Novel hardware
Current projects
Multimodal microscopic platforms
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!
Dual-mode Single-Shot Digital Holographic Microscope
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.
If you want to know more about this research, please read the article here!
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).
Three-dimensional super-resolution light microscopy of thick, unprocessed biological samples
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!
Polarization-sensitive microscope
The insertion of a Fresnel biprism (FB) in the image space of a light microscope potentially turns any commercial system into a DHM system enabling QPI with the five desired features in QPI simultaneously: high temporal sensitivity, high speed, high accuracy, high spatial resolution, and polarization sensitivity. To the best of our knowledge, this is the first FB-based DHM system providing these five features altogether.
Some biological organelle and extracellular matrix components are birefringent; that is, the brightness of their image varies with the polarization state of the illuminating beam of light. To provide polarization-sensitive measurements (e.g., a PS-DHM system), the sample imaged by our FB-based common-path DHM system is now illuminated with linear polarized light whose plane-of-vibration is varied in a controlled way.
Fig. 5 Quantitative phase images of human U87 glioblastoma cells using the proposed PS DHM system: (a) Optical configuration of the simplified PS FB-based DHM system; (b) 2D pseudocolor normalized phase maps at different polarization states (0 and 130 degrees); and (c) ordinary and extraordinary phase maps and the retardance map.
The polarization state of the illuminating beam changes by rotating a linear polarizer inserted before the sample holder, see Fig. 5(a). Note that one can measure the birefringence retardance by illuminating the sample with linearly polarized light and recording the transmitted wavefront without any further analyzer polarizer. Again, we image U87 glioblastoma cells since it has been demonstrated that the glioblastoma cells present higher anisotropy than normal cells, validating the proper use of these cells for PS imaging. Figure 5(b) shows the 2D phase information from U87 glioblastoma cells illuminated with two different polarization angles, 0 and 130 degrees. In these maps, the areas that are enclosed by the dashed lines reveal details quite different. Some features clearly for the 130-deg phase map are hardly seen for the 0-deg phase map which confirms the PS behavior of the glioblastoma cells. To obtain the retardance map24 (e.g., Δϕ = ϕe - ϕo = 2π (ne - no)d/λ being ne and no the refractive indexes of the extraordinary and ordinary waves, and d the thickness of the cells), we obtain the maximum and minimum values of the whole set reconstructed phase image at each pixel. Example phase maps for the extraordinary (e.g., maximum) and the ordinary (e.g., minimum) behavior are displayed in Fig 5(c). The subtraction of these unwrapped maps provides the retardance image, also displayed in Fig. 5(c). Note that on the retardance image, the contrast created is specific to the PS behavior of the sample since the cells and parts without any anisotropy are no longer visible on this image. The color scale bar in the retardance image corresponds to retardance values between 0.4 and 0.9 rad. Note that our phase sensitivity is three orders of magnitude smaller (0.0003 rad), guaranteeing that any divergence on the retardance image is due to differences in the anisotropy of the samples.
A more rigorous research study is currently being under research to analyze more glioblastoma cells and other samples and validating the polarization-sensitive capability achieved by our FB-based DHM system.
Part of this project has been published in JBO. More results will come soon!
Compact digital holographic microscopes
Fresnel-based DHM
C. Hayes-Round, B. Bogue-Jimenez, J. Garcia-Sucerquia, O. Skalli, and A. Doblas, “Advantages of Fresnel biprism-based Digital Holographic Microscopy in Quantitative Phase Imaging,” J. Biomed. Opt. 25(8), 086501 (2020), doi: 10.1117/1.JBO.25.8.086501.
Shearing-based Super-resolution DHM
T. O’Connor, A. Doblas, and B. Javidi, “,” Opt. Letters 44(9), 2326-2329 (2019).
Ronchi-based single-shot inline DHM
S. Hossein, S. Yaghoubi, S. Ebrahimi, M. Dashtdar, A. Doblas, and B. Javidi, “Common-path, single-shot phase-shifting digital holographic microscopy using a Ronchi ruling,” Appl. Phys. Letter 114, 183701 (2019).
Past research projects
Tunable 3D Structured Illumination Microscopy
Related works:
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).
A. Doblas, S. Bedoya, and C. Preza, “Wollaston prism-based structured illumination microscope with tunable-frequency,” Appl. Opt. 58(7), B1-B8(2019).
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).
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).
Supercontinuum parallel OCT
If you want to know more abour this research, click here!
Accurate and diffraction-limited digital holographic microscopes
Related works:
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).
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).
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).
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).
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).
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).
Phase-shifting by means of an electronically tunable lens: quantitative phase imaging of biological specimens with digital holographic microscopy
Related work:
C. Trujillo, A. Doblas, G. Saavedra, M. Martinez-Corral and J. Garcia-Sucerquia, “Phase-shifting by means of an electronically tunable lens: quantitative phase imaging of biological specimens with digital holographic microscopy,” Opt. Lett., 41(7), 1416-1419 (2016).
If you want to read the article of this research, click here!
Static Three-dimensional widefield microscopy
Related works:
M. Martínez-Corral, A. Doblas, E. Sánchez-Ortiga, J. Sola-Pikabea and G. Saavedra, “Static axial scanning in 3D microscopy through electrically controlled liquid lens,” SPIE Newsroom (2015). doi: 10.1117/2.1201503.005832.
2. M. Martínez-Corral, P.-Y. Hsieh, A. Doblas, E. Sánchez-Ortiga, G. Saavedra and Y.-P. Huang, “Fast axial-scanning widefield microscopy with constant magnification and resolution,” J. Display Technol 11, 913-920 (2015). doi: 10.1109/JDT.2015.2404347.
Wavefront Encoding and PSF Engineering in widefield microscopy
Related works:
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).
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).
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).