Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - OptiQ-CanDo (Hybrid Optical Interferometry for Quantitative Cancer Cell Diagnosis)

Teaser

Invasive cancer is a leading cause of death worldwide. Detecting cancer in its early curable stages is a clear and critical unmet need. Late stage metastatic forms of cancer are fatal. Cancer starts with changes in a single cell or a small group of cells, which cause the cells...

Summary

Invasive cancer is a leading cause of death worldwide. Detecting cancer in its early curable stages is a clear and critical unmet need. Late stage metastatic forms of cancer are fatal. Cancer starts with changes in a single cell or a small group of cells, which cause the cells to grow and divide in an uncontrolled manner. Detecting these changes requires new microscopy capabilities. A major challenge in the field of optical imaging of live cells is to achieve label-free but still fully quantitative measurements, which afford high-resolution morphological and mechanical mapping at the single cell level. In particular, developing efficient, non-subjective, quantitative optical imaging technologies for cancer cell diagnosis is a challenging task. The ground-breaking goal of this research project is to establish a robust experimental toolbox for label-free optical diagnosis and monitoring of live cancer cells in-vitro and their potential of metastasis. Optical interferometry is able to provide a platform for imaging live cells quantitatively without the risk of effects caused by using external contrast agents. By overcoming critical technological barriers, I suggest novel hybrid optical interferometric approaches that provide a powerful nano-sensing tool for label-free quantitative measurement of cancer cells. This will be obtained by recording the dynamic quantitative, three-dimensional sub-nanometric structural and mechanical characterization of live cancer cells in different stages. For this aim, I will develop a novel low-noise broadband, common-path, off-axis interferometric system for sub-nanometric physical thickness and mechanical mapping of live cells in thousands of frames per second. Additionally, I will develop rapid tomographic approach for fully capturing the cell three-dimensional refractive-index distribution, as a tool to characterize cancer progression. Interferometry will be combined with multi-trap holographic optical tweezers and dielectrophoresis to enable complete cell manipulations including full rotation, imaging of non-adherent cells, and mechanical measurement validation. New set of interferometry-based quantitative parameters will be developed to enable characterization of cellular transformations, and used to characterize cancer cells with different metastasis potential, for cell lines and for circulating tumor cells.

Work performed

Cell wide-field imaging with 0.2 nm thickness accuracy: I proposed to design wide-field interferometric setups that are able to operate in off-axis, common-path mode with broad-spectral bandwidth light source in reflection mode, providing thickness maps of live cells (in ambient conditions) in accuracy of 0.2 nm. We lately designed and built compact and external off-axis interferometric modules that can achieve interference with low spatial coherence illumination over the entire field of view in reflection mode. Since three mirror retroreflectors are used in both the sample and reference beam paths, the solid angles of the beams stay constant, so that full-field high-visibility interference can be obtained on the entire camera sensor. The spatial thickness accuracy obtained was 1.55 nm, and the temporal thickness accuracy was 0.23 nm, which is very close to the target temporal accuracy.
Off-axis interferometric recording of live cells in 20 KHz with extended FOV: To enable full mapping of cancer-cell fluctuations, we are developing new experimentally-implemented optical interferometric image compression techniques for off-axis interferogram multiplexing to significantly extend the off-axis interferometric FOV or increase the true frame rate of the camera. Therefore, this tool will enable obtaining highly resolved elasticity-related maps for cancer cells, which can be positively correlated with their metastatic potential.

First, we have designed new optical modules that use holographic multiplexing to increase the amount of information that can be acquired in a single off-axis hologram. This was first done for wavelength multiplexing of multiple imaging channels is the following journal papers:

Optics Letters 42, 73-76, 2017
Optics Letters 43, 1943-1946, 2018
Optics Letters 43, 2046-2049, 2018
Then, we presented a new theoretical analysis that is able to compress up to six channels into a single hologram (novel paradigm in the field), called six-pack holography, as well as presented the initial optical setup required, in the following paper:
Optics Letters 42, 4611-4614, 2017.
Furthermore, we have compared the different holographic multiplexing architectures and presented additional uses for rapid extraction of the phase profiles in the following papers:

Optics Express 25, 33400-33415, 2017
Is multiplexed off-axis holography for quantitative phase imaging more spatial bandwidth-efficient than on-axis holography?, Accepted to Journal of Optical Society of America A, 2018
We also purchased a rapid camera, and are currently working on the full implementation of six-pack holography setup for rapid acquisition of the quantitative phase profiles of cells.

Development of interferometry-based mathematical analysis tools for cell characterization:
I proposed to develop parameters extracted from the static (one-frame) optical thickness maps measured by wide-field interferometry. Indeed, we have lately published a peer-review paper about this:
Cytometry A 91, 482-493, 2017.
In this paper, we presented classification of live healthy and cancerous cells by using the spatial morphological and textural information found in the label-free quantitative phase images of the cells. The cells were imaged while unattached to the substrate. After interferometric acquisition, the optical path delay maps of the cells were extracted and then used to calculated 15 static parameters derived from the cellular 3D morphology and texture. We found high statistical significance between the groups, with the same trends for all statistically significant parameters (healthycancermetastatic). Furthermore, a specially designed machine learning algorithm, implemented on the phase map extracted features, classified the correct cell type with 81–93% sensitivity and 81–99% specificity. This quantitative phase imaging approach for liquid biopsies is the basis for grading circulating tumor cells.

Final results

We found new ways to decouple refractive index from cell thickness for cancer cells during flow:

G. Dardikman, Y. N. Nygate, I. Barnea, N. A. Turko, G. Singh, B. Javidi, and N. T. Shaked, “Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy,” Biomedical Optics Express, Vol. 9, Issue 3, pp. 1177-1189, 2018

We found new ways to solve the unwrapping problems in the cell phase profiles measured:

G. Dardikman, S. Mirsky, M. Habaza, Y. Roichman, and N. T. Shaked, “Angular phase unwrapping of optically thick objects with a thin dimension,” Optics Express, Vol. 25, Issue 4, pp. 3347-3357, 2017

G. Dardikman*, G. Singh*, and N. T. Shaked, “Four dimensional phase unwrapping of dynamic objects in digital holography,” Optics Express, Vol. 26, Issue 4, pp. 3772-3778, 2018

We also demonstrated multiplexing of holography and fluorescence onto the same camera (was not in the original plan, but can help in the project for cancer cell imaging using multimodal microscopy):
Y. Nygate, G. Singh, N. A. Turko, and N. T. Shaked, “Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells,” Optics Letters, Vol. 43, No. 1, pp. 2587-2590, 2018

Furthermore, we have compared the different holographic multiplexing architectures and presented additional uses for rapid extraction of the phase profiles in the following papers:
G. Dardikman, N. A. Turko, N. Nativ, S. K. Mirsky, and N. T. Shaked, “Optimal spatial bandwidth capacity in multiplexed off-axis holography for rapid quantitative phase reconstruction and visualization,” Optics Express, Vol. 25, No. 26, pp. 33400-33415, 2017
G. Dardlkman and N. T. Shaked, “Is multiplexed off-axis holography for quantitative phase imaging more spatial bandwidth-efficient than on-axis holography?,” Accepted to Journal of Optical Society of America A, 2018
These works help in making the ERC project goals become more useful for clinical implementations, allowing the rapid analysis of the quantitative phase maps of cancer cells.

Website & more info

More info: http://www.eng.tau.ac.il/.