Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity
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Evaluation Summary:
In this interesting study, the authors combined Brillouin microscopy with Optical Diffraction Tomography and epi-fluorescence imaging to investigate physical properties of biological materials including nucleoplasm, cytoplasm, phase-separated organelles, and adipocytes. The results are largely convincing and offer interesting insights into the material properties of these subcellular structures.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples − so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample − a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.
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Author Response:
Reviewer #1:
There is a critical need for new methodologies to study the physical properties of biomolecular condensates in living cells under normal and pathological conditions. To address this, Schlüßler et al innovatively combined Brillouin microscopy with Optical Diffraction Tomography (ODT) and epi-fluorescence imaging. The current study can have a significant impact on the community. A major strength of the study resides in the application of Brillouin microscopy, which offers a label-free and nondestructive approach to investigate the complex viscoelastic behavior of biological materials. The study initially attempts to benchmark their new methodology using control samples, called cell phantoms. Subsequently, the authors apply their new method to study the physical properties of biological materials including …
Author Response:
Reviewer #1:
There is a critical need for new methodologies to study the physical properties of biomolecular condensates in living cells under normal and pathological conditions. To address this, Schlüßler et al innovatively combined Brillouin microscopy with Optical Diffraction Tomography (ODT) and epi-fluorescence imaging. The current study can have a significant impact on the community. A major strength of the study resides in the application of Brillouin microscopy, which offers a label-free and nondestructive approach to investigate the complex viscoelastic behavior of biological materials. The study initially attempts to benchmark their new methodology using control samples, called cell phantoms. Subsequently, the authors apply their new method to study the physical properties of biological materials including nucleoplasm, cytoplasm, phase-separated organelles, and adipocytes. The results are largely convincing and offer interesting insights into the complex material properties of these subcellular fluids and organelles.
Thank you very much for this encouraging assessment.
Reviewer #2:
The multimodal instrument presented here provides an independent measurement of the spatially dependent cellular refractive index, which yields a more quantitative extraction of the longitudinal modulus from Brillouin spectroscopy. To my knowledge, this instrument is unique, and its capability addresses unresolved problems in Brillouin studies. The method was judiciously validated on standard samples. The experiments and analysis were carefully performed, and the statistics seems solid. The manuscript is very well written and clear.
The results highlight some discrepancies between the generally accepted assumptions regarding the cell density and refractive index. One striking example is the finding that the nuclear matter exhibits lower mass density but higher longitudinal modulus. Using the fluorescence channel for specificity, the authors investigated successfully other cellular compartments.
While the objectives of the study seem to have been achieved, I wonder how large an impact this development will have in the field. At the end of the day, the method yields the longitudinal modulus at GHz frequencies. Cell mechanics is indeed very important, but at much lower frequencies. For example, actin filament lifetime is of the order of minutes. It seems very difficult to infer cell mechanics information relevant to its function, from the GHz range, as the dispersion of the material is unknown.
Thank you very much for this positive and accurate feedback.
Indeed, Brillouin microscopy measures mechanical properties at a fundamentally different (higher) frequency range than common methods for accessing cell mechanics, such as atomic force microscopy, microrheology, deformability cytometry, and others, and converting between elastic and longitudinal modulus is generally not possible as the Poisson’s ratio and its dispersion is unknown. However, differences in mechanical properties measured should not be considered insignificant just because the underlying model might remain yet unknown. Hence, Brillouin microscopy results in the GHz and GPa range can still indicate differences in the mechanical properties important to cells, even though common cell mechanics happens at much lower time and frequency scales. In fact, Brillouin microscopy was shown to be sensitive to e.g. actin polymerization and branching of actin fibers (see Scarcelli et al. Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy. Nat. Methods 12, 1132–1134 (2015)) and empirical correlations to the elastic (Young’s) modulus have been found (Scarcelli et al. In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy. Biophys. J. 101, 1539–1545 (2011); Schlüßler et al. Mechanical mapping of spinal cord growth and repair in living zebrafish larvae by Brillouin imaging. Biophys. J. 115, 911–923 (2018)).
Reviewer #3:
In the manuscript by Kim et al, the authors present a combined optical system, termed FOB microscopy which bring together the epi-fluorescence, optical diffraction tomography (ODT), and Brillouin microscopy. The main purpose of FOB is to establish a colocalized measurement of Brillouin shift and the refractive index (RI) to calculate absolute densities of biological sample, especially the biomolecular condensate.
The major strength of this paper (method development) is the added measurement of ODT which can correct for and thus provide a more precise RI and density of a given sample. If the RI and densities can be reliably measured in in vitro and cell samples, it will be a great tool that can complement other microrheological measurement. The major weakness is the lack of appropriate controls and lack of comparisons to other conventional methods (microrheological), which together lead to questionable outcomes from various measurements shown throughout the manuscript. Another concern is the pervasiveness of the method which involves excessive level of illumination and vibrational excitation.
If the work could be revised to present careful calibration with samples that are pertinent to biological systems (both in vitro and cellular) and make a comparison to other conventional methods in every possible case, the strength and limitations of the combined microscopy will be clear, making it very helpful for the researchers in the field.
Thank you very much for this review.
The accuracy of optical diffraction tomography (ODT) has been shown in previous publications already and the combination with Brillouin microscopy does not affect this, since the basic working principle remains untouched. E.g. in McCall et. al. “Quantitative Phase Microscopy Enables Precise and Efficient Determination of Biomolecular Condensate Composition” bioRxiv, 2020 Oct; p. 2020.10.25.352823. doi: 10.1101/2020.10.25.352823 we show that the protein concentrations acquired with ODT/QPM agree well with results acquired with a volume-based approach and did not suffer from uncertainties of the fluorescent dye quantum yield, as it can happen for concentration measurements based on the fluorescence intensity ratio. Further publications tested the correctness of ODT by measuring samples with known geometry and RI value, such as microspheres (Y. Sung et al., Opt. Express. 17, 266–277 (2009); K. Kim et al., Opt. Lett. 39, 6935 (2014); A. Kuś et al., J. Biomed. Opt. 20, 111216 (2015); S. Chowdhury et al., Optica. 4, 537 (2017)). Also, hemoglobin concentration of red blood cells calculated from ODT measurements is consistent with mean corpuscular hemoglobin concentration (MCHC) measured independently by complete blood count (CBC) test (Y. Kim et al., Sci. Rep. 4, 6659 (2014)).
Due to the low scattering efficiency of Brillouin scattering, Brillouin microscopy requires higher laser illumination powers (typically around 10 mW) than e.g. fluorescence microscopy. While certain illumination strategies, such as focusing the nucleus, can substantially damage certain cell types, such as GFP-FUS HeLa cells, adjusting the illumination strategy proved to reduce the negative effect on the cells, and wild-type HeLa cells were not affected in the first place. Furthermore, larger laser wavelengths in the near-infrared with less photon energy are known to be less phototoxic and would work similarly well for FOB microscopy. Hence, we think the method can be considered non-invasive, especially when realized with adjusted laser sources.
Furthermore, the presented Brillouin microscopy makes use of spontaneous Brillouin phonons, which are – in difference to the phonons excited when using stimulated Brillouin microscopy – intrinsic to the sample. Hence, no vibrational excitation possibly altering the sample occurs when using the FOB microscope.
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Evaluation Summary:
In this interesting study, the authors combined Brillouin microscopy with Optical Diffraction Tomography and epi-fluorescence imaging to investigate physical properties of biological materials including nucleoplasm, cytoplasm, phase-separated organelles, and adipocytes. The results are largely convincing and offer interesting insights into the material properties of these subcellular structures.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
There is a critical need for new methodologies to study the physical properties of biomolecular condensates in living cells under normal and pathological conditions. To address this, Schlüßler et al innovatively combined Brillouin microscopy with Optical Diffraction Tomography (ODT) and epi-fluorescence imaging. The current study can have a significant impact on the community. A major strength of the study resides in the application of Brillouin microscopy, which offers a label-free and nondestructive approach to investigate the complex viscoelastic behavior of biological materials. The study initially attempts to benchmark their new methodology using control samples, called cell phantoms. Subsequently, the authors apply their new method to study the physical properties of biological materials including …
Reviewer #1 (Public Review):
There is a critical need for new methodologies to study the physical properties of biomolecular condensates in living cells under normal and pathological conditions. To address this, Schlüßler et al innovatively combined Brillouin microscopy with Optical Diffraction Tomography (ODT) and epi-fluorescence imaging. The current study can have a significant impact on the community. A major strength of the study resides in the application of Brillouin microscopy, which offers a label-free and nondestructive approach to investigate the complex viscoelastic behavior of biological materials. The study initially attempts to benchmark their new methodology using control samples, called cell phantoms. Subsequently, the authors apply their new method to study the physical properties of biological materials including nucleoplasm, cytoplasm, phase-separated organelles, and adipocytes. The results are largely convincing and offer interesting insights into the complex material properties of these subcellular fluids and organelles.
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Reviewer #2 (Public Review):
The multimodal instrument presented here provides an independent measurement of the spatially dependent cellular refractive index, which yields a more quantitative extraction of the longitudinal modulus from Brillouin spectroscopy. To my knowledge, this instrument is unique, and its capability addresses unresolved problems in Brillouin studies. The method was judiciously validated on standard samples. The experiments and analysis were carefully performed, and the statistics seems solid. The manuscript is very well written and clear.
The results highlight some discrepancies between the generally accepted assumptions regarding the cell density and refractive index. One striking example is the finding that the nuclear matter exhibits lower mass density but higher longitudinal modulus. Using the fluorescence …
Reviewer #2 (Public Review):
The multimodal instrument presented here provides an independent measurement of the spatially dependent cellular refractive index, which yields a more quantitative extraction of the longitudinal modulus from Brillouin spectroscopy. To my knowledge, this instrument is unique, and its capability addresses unresolved problems in Brillouin studies. The method was judiciously validated on standard samples. The experiments and analysis were carefully performed, and the statistics seems solid. The manuscript is very well written and clear.
The results highlight some discrepancies between the generally accepted assumptions regarding the cell density and refractive index. One striking example is the finding that the nuclear matter exhibits lower mass density but higher longitudinal modulus. Using the fluorescence channel for specificity, the authors investigated successfully other cellular compartments.
While the objectives of the study seem to have been achieved, I wonder how large an impact this development will have in the field. At the end of the day, the method yields the longitudinal modulus at GHz frequencies. Cell mechanics is indeed very important, but at much lower frequencies. For example, actin filament lifetime is of the order of minutes. It seems very difficult to infer cell mechanics information relevant to its function, from the GHz range, as the dispersion of the material is unknown.
-
Reviewer #3 (Public Review):
In the manuscript by Kim et al, the authors present a combined optical system, termed FOB microscopy which bring together the epi-fluorescence, optical diffraction tomography (ODT), and Brillouin microscopy. The main purpose of FOB is to establish a colocalized measurement of Brillouin shift and the refractive index (RI) to calculate absolute densities of biological sample, especially the biomolecular condensate.
The major strength of this paper (method development) is the added measurement of ODT which can correct for and thus provide a more precise RI and density of a given sample. If the RI and densities can be reliably measured in in vitro and cell samples, it will be a great tool that can complement other microrheological measurement. The major weakness is the lack of appropriate controls and lack of …
Reviewer #3 (Public Review):
In the manuscript by Kim et al, the authors present a combined optical system, termed FOB microscopy which bring together the epi-fluorescence, optical diffraction tomography (ODT), and Brillouin microscopy. The main purpose of FOB is to establish a colocalized measurement of Brillouin shift and the refractive index (RI) to calculate absolute densities of biological sample, especially the biomolecular condensate.
The major strength of this paper (method development) is the added measurement of ODT which can correct for and thus provide a more precise RI and density of a given sample. If the RI and densities can be reliably measured in in vitro and cell samples, it will be a great tool that can complement other microrheological measurement. The major weakness is the lack of appropriate controls and lack of comparisons to other conventional methods (microrheological), which together lead to questionable outcomes from various measurements shown throughout the manuscript. Another concern is the pervasiveness of the method which involves excessive level of illumination and vibrational excitation.
If the work could be revised to present careful calibration with samples that are pertinent to biological systems (both in vitro and cellular) and make a comparison to other conventional methods in every possible case, the strength and limitations of the combined microscopy will be clear, making it very helpful for the researchers in the field.
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