Prof. Natan T. Shaked has received the

Chair (Cathedra) of Advanced Biomedical Optical Microscopy

in Tel Aviv University

Prof. Shaked was honored to give a Plenary Lecture in SPIE Photonics West, San Francisco, CA, USA, on 29 January 2023

Lecture title:
Deep2Deep: AI and deep learning for label-free 3D cell classification

https://spie.org/photonics-west/event/biophotonics-focus-ai-ml-dl-plenary/2656964

Prof. Shaked has been appointed as the Chair of the Department of Biomedical Engineering in Tel Aviv University (containing 15 research groups and ~500 undergraduate & graduate students).

Prof. Natan T. Shaked has been nominated to a Fellow in the SPIE.

[Link]

High-resolution 4D acquisition of freely swimming human sperm cells without staining

Gili Dardikman-Yoffe, Simcha K. Mirsky, Itay Barnea, and Natan T. Shaked

Abstract: We present a new acquisition method that enables high-resolution, fine-detail full reconstruction of the three-dimensional movement and structure of individual human sperm cells swimming freely. We achieve both retrieval of the three-dimensional refractive-index profile of the sperm head, revealing its fine internal organelles and time-varying orientation, and the detailed four-dimensional localization of the thin, highly-dynamic flagellum of the sperm cell. Live human sperm cells were acquired during free swim using a high-speed off-axis holographic system that does not require any moving elements or cell staining. The reconstruction is based solely on the natural movement of the sperm cell and a novel set of algorithms, enabling the detailed four-dimensional recovery. Using this refractive-index imaging approach, we believe we have detected an area in the cell that is attributed to the centriole. This method has great potential for both biological assays and clinical use of intact sperm cells.

Science Advances, Vol. 6, No. 15, eaay7619, 2020 [Link]

Download: [PDF, Supp Mat, Video 1, Video 2, Video 3, Video 4, Video 5]

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Rapid label-free refractive-index tomography of a sperm cell during free swim.

Congrats to Maysam Nasser for winning the prestigious Neuberger Foundation Fellowship for excellent PhD Arab Students!

Mayssam Nassir is a PhD student in the Department of Biomedical Engineering at Tel Aviv University. Mayssam received her BSc in the Department of Biomedical Engineering in Tel Aviv University (2011). She completed her MSc thesis on modeling the mechanical behavior of Mesenchymal Stem Cells based on phase-contrast microscopy method in the Department of Mechanical Engineering at Tel Aviv University (2016). Mayssam filled a position of Mechanical Engineer in a growing startup for a few years. Then, she started her PhD  in the OMNI group, studying the mechanical behavior of sperm cells through computerized modeling techniques and methods.

Prof. Shaked has been promoted to a Full Professor Rank.

New paper in Advanced Science, 2016:

Rapid three-dimensional refractive-index imaging of live cells in suspension without labeling using dielectrophoretic cell rotation

Mor Habaza, Michael Kirschbaum, Christian Guernth-Marschner, Gili Dardikman, Itay Barnea, Rafi Korenstein, Claus Duschl, and Natan T. Shaked

Abstract:

A major challenge in the field of optical imaging of live cells is achieving rapid, three-dimensional (3-D) and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved.  We present a new label-free tomographic interferometry approach that provides rapid capturing of the 3-D refractive index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped and rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3-D refractive index map.  Uniquely, this approach provides full (360^o) coverage of the rotation angular range on any axis, and knowledge on the viewing angle. Our experimental demonstrations include 3-D, label-free imaging of both large cancer cells and three-types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of contain cellular types in blood or other body fluids.

(a,b) Refractive-index maps of an MCF-7 cell at the mid-sagittal slice for using a full cell rotation on a single axis (a) and on two axes (b). The arrow indicate details that are clearer when rotating the cell on two axes.  (c,d) 3-D renderings (c) and rendered iso-surface plot (d) of the refractive-index map of the reconstructed refractive index map of an MCF-7 cancer cell. (e-j) Refractive index maps of three types of white blood cells at the mid-axial positions (e-g), and the coinciding rendered iso-surface plots of the refractive-index maps (h-j); (e,h) T cell, see also Supplementary Video 4; (f,i) Monocyte, see also Supplementary Video 5; (g,j) Neutrophil, see also Supplementary Video 6.

Nov. 2015

Grant title:

OptiQ-CanDo: Hybrid Optical Interferometry for Quantitative Cancer Cell Diagnosis
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First internal marriage in our group:

Pinhas and Irena got married.

Congrats!!

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New paper in Fertility and Sterility, 2015:

Interferometric phase microscopy for label-free morphological evaluation of sperm cells

Miki Haifler, Pinhas Girshovitz, Gili Band, Gili Dardikman, Igal Madjar, and Natan T. Shaked

Abstract:

Objective: To compare label-free interferometric phase microscopy (IPM) to label-free and label-based bright-field microscopy (BFM) in evaluating sperm cell morphology. This comparison helps in evaluating the potential of IPM for clinical sperm analysis without staining.

Design: Comparison of imaging modalities.

Setting: University laboratory.

Patient(s): Sperm samples were obtained from healthy sperm donors. Intervention(s): We evaluated 350 sperm cells, using portable IPM and BFM, according to World Health Organization (WHO) criteria. The parameters evaluated were length and width of the sperm head and midpiece; size and width of the acrosome; head, midpiece, and tail configuration; and general normality of the cell.

Main Outcome Measure(s): Continuous variables were compared using the Student’s t test. Categorical variables were compared with the X^2 test of independence. Sensitivity and specificity of IPM and label-free BFM were calculated and compared with label-based BFM.

Result(s): No statistical differences were found between IPM and label-based BFM in the WHO criteria. In contrast, IPM measurements of head and midpiece width and acrosome area were different from those with label-free BFM. Sensitivity and specificity of IPM were higher than those with label-free BFM for the WHO criteria.

Conclusion(s): Label-free IPM can identify sperm cell abnormalities, with an excellent correlation with label-based BFM, and with higher accuracy compared with label-free BFM. Further prospective clinical trials are required to enable IPM as part of clinical sperm selection procedures.

[Link]


(a) An interferogram obtained using IPM. The sample is still barely seen. However, the OPD information is encoded into the bend of the interference fringes, as can be seen from the enlarged region of interest in (b). (c) The OPD map of the same sperm cell, as digitally calculated from the interferogram shown in (a). All the main morphological features of the cell are discernable. (c, e) Imaging of a sperm cell with an acrosomal vacuole, using: (d) label-based BFM, and (e) label-free IPM. The vacuole is clearly seen as a defect in the OPD map of the IPM image. BFM = bright-field microscopy; IPM = interferometric phase microscopy; OPD = optical path delay. Elsevier 2015(c).

New paper in Optics Letters, Vol. 40, Issue 10, pp. 2273-2276, 2015:

Off-axis interferometer with adjustable fringe contrast based on polarization encoding

Sharon Karepov, Natan T. Shaked, and Tal Ellenbogen

Abstract: We propose a compact, close-to-common-path, off-axis interferometric system for low polarizing samples based on a spatial polarization encoder that is placed at the Fourier plane of a conventional transmission microscope. The polarization encoder erases the sample information from one polarization state and maintains it on the orthogonal polarization state, while retaining the low spatial frequencies of the sample, and thus enabling quantitative phase acquisition. In addition, the interference fringe visibility can be controlled by polarization manipulations. We demonstrate this concept experimentally by quantitative phase imaging of a USAF 1951 phase test target and human red blood cells with optimal fringe visibility and single-exposure phase reconstruction.

[PDF] [Link]

Imaging of USAF 1951 phase test target using the polarization encoding interferometer. (a) Intensity image with co-polarized analyzer (at 90°), associated with the sample arm, and (b) with cross-polarized analyzer (at 0°), associated with the reference arm. (c) The off-axis interference obtained on a small area of the background. (d) The reconstructed quantitative phase map. Scale bars are 26.7 μm in (a), (b) and (d), and 6.8 μm in (c).

New paper accepted to Nature – Light Science & Applications (Nature LSA), 2015:

Omry Blum and Natan T. Shaked

Abstract: We present a new approach for predicting spatial phase signals originated from photothermally excited metallic nanoparticles of arbitrary shapes and sizes. Heat emitted from the nanoparticle affects the measured phase signal via both the nanoparticle surrounding refractive index and thickness changes. Since these particles can be bio-functionalized to bind certain biological cell components, they can be used for biomedical imaging with molecular specificity, as new nanoscopy labels, and for photothermal therapy. Predicting the ideal nanoparticle parameters requires a model that computes the thermal and phase distributions around the particle, enabling more efficient phase imaging of plasmonic nanoparticles, and sparing trial and error experiments of using unsuitable nanoparticles. For the first time to our knowledge, using the proposed model, one can predict phase signatures from nanoparticles with arbitrary parameters. The proposed nonlinear model is based on a finite-volume method for geometry discretization, and an implicit backward Euler method for solving the transient inhomogeneous heat equation. To validate the model, we correlate its results with experimental results obtained for gold nanorods of various concentrations, which we acquired by a custom-built wide-field interferometric phase microscopy system.

[Link] [PDF, Video 1, Video 2, Video 3, Video 4, Video 5, Video 6, Video 7]

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Demonstration of using the proposed model for simulating the thermal and phase distributions of a structure of four gold nanoparticles. Top: optical excitation profile. Bottom left: heat distribution. Bottom right: optical path delay distribution.

New paper in Optics Letters, Vol. 40, Issue 8, pp. 1881-1884 (2015):

Tomographic phase microscopy with 180° rotation of live cells in suspension by holographic optical tweezers

Mor Habaza, Barak Gilboa, Yael Roichman, and Natan T. Shaked

Abstract: We present a new tomographic phase microscopy (TPM) approach that allows capturing the three-dimensional refractive-index structure of single cells in suspension without labeling using 180° rotation of the cells. This is obtained by integrating an external off-axis interferometer for wide-field wave front acquisition with holographic optical tweezers (HOTs) for trapping and micro-rotation of the suspended cells. In contrast to existing TPM approaches for cell imaging, our approach does not require anchoring the sample to a rotating stage nor is it limited in angular range as is the illumination rotation approach, and thus it allows TPM of suspended live cells in a wide angular range. The proposed technique is experimentally demonstrated by capturing the three-dimensional refractive-index map of yeast cells while collecting interferometric projections at an angular range of 180° with 5° steps. The interferometric projections are processed by both the filtered back-projection method and the diffraction-theory method. The experimental system is integrated with spinning-disk confocal fluorescent microscope for validation of the label-free TPM results.

[PDF, Media 1, Media 2, Media 3, Media 4] [Link]

3-D refractive index map of yeast cells obtained by the proposed TPM-HOTs technique.

Congratulations to Omry for winning the Rector Award of Excellence (first place), to Mor for winning the Dean Award of Excellence, and to Noa for winning the Dean Award of Excellence!

Doubling the field of view in off-axis low-coherence interferometric imaging

Pinhas Girshovitz and Natan T. Shaked

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Abstract: We present a new interferometric and holographic approach, named interferometry with doubled imaging area (IDIA), with which it is possible to double the camera field of view while performing off-axis interferometric imaging, without changing the imaging parameters, such as the magnification and the resolution. This technique enables quantitative amplitude and phase imaging of wider samples without reducing the acquisition frame rate due to scanning. The method is implemented using a compact interferometric module that connects to a regular digital camera, and is useful in a wide range of applications in which neither the field of view nor the camera frame rate can be compromised. Specifically, the IDIA principle allows doubling the off-axis interferometric field of view, which might be narrower than the camera field of view due to low-coherence illumination. We demonstrate the proposed technique for scan-free quantitative optical thickness imaging of microscopic biological samples, including live neurons, and rapid human sperm cell in motion under large magnification. In addition, we used the IDIA principle to perform non-destructive profilometry during a rapid lithography process of transparent structures.

Quantitative optical thickness maps of a human spermatozoon swimming,  as recorded by IDIA under low coherence illumination, enabling the acquisition of the fast dynamics of the spermatozoon with fine details on a doubled field of view. The white dashed line indicates the location of the stitching between the two fields of view. The color bar represents the quantitative optical thickness.

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N. A. Turko, A. Peled, and N. T. Shaked, “Wide-field interferometric phase microscopy with molecular specificity using plasmonic nanoparticles,” Journal of Biomedical Optics, Vol. 18, No. 11, 111414:1-8, 2013.
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Abstract: We present a method for adding molecular specificity to wide-field interferometric phase microscopy (IPM) by recording the phase signatures of gold nanoparticles (AuNPs) labeling targets of interest in live cells. The AuNPs are excited by light at a wavelength corresponding to their absorption spectral peak, evoking a photothermal (PT) effect due to their plasmonic resonance. This effect induces a local temperature rise, resulting in local refractive index and phase changes that can be detected optically. Using a wide-field interferometric phase microscope, we acquired an image sequence of the AuNPs sample phase profile without requiring lateral scanning, and analyzed the time-dependent profile of the entire field of view using a Fourier analysis, creating a map of the locations of AuNPs in the sample. The system can image a wide-field PT phase signal from a cluster containing down to 16 isolated AuNPs. AuNPs were then conjugated to epidermal growth factor receptor (EGFR) antibodies and inserted to an EGFR-overexpressing cancer cell culture, which was imaged using IPM, and verified by confocal microscopy. To the best of our knowledge, this is the first time wide-field interferometric PT imaging is performed at the subcellular level without the need for a total-internal-reflection effects or scanning.

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Imaging of MDA-MB 468 (EGFR+) cancer cell with conjugated AuNPs:

(a) Confocal imaging: Six consecutive confocal images of the AuNPs in the cell, from top-left image, representing the bottom of the cell, to bottom-right image, representing the top of the cell, in axial steps of 0.5 µm apart.

(b) Confocal plus DIC imaging: Cumulative confocal image of the AuNPs (colored), as obtained by summing the six consecutive confocal images shown in (a), overlaying a DIC image of the same cel (grayscale).

(c) PT IPM plus regular IPM imaging: PT phase image of the AuNPs (colored), overlaying a wide-field IPM phase image of the same cell (grayscale).

Figure is modified from Journal of Biomedical Optics [PDF].

Haniel Gabai, Maya Baranes-Zeevi, Meital Zilberman and Natan T. Shaked

Abstract: We obtained continuous, noncontact wide-field imaging and characterization of drug release from a polymeric device invitro by uniquely using off-axis interferometric imaging. Unlike the current gold-standard methods in this field, which are usually based on chromatography and spectroscopy, our method requires no user intervention during the experiment, and involves less lab consumable instruments. Using a simplified interferometric imaging system, we experimentally demonstrate the characterization of anesthetic drug release (Bupivacaine) from a soy-based protein matrix, used as skin substitute for wound dressing. Our results demonstrate the potential of interferometric imaging as an inexpensive and easy-to-use alternative for characterization of drug release in-vitro.

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Left - Quantitative phase profile resulted from drug release during 12 hours as measured by continuous wide-field interferometric imaging. The right lower frame illustrates the DDD location relatively to the FOV.

Right - Cumulative drug release profiles obtained from HPLC (solid line), off-axis interferometry (dotted line) and Weibull model (crossed line) for drug release period of 12 hours.

Figure is modified from Optic Letters [PDF].

Congratulations to Itay Shock for receiving his MSc yesterday, to Pini for his BSc with Distinction, and to Irena for her BSc.

Congrats to Pini and Haniel for winning the Excellent Graduate-Student Award (and 4000 NIS).

Two prizes in the same lab is a good thing for all of us.

• I. Frenklach, P. Girshovitz and N. T. Shaked,
  ”Quantitative cell nucleus analysis obtained by integrating simultaneous interferometric phase and fluorescence microscopy,”
  OSA European Conferences on Biomedical Optics (ECBO)
  Munich, Germany
  May 16, 2013
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• P. Girshovitz and N. T. Shaked,
  ”Compact interferometric module for quantitative phase microscopy of biological cells,”
  OSA European Conferences on Biomedical Optics (ECBO)
  Munich, Germany
  May 16, 2013
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• N. T. Shaked, Y. Bishitz, H. Gabai, and P. Girshovitz,
  “Optical-mechanical properties of diseased cells measured by interferometry,”
  SPIE Optical Metrology 2013
  Conference 8792: Optical Methods for Inspection, Characterization, and Imaging of Biomaterials
  Munich, Germany
  May 16, 2013
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• H. Gabai, M. Baranes-Zeevi, M. Zilberman, and N. T. Shaked,
  “Noninvasive continuous imaging of drug release from soy-based skin equivalent using wide-field interferometry,”
  SPIE Optical Metrology 2013
  Conference 8792: Optical Methods for Inspection, Characterization, and Imaging of Biomaterials
  Munich, Germany
  May 16, 2013

[Invited]

N. T. Shaked, “Low-coherence, common-path, and dynamic holographic microscopy and nanoscopy for biomedicine using portable systems,” OSA Digital Holography and 3D Imaging, Kohala Coast, Hawaii, USA, April 21-25, 2013.

N. T. Shaked and P. Girshovitz, “Portable low-coherence interferometer for quantitative phase microscopy of live cells,” SPIE Photonics West, Biomedical Optics (BiOS), San Francisco, California USA, February 2-7, 2013. 
 
N.  Turko and N. T. Shaked, “Wide-field interferometric phase imaging of plasmonic nanoparticles at the subcellular level,” SPIE Photonics West, Biomedical Optics (BiOS), San Francisco, California USA, February 2-7, 2013.

Dual-channel low-coherence interferometry and its application to quantitative phase imaging of fingerprints

H. Gabai and N. T. Shaked

Abstract:

We introduce a low-coherence, dual channel and common-path interferometric imaging system for three dimensional imaging of fingerprints for  both biomedical and biomertical applications.The proposed system is simple to align, and requires no alignmet in order to obtain interference with low-coherence light source, thus enabling non-expert useres to benifit from the attractive advantages of low-coherence, common-path and dual-channel interferometry. In our case, we have used the dual-channel property in order to create a noise reduced and DC supressed equivalent hologram, from which we were able to derive a high quality, with nano-meter resolution depth profile of fingerprints

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The figure shows measurements of thick samples (up to 100 microns) using low-coherence, common-path, wide-field phase interferometry with two-wavelength unwrapping. (a) Two 180°-phase-shifted interferograms of a finger-print template, acquired simultaneously (dual imaging channel). The dashed white square indicates on the scar location. (b) Depth profile distorted by blurring (in blue). Significant distortion can be seen in the upper-right part of the image. (c) Final result with improved contrast obtained by using the two interferograms  to decrease noise. (d) Final result in three-dimensional view.
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[Download PDF] [Journal ink].

Presented in OSA Frontiers in Optics, Rochester, NY, USA, October 2010:

Cell life cycle characterization based on generalized morphological parameters for interferometric phase microscopy. 
P. Girshovitz and N. T. Shaked.

Dual-channel digital holography imaging for fingerprint biometrics.
H. Gabai and N. T. Shaked.

Optical-mechanical signature of cancer cells measured by interferometry.
Y. Bishitz, H. Gabai and N. T. Shaked.

Our paper got the cover page in Biomedical Optics Express:

P. Girshovitz and N. T. Shaked, “Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization,” Biomedical Optics Express, Vol. 3, No. 8, pp. 1757-1773, 2012 [PDF, Media 1] [Link].