Quantitative Phase Imaging & Optical Diffraction Tomography

Quantitative Phase Imaging

Quantitative Phase Imaging (QPI) is a marker-free technique for quantifying the phase retardation of samples, which represents the integration of refractive index (RI) contrast along the illumination axis. QPI is commonly performed using digital holographic microscopy (DHM), and we employ the technique to measure dry mass and averaged RI values for single cells in suspension [1] with an analysis pipeline developed by our group under the assumption of the spherical object [2,3].

Optical Diffraction Tomography

Furthermore, we have developed and applied Optical Diffraction Tomography (ODT), a technique that quantitatively measures the 3D RI distribution within cells. ODT utilizes QPI techniques to record 2D phase images from multiple illumination angles, which are then used for tomographic 3D reconstruction. Since the RI of most biological samples is linearly proportional to their mass density, ODT offers an unbiased and label-free view of the quantitative mass density distributions in living cells and organisms.

Our group has developed optical setups [5], tomogram reconstruction algorithms [6], and theoretical frameworks that relates mass density to RI of various substances [7]. These advancements have been applied to study the mass density distributions in living cells and organisms. Notable studies include the investigation of lower mass density in nuclei compared to cytoplasm in adherent cells [8], Xenopus egg extract and its mitotic spindle [9,10], and cells under osmotic stress [11]. Additionally, we have quantitatively characterized the mass density of protein condensates generated by liquid-liquid phase separation (LLPS) [12], and observed significant increases in mass density of yeast [13] and C. elegans larvae [14] as they enter dormancy to survive harsh environment. ODT has also been utilized for the quantitative characterization of various interesting samples such as zebrafish larvae [15] and microgels beads [16] and rods [17].

Operation principle of Optical Diffraction Tomography (ODT). (a) Schematic overview of an ODT setup employing Mach-Zehnder interferometry. (b) Measured spatially modulated hologram encoding complex optical field. (c) Retrieved optical phase delay maps of HeLa cells illuminated from various incident angles and their 2D Fourier spectra. (d) The 3D Fourier spectra where 2D Fourier spectra from various incident angles are mapped by the Fourier diffraction theorem. (e) The reconstructed RI tomogram of HeLa cells.

The ODT study for various biological samples. (a, b) The epi-fluorescence image (upper row) and the cross-sectional slice of an RI tomogram of (a) adherent HeLa cells [8] and (b) Xenopus mitotic spindle [9]. The cross-sectional slice of an RI tomogram of (c) an adipocyte [18] and (d) C. elegans embryo in the early embryogenesis [10].

The mass density changes of C. elegans larvae during dormancy. (a) The cross-sectional slices of the RI tomograms of C. elegans larvae at the reproductive cycle (L3) and the dauer state. The cross-sectional slices of the RI tomograms of C. elegans dauer larvae of (b) controls and (c) lea-1 deletion mutant after harsh desiccation (60% RH). Figure reproduced from [14].

[1] M. Schürmann, J. Scholze, P. Müller, C. J. Chan, A. E. Ekpenyong, K. J. Chalut, and J. Guck, "Refractive index measurements of single, spherical cells using digital holographic microscopy," Methods Cell Biol. 125, 143–159 (2015).

[2] P. Müller, G. Cojoc, and J. Guck, "DryMass: handling and analyzing quantitative phase microscopy images of spherical, cell-sized objects," BMC Bioinformatics 21(1), 226 (2020).

[3] P. Müller, M. Schürmann, S. Girardo, G. Cojoc, and J. Guck, "Accurate evaluation of size and refractive index for spherical objects in quantitative phase imaging," Opt. Express 26(8), 10729 (2018).

[4] P. Müller, "Optical Diffraction Tomography for Single Cells," PhD Thesis, (2016).

[5] P. Müller, M. Schürmann, C. J. Chan, and J. Guck, "Single-cell diffraction tomography with optofluidic rotation about a tilted axis," 9548, 95480U (2015).

[6] P. Müller, M. Schürmann, and J. Guck, "The Theory of Diffraction Tomography," arXiv Prepr. 1507.00466, (2015).

[7] C. Möckel, T. Beck, S. Kaliman, S. Abuhattum, K. Kim, J. Kolb, D. Wehner, V. Zaburdaev, and J. Guck, "Estimation of the mass density of biological matter from refractive index measurements," Biophys. Reports 4(2), 100156 (2024).

[8] K. Kim and J. Guck, "The Relative Densities of Cytoplasm and Nuclear Compartments Are Robust against Strong Perturbation," Biophys. J. 119(10), 1946–1957 (2020).

[9] A. Biswas, K. Kim, G. Cojoc, J. Guck, and S. Reber, "The Xenopus spindle is as dense as the surrounding cytoplasm," Dev. Cell 56(7), 967-975.e5 (2021).

[10] A. Biswas, O. Muñoz, K. Kim, C. Hoege, B. M. Lorton, D. Shechter, J. Guck, V. Zaburdaev, and S. Reber, "Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes," bioRxiv 1–81 (2023).

[11] C. Roffay, G. Molinard, K. Kim, M. Urbanska, V. Andrade, V. Barbarasa, P. Nowak, V. Mercier, J. García-Calvo, S. Matile, R. Loewith, A. Echard, J. Guck, M. Lenz, and A. Roux, "Passive coupling of membrane tension and cell volume during active response of cells to osmosis," Proc. Natl. Acad. Sci. 118(47), e2103228118 (2021).

[12] AJ. Guillén-Boixet, A. Kopach, A. S. A. S. Holehouse, S. Wittmann, M. Jahnel, R. Schlüßler, K. Kim, I. R. E. A. I. R. E. A. Trussina, J. Wang, D. Mateju, I. Poser, S. Maharana, M. Ruer-Gruß, D. Richter, X. Zhang, Y.-T. Y.-T. Chang, J. Guck, A. Honigmann, J. Mahamid, A. A. A. A. Hyman, R. V. R. V. Pappu, S. Alberti, and T. M. T. M. Franzmann, "RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation," Cell 181(2), 346-361.e17 (2020).

[13] S. Abuhattum, K. Kim, T. M. Franzmann, A. Eßlinger, D. Midtvedt, R. Schlüßler, S. Möllmert, H.-S. Kuan, S. Alberti, V. Zaburdaev, and J. Guck, "Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells," Front. Phys. 6, 131 (2018).

[14] K. Kim, V. R. Gade, T. V Kurzchalia, and J. Guck, "Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition," Biophys. J. 121(7), 1219–1229 (2022).

[15] J. Kolb, V. Tsata, N. John, K. Kim, C. Möckel, G. Rosso, V. Kurbel, A. Parmar, G. Sharma, K. Karandasheva, S. Abuhattum, O. Lyraki, T. Beck, P. Müller, R. Schlüßler, R. Frischknecht, A. Wehner, N. Krombholz, B. Steigenberger, D. Beis, A. Takeoka, I. Blümcke, S. Möllmert, K. Singh, J. Guck, K. Kobow, and D. Wehner, "Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment," Nat. Commun. 14(1), 6814 (2023).

[16] S. Girardo, N. Träber, K. Wagner, G. Cojoc, C. Herold, R. Goswami, R. Schlüßler, S. Abuhattum, A. Taubenberger, F. Reichel, D. Mokbel, M. Herbig, M. Schürmann, P. Müller, T. Heida, A. Jacobi, E. Ulbricht, J. Thiele, C. Werner, and J. Guck, "Standardized microgel beads as elastic cell mechanical probes," J. Mater. Chem. B 6(39), 6245–6261 (2018).

[17] Y. Kittel, L. P. B. Guerzoni, C. Itzin, D. Rommel, M. Mork, C. Bastard, B. Häßel, A. Omidinia‐Anarkoli, S. P. Centeno, T. Haraszti, K. Kim, J. Guck, A. J. C. Kuehne, and L. De Laporte, "Varying the Stiffness and Diffusivity of Rod‐Shaped Microgels Independently through Their Molecular Building Blocks," Angew. Chemie Int. Ed. 62(44), (2023).

[18] R. Schlüßler, K. Kim, M. Nötzel, A. Taubenberger, S. Abuhattum, T. Beck, P. Müller, S. Maharana, G. Cojoc, S. Girardo, A. Hermann, S. Alberti, and J. Guck, "Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity," Elife 11, (2022).

Data Collection