Coherent Raman microscopy detects nucleolar defects through amide I peak shifts originating from β-sheets: an application to visualizing ongoing cellular senescence
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
Cellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence by obtaining molecular fingerprint signals in nucleoli using a CARS microspectroscopic system. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.
Article activity feed
-
The ultra-multiplex CARS microscope has been described in detail in a previous study [29].
How was the instrument calibrated prior to use? Were polystyrene beads the main standard sample used, as described in 29?
-
CARS
More details about determining what constitutes a significant shift, particularly in comparison to the expected error of the instrument, would be welcome!
-
We focused our analysis on two protein secondary structures, the α-helix and the β-sheet. The reason is that the spectral changes were caused mainly by the formation of unfolded/misfolded proteins and their aggregation. Amyloid-like aggregates induce extensive conformational changes from α-helices to β-sheets during their formation. Images were created for the ratios 𝑔2, 𝕩/(𝑔1, 𝕩 + 𝑔2, 𝕩).
Out of curiosity, would the higher wavenumber shift be due to the secondary structure creating a more constrained conformation, and smaller bond length? Not sure if I have the right logic but am curious!
-
These are: (1) the peak wavenumbers were higher than those of the control cells, and (2) these peak wavenumbers were also higher than those in the cytoplasm of the same cell (typically at 1653 cm-1). In detail, the peak shifts in the nucleoplasm were not distinct compared to those in the nucleoli.
How much higher were they (cm-1)?
-
In this study, we observed binuclear senescent cells with CARS microscopy and found the peak of the nucleolar amide I band shifted to a higher wavenumber in the spectra.
Did you also notice or evaluate shifts in other peaks (e.g. Amide III)? Why or why not? Reference 45 mentions Amide III, for instance.
Also, what was the threshold to determine if a shift was real, especially in comparison to the expected error (cm-1) of the instrument?
-
The vibrational mode of water (1640 cm-1) had already been removed.
Did removing this mode increase the noise of this region of the spectrum (if by subtraction)?
-
One interesting use case would be to be able to detect cells on the pathway to growth arrest or cell death before a dramatic cellular phenotype as a way to evaluate phenotypes of only the "healthy" cells (if that is even a thing given we don't traditionally assay for more than a few dimensions). That said, even with the dramatic cellular phenotype, the shift is minor so perhaps early signals of poor cell health would not be detectable?
-
However, the molecular basis of this technique is not well understood.
I find this sentence to be a bit vague. What do you mean by "molecular basis"? While the setup and hardware for the types of Raman are different, in all cases, molecular vibrations are interrogated.
-
-