Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity

  1. Raimund Schlüßler  Is a corresponding author
  2. Kyoohyun Kim  Is a corresponding author
  3. Martin Nötzel
  4. Anna Taubenberger
  5. Shada Abuhattum
  6. Timon Beck
  7. Paul Müller
  8. Shovamaye Maharana
  9. Gheorghe Cojoc
  10. Salvatore Girardo
  11. Andreas Hermann
  12. Simon Alberti
  13. Jochen Guck  Is a corresponding author
  1. Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität, Germany
  2. Max Planck Institute for the Science of Light and Max-Planck-Zentrum für Physik und Medizin, Germany
  3. Department of Microbiology and Cell Biology, Indian Institute of Science, India
  4. Translational Neurodegeneration Section "Albrecht Kossel", University Rostock, and German Center for Neurodegenerative Diseases (DZNE), Germany
  5. Physics of Life, Technische Universität Dresden, Germany
5 figures and 5 additional files

Figures

Figure 1 with 1 supplement
Combined fluorescence, optical diffraction tomography (ODT), and Brillouin microscopy.

(a) Optical setup. The beam paths for epifluorescence/brightfield imaging, ODT, and Brillouin microscopy are shown in light yellow, dark green, and light green, respectively. The laser light …

Figure 1—figure supplement 1
Absolute density of a cell phantom consisting of a polydimethylsiloxane (PDMS) bead inside a polyacrylamide (PAA bead).

Scale bar 10 µm.

Figure 2 with 1 supplement
Cell nucleoplasm has lower refractive index (RI) but higher longitudinal modulus than cytoplasm.

(a–c) Representative maps of the (a) epifluorescence intensity distribution, (b) longitudinal moduli, and (c) RIs of a HeLa cell. Nuclei are stained with Hoechst (blue), and the nucleolar protein in …

Figure 2—source data 1

Refractive index, longitudinal modulus, Brillouin shift, and absolute density values of different compartments in HeLa cells.

https://cdn.elifesciences.org/articles/68490/elife-68490-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Brillouin shift and absolute density of cytoplasm, nucleoplasm, and nucleoli in HeLa cells.

Representative maps of the (a) Brillouin shift and (b) absolute density of a HeLa cell. Quantitative analysis of (c) the Brillouin shift υB, (d) the absolute density ρ, and (e) peak linewidth ΔB. **p

Polyglutamine (polyQ) aggregates have a higher refractive index, Brillouin shift, and longitudinal modulus than the peripheral cytoplasm.

(a–c) Representative maps of (a) the epifluorescence intensity distribution, (b) the refractive indices, and (c) the longitudinal moduli of a HeLa cell transfected with a plasmid encoding HttQ103. …

Figure 3—source data 1

Refractive index, Brillouin shift, and longitudinal modulus values of polyglutamine (polyQ) aggregates and their periphery.

https://cdn.elifesciences.org/articles/68490/elife-68490-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
FUS-GFP-labeled stress granules induced by oxidative stress in living P525L FUS HeLa cells show a similar refractive index (RI), and longitudinal modulus as the peripheral cytoplasm.

Representative example of (a–c) the fluorescence intensity and (d−f) the RI distribution under control conditions without arsenite, with arsenite, and with arsenite after fixation, respectively. …

Figure 4—source data 1

Refractive index and longitudinal modulus values of cytoplasm, nucleoplasm, and stress granules (SGs) in P525L HeLa cells after different treatments.

https://cdn.elifesciences.org/articles/68490/elife-68490-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Comparison of the influence of different acquisition schemes and laser wavelengths on the viability of P525L HeLa cells that express GFP-tagged FUS.

For better comparability, the Brillouin shift υB measured with the 780 nm setup is normalized to a wavelength of 532 nm. Scale bars 10 μm.

Figure 4—figure supplement 2
Evaluation of the refractive index (RI) of P525L FUS HeLa cells taking into account the complete cell.

FUS-GFP-labeled stress granules induced by oxidative stress show a similar RI as the peripheral cytoplasm. *p<0.05; ****p<0.0001.

Despite a higher refractive index (RI) and Brillouin shift, the longitudinal modulus of lipid droplets is lower than that of the surrounding cytoplasm.

(a–d) Representative maps of the (a) epifluorescence intensities, (b) brightfield intensities, (c) RIs, and (d) Brillouin shifts of an adipocyte cell. The nucleus is stained with Hoechst (blue in a) …

Additional files

Supplementary file 1

Average values and standard errors of the mean of the refractive index (RI) n, Brillouin shift νB, absolute density ρ, longitudinal modulus M, and linewidth ΔB for the cytoplasm (cyto), nucleoplasm (np), and nucleoli (nl) of 139 wild-type HeLa cells.

https://cdn.elifesciences.org/articles/68490/elife-68490-supp1-v2.docx
Supplementary file 2

Kruskal−Wallis p-values when comparing the refractive index (RI) n, Brillouin shifts νB, mass densities ρ, longitudinal moduli M, and linewidths ΔB of the cytoplasm (cyto), nucleoplasm (np), and nucleoli (nl) of 139 wild-type HeLa cells, respectively.

https://cdn.elifesciences.org/articles/68490/elife-68490-supp2-v2.docx
Supplementary file 3

Average values and standard errors of the mean of the refractive index (RI) n, Brillouin shift νB, absolute density ρ, and longitudinal modulus M for the cytoplasm and polyglutamine (polyQ) aggregates of 22 wild-type HeLa cells.

https://cdn.elifesciences.org/articles/68490/elife-68490-supp3-v2.docx
Supplementary file 4

Average values and standard errors of the mean of the refractive index (RI) n and longitudinal modulus M for different conditions and compartments of P525L HeLa cells.

https://cdn.elifesciences.org/articles/68490/elife-68490-supp4-v2.docx
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https://cdn.elifesciences.org/articles/68490/elife-68490-transrepform1-v2.pdf

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