Lensless digital holographic microscopy (LDHM), as one of key computational microscopy techniques, performs high throughput in silico imaging. Numerical propagation of digitally recorded in-line Gabor holograms allows for accessing both amplitude (absorption) and phase (refraction) contrast, devoid of microscope objective limitations, e.g., in depth of field and field of view. The in-line coherent holographic framework induces inherent twin image errors and various coherent artifacts, however. The signal-to-noise ratio of reconstructed holograms additionally deteriorates due to low photon budget environment, favorable in terms of time-lapse photostimulation-free bioimaging of live cells. In this contribution, we discuss several techniques for minimization of LDHM reconstruction errors, with the emphasis on simultaneous validation of phase measurement fidelity via calibration target testing. Crafted using two-photon polymerization, our targets enable large field of view phase imaging verification and assess the efficacy of the 3D printing method itself. We also present bio-applications of enhanced LDHM in dynamic (migrating neural cells) and static (brain tissue slices) scenarios.
This research presents the WUTScope, a novel interferometric microscope developed by the Quantitative Computational Imaging group at Warsaw University of Technology. This system, leveraging Quantitative Phase Microscopy and Optical Diffraction Tomography, provides insightful three-dimensional reconstructions of the refractive index distribution in semi-transparent objects. The WUTScope is distinguished by its compact design and capability to operate under partially coherent illumination, using polarization diffraction gratings for beam splitting and recombination. This approach allows for efficient phase shifting and reduces speckle noise, enhancing image signal-to-noise ratio. The system's achromatic nature, due to the identical optical paths of the diffraction orders, facilitates the use of less coherent light sources, a distinct advantage over traditional holographic methods. Its effectiveness is demonstrated through tomographic reconstruction of a 3D-printed brain sample and analysis of refractive index changes in HeLa cells' lipid droplets, revealing the impact of cholesterol accumulation.
Quantitative phase imaging techniques (QPI) enable to observe transparent samples with high contrast and with quantitative information about their optical thickness. Among the vast family of QPI methods, two of them: (1) transport of intensity equation (TIE) and (2) digital in-line holographic microscopy (DIHM), rely on retrieving phase from several images collected with different defocus. In this work, we preliminarily investigate, mainly numerically under simulated conditions, the differences between those two methods, showing that TIE is more suitable for imaging lower-frequency objects with small defocus distance difference between collected images (around several micrometers). On the other hand, DIHM performs better for higher frequency objects and images collected with larger defocus distance difference (hundreds of micrometers). Moreover, our results show that DIHM achieves better results, when all collected images are relatively far from focal plane (in the millimeter range rather than in micrometer range), while for TIE this parameter does not have a significant influence onto the retrieved phase.
This study introduces a methodology for quantitative assessment of phase measurement sensitivity in lensless digital holographic microscopy (LDHM) setups, incorporating an immersion medium between the object and the detector. Utilizing two setup configurations, we systematically investigated the influence of immersion conditions on phase accuracy, numerical reconstruction, and twin-image artefacts. Employing the Angular Spectrum and iterative Gerchberg-Saxton methods, we reconstructed phase maps for varying immersion thicknesses. Results demonstrate that immersion medium has minimal influence on phase sensitivity but significantly reduces twin-image artifacts when in direct contact with the object, providing valuable insights for developing LDHM biological applications.
KEYWORDS: Digital holography, Microscopy, Holography, Biological imaging, Printing, Design and modelling, Spatial resolution, Phase imaging, Two photon polymerization, Refractive index
Lensless digital holographic microscopy (LDHM) as a rapidly developing technique of microscale objects investigation requires precise metrological verification examining the accuracy of the novel solutions. LDHM method finds numerous applications in biological specimen and technical objects studies that show diverse optical and geometrical characteristics. Hereby, we introduce custom-designed resolution targets providing an extended quantitative experimental examination of LDHM imaging capabilities in its uniquely wide field-of-view. Proposed structures, manufactured via two-photon polymerization, incorporate the axial thickness and refractive index variation to the qualitative and quantitative imaging analysis.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.