Lateral resolution plays a crucial role in the effectiveness of microscopy systems, such as Digital Holographic Microscopes (i.e., DHM from now on), determining their ability to discern fine details and structures within samples, thus directly impacting the clarity and accuracy of the imaged objects. Defined as the minimum distance at which two distinct points in an object plane are still imaged separately the Abbe diffraction principle governs the lateral resolution. This principle correlates the Numerical Aperture (NA) of the microscope objective (MO) with the wavelength (λ) of the light source. However, various factors, such as system misalignment and optical imperfections, can compromise this parameter. Although in typical microscope systems, their lateral resolution allows observing certain biological structures like red blood cells or yeast spores, it falls short when attempting to visualize smaller organelles or large proteins. Addressing this limitation by altering the recording wavelength or increasing the NA of the MO can lead to undesirable consequences such as phototoxicity or reduced field-of-view (FOV). Numerous techniques have been proposed to overcome these challenges and enhance resolution in DHM, with Structured Illumination (SI) emerging as a promising approach. Despite its potential, SI in DHM has predominantly been applied to transmission samples, leaving a significant gap in research when it comes to reflection samples. This research project, which was the Master dissertation from Sofia Obando-Vasquez in 2024, presents advancements in Digital Holographic Microscopy (DHM) by integrating Structured Illumination (SI) techniques. Two novel optical setups are proposed for SI-DHM for both reflection and transmission scenarios. These setups offer distinct advantages over conventional SI-DHM systems found in literature, primarily in the ease of adjusting the frequency of the periodic pattern required for SI. Moreover, the proposed SI-DHM systems boast an enlarged field of view, facilitating efficient examination of large samples without requiring sequential time-lapse imaging

This project is currently going – stay tuned for its results!

 Despite the emergence of various methods for Mueller matrix recovery, achieving complete volumetric Mueller matrix retrieval remains a challenge. An alternative approach that leverages in-line Gabor holography to comprehensively extract polarization information from volumetric samples is introduced in this context. The proposed polarization-sensitive in-line Gabor holographic setup enables the recovery of the complete Mueller matrix of three-dimensional (3D) samples after the numerical repropagation of the holographically rendered complex field to various sample planes. This proposal is validated using a calibrated birefringent polarization test target, a sample of Calcium Oxalate crystals, and a volumetric sample containing microplastics, providing the 3D measurement of polarimetric parameters such as diattenuation, polarizance, depolarization, and retardance. The results agree with those obtained through reference methods based on image-plane brightfield polarimetry. The in-line Gabor holographic system proposed is sensitive to the axial variations in polarimetric information within volumetric samples without any mechanical movement nor optical adjustments— an accomplishment that remains elusive to conventional image-plane reference methods and non-holographic/interferometric systems. These findings emphasize the versatility and potential of this alternative approach in recovering the intricate polarization characteristics of 3D specimens, offering the first in-line holographic Mueller imaging to the best of the authors’ knowledge. 

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Citation:

M. J. Lopera, M. Trusiak, A. Doblas, H. Ottevaere, and C. Trujillo, “Mueller-Gabor holographic microscopy,” Optics and Lasers in Engineering 178, 19191 (2024).

All imaging systems present a tradeoff between the imaging depth and the resolution limitation making it impossible to capture an image of thick samples. A multimodal microscopic platform will enable overcoming this tradeoff while imaging stained or unstained samples of different thicknesses. imaging techniques for retrieving three-dimensional (3D) specimen attributes over time (4D) at different imaging depths with a range of resolution capability and information specificity. The multimodal instrument is motivated by the medical imaging multi-modality paradigm where PET, MRI, and CT are used together for improved diagnosis. Synergism in the different imaging capabilities will be achieved by integrating the three microscopic techniques, enabling the simultaneous acquisition of multiple specimen’s information. The sample will be placed in the same platform to be imaged by any of the implemented imaging modalities. Using new computational imaging approaches, one could extract and fuse sample information from the different imaging modalities to create images with rich content of specimen features and attributes, improving the current understanding of the underlying sample and its dynamic processes. The benefit of integrating the multiple imaging modalities is imaging of samples with a range of thickness (thin-thick) with specificity at different resolutions, advancing research studies relevant to cancer, fertility, regenerative medicine, material sciences, and living plants among many others.

This project is currently in its early stages – stay tuned for more updates!

Common path DHM systems are the most robust DHM systems as they are based on self-interference and are thus less prone to external fluctuations. A common issue amongst these DHM systems is that the two replicas of the sample’s information overlay due to self-interference, makingthem only suitable for imaging sparse samples. This overlay has restricted the use of common-pathDHM systems in material science. The overlay can be overcome by limiting the sample’s field of viewto occupy only half of the imaging field of view or by using an optical spatial filter. In this work, wehave implemented optical spatial filtering in a common-path DHM system using a Fresnel biprism.We have analyzed the optimal pinhole size by evaluating the frequency content of the reconstructedphase images of a star target. We have also measured the accuracy of the system and the sensitivity tonoise for different pinhole sizes. Finally, we have proposed the first dual-mode common-path DHMsystem using a Fresnel biprism. The performance of the dual-model DHM system has been evaluatedexperimentally using transmissive and reflective microscopic samples. 

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If you want to download the alignment protocol,  please go to our alignment protocols!

Citation:

A. Doblas, C. Hayes-Rounds, R. Isaac, and F. Perez, “Single-shot 3D topography of transmissive and reflective samples with a dual-mode telecentric-based digital holographic microscope,” Sensors 22, 3793 (2022).

Available methods of optical microscopy do not enable 3D super-resolution (SR) imaging of thick (>50 μm) biological samples with phase-contrast methods. This limitation results in a significant gap in our understanding of dynamic changes occurring in the behavior and 3D shape of cells in unstained specimens. The proposed imaging system addresses the unmet need for 3D SR imaging in thick, unstained tissues. The PI proposes to develop an innovative, quantitative imaging system for unstained biological specimens that combines high imaging depth and SR capability along the lateral (xy) and axial (z) directions. The unique specific aim focuses on developing this imaging instrument and evaluating its performance using calibrated manufactured objects and relevant biological specimens such as human neuroblastoma cells on plastic or embedded in collagen gel, primary murine stem cells, and murine blastocysts. Dr. Doblas and her biologist collaborators (Drs. Abell and Skalli) will evaluate the proposed system through proof-of-concept studies and assess its potential to generate high-impact biological 3D imaging of thick specimens. The intellectual merit of the proposed imaging system includes advances in hardware and two computational methods, which have never been demonstrated. The hardware consists of the design of a DHM imaging system using an innovative structured illumination module. The first computational method will improve the axial resolution in the reconstructed phase images. Finally, the second computational method is a robust computational approach for reproducing accurate 3D, refractive index (RI) maps. Our proposed system is therefore based on the innovative combination of these three advances. The proposed 3D SR optical microscope will enable, for the first time, imaging of thick, unstained biological samples with subcellular accuracy. This novel capacity will significantly enhance the imaging infrastructure for biological and biomedical research, expanding our knowledge of cell behavior in 3D systems. 

This project is currently funded by NSF through an NSF CAREER. So, stay tuned for getting more information!

Related works:

1. H. Shabani, A. Doblas, G. Saavedra, and C. Preza, “Optical transfer function engineering using a tunable 3D structured illumination microscope,” Opt. Letters 44(7), 1560-1563 (2019).

2. A. Doblas, S. Bedoya, and C. Preza, “Wollaston prism-based structured illumination microscope with tunable-frequency,” Appl. Opt. 58(7), B1-B8(2019).

3. A. Doblas, H. Shabani, G. Saavedra, and C. Preza, “Tunable-frequency three-dimensional structured illumination microscopy with reduced data-acquisition,” Opt. Express 26(23), 30492-30505 (2018).

4. H. Shabani, A. Doblas, G. Saavedra, E. Sanchez-Ortiga and C. Preza, “Improvement of two-dimensional structured illumination microscopy with an incoherent illumination pattern” Appl. Optics 57(7), B92-B101 (2018).

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Related works:

1. A. Doblas, D. Hincapie-Zuluaga, G. Saavedra, M. Martínez-Corral and J. Garcia-Sucerquia, “Physical compensation of phase curvature in digital holographic microscopy by use of programmable liquid lens,” Appl. Opt., 54, 5229-5233 (2015).

2. A. Doblas, E. Sánchez-Ortiga, M. Martínez-Corral and J. Garcia-Surcerquia, “Study of spatial lateral resolution in off-axis digital holographic microscopy,” Opt. Commun. 352, 63-69 (2015).

3. A. Doblas, E. Sánchez-Ortiga, M. Martínez-Corral, G. Saavedra and J. Garcia-Surcerquia, “Accurate single-shot quantitative phase imaging of biological specimens with telecentric digital holographic microscopy,” J. Biomed. Opt. 19(4), 046022- (2014).

4. E. Sánchez-Ortiga, A. Doblas, G. Saavedra, M. Martínez-Corral and J. Garcia-Sucerquia, “Off-axis Digital Holographic Microscopy: practical design parameters for operating at diffraction limit,” Appl. Opt. 53(10), 2058-2066 (2014).

5. A. Doblas, E. Sánchez-Ortiga, M. Martínez-Corral, G. Saavedra, P. Andres, and J. Garcia-Sucerquia, “Shift-variant digital holographic microscopy: innacuracies in quantitative phase imaging,” Opt. Lett. 38(8), 1352-1354 (2013).

6. 26. E. Sánchez-Ortiga, Pietro Ferraro, M. Martínez-Corral, G. Saavedra and A. Doblas, “Digital holographic microscopy with pure-optical spherical phase compensation”, J. Opt. Soc. Am. A 28(7), 1410-1417(2011).

Related works:

1. N. Patwary, H. Shabani, A. Doblas, G. Saavedra, and C. Preza, “Experimental validation of a customized phase mask designed to enable efficient computational optical sectioning microscopy through wavefront encoding” Appl. Optics 56(9), D14-D23 (2017).

2. S. V. King, A. Doblas, N. Patwary, G. Saavedra, M. Martínez-Corral, and C. Preza, “Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy,” Appl. Optics 54, 8587-8595 (2015).

3. I. Escobar, G. Saavedra, M. Martínez-Corral, A. Calatayud and A. Doblas, “Shaded-Mask Filtering for Extended Depth-of-Field Microscopy,” J. Inf. Commun. Converg. Eng 11(2), 139-146, (2013).