Microscopic images of biological phase specimens of various optical thickness, acquired under off-axis illumination and apodized/conventional phase-contrast are compared. The luminance profiles in appropriately filtered apodized phase-contrast images compare well with those in the original off-axis illumination images. The two unfiltered image types also yield similar results in terms of quasi-three-dimensional surface (pseudo-relief) rendering, and thus are comparable in terms of the information contents (optical thickness map). However, the overall visual impression is very different as the visual cues to depth structure are present in the off-axis illumination images only. The comparison demonstrated in the present paper was made possible owing to apodization, which substantially reduces the "halo"/shade-off artifacts in the phase-contrast images. The results imply the possibility of combining the off-axis illumination and apodized phase-contrast imaging to examine specimens of medium optical thickness, in which the phase visualization capability of the two imaging modes substantially overlaps (e.g., larger cells or cell clusters).

• MeSH
• algoritmy * MeSH
• interpretace obrazu počítačem metody   MeSH
• mikroskopie fázově kontrastní metody   MeSH
• osvětlení metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• statistika jako téma MeSH
• vylepšení obrazu metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The paper deals with quantitative phase imaging of two-height-level surface reliefs. The imaging is considered to be a linear system and, consequently, the Fourier transform of the image is the product of the Fourier transform of a 2D function characterizing the surface and a specific 2D coherent transfer function. The Fourier transform of functions specifying periodic surface reliefs is factorized into two functions similar to lattice and structure amplitudes in crystal structure analysis. The approach to the imaging process described in the paper enables us to examine the dependence of the phase image on the surface geometry. Theoretical results are verified experimentally by means of a digital holographic microscope.

• MeSH
• design vybavení MeSH
• Fourierova analýza MeSH
• holografie metody   MeSH
• interferometrie metody   MeSH
• křemík chemie   MeSH
• krystalizace MeSH
• mikroskopie atomárních sil metody   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• mikroskopie fázově kontrastní metody   MeSH
• počítačové zpracování obrazu MeSH
• povrchové vlastnosti MeSH
• statistické modely MeSH
• světlo MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• křemík MeSH

Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.

• MeSH
• časové faktory MeSH
• interferenční mikroskopie metody   statistika a číselné údaje   MeSH
• kovové nanočástice ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• mikroskopie fázově kontrastní metody   statistika a číselné údaje   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• nanotechnologie MeSH
• nanotrubičky ultrastruktura   MeSH
• optické jevy MeSH
• počítačová simulace MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny metabolismus   MeSH
• světlo MeSH
• tubulin metabolismus   MeSH
• zlato MeSH
• zobrazování trojrozměrné metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ase1 protein, S pombe MeSH Prohlížeč
• PRC1 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• tubulin MeSH
• zlato MeSH

Quantitative Phase Imaging is becoming an important tool in the objective evaluation of cellular responses to experimental treatment. The technique is based on interferometric measurements of the optical thickness of cells in tissue culture reporting on the distribution of dry mass inside the cells. As the measurement of the optical thickness is interferometric, it is not subjected to the Abbe resolution limit, and the use of an incoherent-light source further increases the accuracy practically achieving 0.93nm in optical path difference corresponding to 4.6 femtograms/μm2. Holographic mode reduces the exposure in comparison to phase-shifting or phase-stepping interference microscopy and allows observation of faster dynamics. An attractive application is in the development of novel anti-cancer drugs and there is an important potential for pretesting chemotherapeutic drugs with biopsy material for personalized cancer treatment. The procedure involves the preparation of live cells in tissue culture, seeding them into suitable observation chambers, and time-lapse recording with an adjusted microscope. Subsequent image processing and statistical analysis are essential last steps producing the results, which include rapid measurements of cell growth in terms of dry-mass increase in individual cells, speed of cell motility and other dynamic morphometric parameters.

• Klíčová slova
• Cancer cells, Holographic quantitative phase imaging, Light microscopy, Measurements of cell growth and motility,
• MeSH
• holografie * metody   MeSH
• mikroskopie metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• pohyb buněk MeSH
• protinádorové látky * farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protinádorové látky * MeSH

Optical metasurfaces have emerged as a new generation of building blocks for multifunctional optics. Design and realization of metasurface elements place ever-increasing demands on accurate assessment of phase alterations introduced by complex nanoantenna arrays, a process referred to as quantitative phase imaging. Despite considerable effort, the widefield (nonscanning) phase imaging that would approach resolution limits of optical microscopy and indicate the response of a single nanoantenna still remains a challenge. Here, we report on a new strategy in incoherent holographic imaging of metasurfaces, in which unprecedented spatial resolution and light sensitivity are achieved by taking full advantage of the polarization selective control of light through the geometric (Pancharatnam-Berry) phase. The measurement is carried out in an inherently stable common-path setup composed of a standard optical microscope and an add-on imaging module. Phase information is acquired from the mutual coherence function attainable in records created in broadband spatially incoherent light by the self-interference of scattered and leakage light coming from the metasurface. In calibration measurements, the phase was mapped with the precision and spatial background noise better than 0.01 and 0.05 rad, respectively. The imaging excels at the high spatial resolution that was demonstrated experimentally by the precise amplitude and phase restoration of vortex metalenses and a metasurface grating with 833 lines/mm. Thanks to superior light sensitivity of the method, we demonstrated for the first time to our knowledge the widefield measurement of the phase altered by a single nanoantenna while maintaining the precision well below 0.15 rad.

• Klíčová slova
• Pancharatnam−Berry phase, Plasmonic metasurfaces, holographic microscopy, plasmonic nanoantennas, quantitative phase imaging, vortex metalens,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope.

• Klíčová slova
• Aberration compensation, cell tracking, digital image processing, quantitative phase imaging, real-time holography,
• MeSH
• algoritmy MeSH
• fibrosarkom patologie   MeSH
• holografie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• krysa rodu Rattus MeSH
• mikroskopie fázově kontrastní metody   MeSH
• nádorové buněčné linie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We demonstrate the formation of the near field with non-trivial phase distribution using surface plasmon interference devices, and experimental quantitative imaging of that phase with near-field phase microscopy. The phase distribution formed with a single device can be controlled by the polarization of the external illumination and the area of the device assigned to the object wave. A comparison of the experimental data to a numerical electromagnetic model and an analytical model assigns the origin of the near-field phase to the out-of-plane electric component of surface plasmon polaritons, and also verifies the predictive power of the models. We demonstrate a formation of near-field plane waves with different propagation directions on a single device, or even simultaneously at distinct areas of a single device. Our findings open the way to the imaging and tomography of phase objects in the near field.

• Klíčová slova
• SNOM, SPP waves, interference nanostructures, near-field, phase imaging,
• Publikační typ
• časopisecké články MeSH

We report on recent developments that enable megahertz hard X-ray phase contrast imaging (MHz XPCI) experiments at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL facility (EuXFEL). We describe the technical implementation of the key components, including an MHz fast camera and a modular indirect X-ray microscope system based on fast scintillators coupled through a high-resolution optical microscope, which enable full-field X-ray microscopy with phase contrast of fast and irreversible phenomena. The image quality for MHz XPCI data showed significant improvement compared with a pilot demonstration of the technique using parallel beam illumination, which also allows access to up to 24 keV photon energies at the SPB/SFX instrument of the EuXFEL. With these developments, MHz XPCI was implemented as a new method offered for a broad user community (academic and industrial) and is accessible via standard user proposals. Furthermore, intra-train pulse diagnostics with a high few-micrometre spatial resolution and recording up to 128 images of consecutive pulses in a train at up to 1.1 MHz repetition rate is available upstream of the instrument. Together with the diagnostic camera upstream of the instrument and the MHz XPCI setup at the SPB/SFX instrument, simultaneous two-plane measurements for future beam studies and feedback for machine parameter tuning are now possible.

• Klíčová slova
• European XFEL, X-ray phase contrast imaging, indirect MHz X-ray detector, megahertz sampling, pulse-resolved imaging,
• Publikační typ
• časopisecké články MeSH

Spatial phase modulation in an imaging interferometer is utilized in surface plasmon resonance (SPR) based sensing of liquid analytes. In the interferometer, a collimated light beam from a laser diode irradiating at 637.1 nm is passing through a polarizer and is reflected from a plasmonic structure of SF10/Cr/Au attached to a prism in the Kretschmann configuration. The beam passes through a combination of a Wollaston prism, a polarizer and a lens, and forms an interference pattern on a CCD sensor of a color camera. Interference patterns obtained for different liquid analytes are acquired and transferred to the computer for data processing. The sensing concept is based on the detection of a refractive index change, which is transformed via the SPR phenomenon into an interference fringe phase shift. By calculating the phase shift for the plasmonic structure of SF10/Cr/Au of known parameters we demonstrate that this technique can detect different weight concentrations of ethanol diluted in water, or equivalently, different changes in the refractive index. The sensitivity to the refractive index and the detection limit obtained are -278 rad/refractive-index-unit (RIU) and 3.6 × 10 - 6 RIU, respectively. The technique is demonstrated in experiments with the same liquid analytes as in the theory. Applying an original approach in retrieving the fringe phase shift, we revealed good agreement between experiment and theory, and the measured sensitivity to the refractive index and the detection limit reached -226 rad/RIU and 4.4 × 10 - 6 RIU, respectively. These results suggest that the SPR interferometer with the detection of a fringe phase shift is particularly useful in applications that require measuring refractive index changes with high sensitivity.

• Klíčová slova
• Kretschmann configuration, aqueous solutions of ethanol, fringe phase shift, imaging interferometer, sensitivity, spatial phase modulation, surface plasmon resonance,
• Publikační typ
• časopisecké články MeSH

The 255th ENMC workshop on Muscle Imaging in Idiopathic Inflammatory myopathies (IIM) aimed at defining recommendations concerning the applicability of muscle imaging in IIM. The workshop comprised of clinicians, researchers and people living with myositis. We aimed to achieve consensus on the following topics: a standardized protocol for the evaluation of muscle images in various types of IIMs; the exact parameters, anatomical localizations and magnetic resonance imaging (MRI) techniques; ultrasound as assessment tool in IIM; assessment methods; the pattern of muscle involvement in IIM subtypes; the application of MRI as biomarker in follow-up studies and clinical trials, and the place of MRI in the evaluation of swallowing difficulty and cardiac manifestations. The following recommendations were formulated: In patients with suspected IIM, muscle imaging is highly recommended to be part of the initial diagnostic workup and baseline assessment. MRI is the preferred imaging modality due to its sensitivity to both oedema and fat accumulation. Ultrasound may be used for suspected IBM. Repeat imaging should be considered if patients do not respond to treatment, if there is ongoing diagnostic uncertainty or there is clinical or laboratory evidence of disease relapse. Quantitative MRI is established as a sensitive biomarker in IBM and could be included as a primary or secondary outcome measure in early phase clinical trials, or as a secondary outcome measure in late phase clinical trials. Finally, a research agenda was drawn up.

• MeSH
• biologické markery MeSH
• kosterní svaly patologie   MeSH
• lidé MeSH
• magnetická rezonanční tomografie metody   MeSH
• myozitida * diagnóza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• kongresy MeSH
• Geografické názvy
• Nizozemsko MeSH
• Názvy látek
• biologické markery MeSH