in experiments with certified phase targets and biological cells. The study's relevance is underscored by results showing that the phase accuracy of some structures is lost (a relative phase error of -40%) even though they are spatially resolved (a phase visibility of 0.5). A reliable procedure is used to estimate phase errors in given experimental conditions, opening the way to mitigate errors' impact through data post-processing. Finally, the phase accuracy enhancement in super-resolution QPI is discovered, which has not been previously reported.
- ItemMultimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis(PLOS, 2015-03-24) Balvan, Jan; Křížová, Aneta; Gumulec, Jaromír; Raudenská, Martina; Sládek, Zbyšek; Sedláčková, Miroslava; Babula, Petr; Svobodová, Markéta; Kizek, René; Chmelík, Radim; Masařík, MichalIdentification 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.
- ItemSilicon waveguides with graphene: coupling of waveguide mode to surface plasmons(IOP Publishing, 2020-08-18) Čtyroký, Jiří; Petráček, Jiří; Kuzmiak, Vladimír; Kwiecien, Pavel; Richter, IvanSilicon waveguides with graphene layers have been recently intensively studied for their potential as fast and low-power electro-optic modulators with small footprints. In this paper we show that in the optical wavelength range of 1.55 mu m, surface plasmons supported by the graphene layer with the chemical potential exceeding similar to 0.5 eV can couple with the guided mode of the silicon waveguide and affect its propagation. On the other hand, this effect might be possibly utilized in technical applications like a very low-power amplitude modulation, temperature sensing, etc.
- ItemMultimode fibre: Light-sheet microscopy at the tip of a needle(Springer Nature, 2015-12-14) Plöschner, Martin; Kollárová, Věra; Dostál, Zbyněk; Nylk, Jonathan; Barton-Owen, Thomas; Ferrier, David E.K.; Chmelík, Radim; Dholakia, Kishan; Čižmár, TomášLight-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.