- Last 7 days
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www.biorxiv.org www.biorxiv.org
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A
what is the box on the bottom right in A? Presumably it is the camera? A label would be helpful.
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Large-field tiled acquisitions highlight nuclei and clear cellboundaries, enabling the simultaneous observation of numerous cells.
how does this compare to just using standard brightfield imaging or other label-free techniques (e.g. DIC)? are there any features that quantitative phase picks up that other techniques miss?
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The precipitous drop in RI valuesmay be attributed to changes in cell density and volume as cells spread out,
Is it possible to determine computationally whether the drop in RI is only attributed to density change or something else?
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- Nov 2024
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www.biorxiv.org www.biorxiv.org
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acoustic focusing from an attached pi-ezoelectric transducer to confine cell motion to the center ofthe capillary
a citation describing acoustic focusing may be useful. is it known how well the particles are confined using this technique?
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Conclusion
very impressive technology!
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-865, nac Image Tech-nology
i may have missed it but what is the CMOS camera used for in the study?
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depending laser out-put
typo: depending on laser output
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0.26 – 3.2 mW/μm
this seems like a large amount of power, is it possible to theoretically determine the temperature increase that this light could induce in a transiting object?
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- Oct 2024
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www.biorxiv.org www.biorxiv.org
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RepresentativeEGFP and mStayGold nanocage photobleaching images. Images are scaled to the same absolute intensities
these are not the same nanocages being imaged at the three different timepoints, correct? would it be possible to image the same immobilized nanocages over time?
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we present a new method to compare fluorescent proteins on a molecule-by-molecule basis inphysiological conditions independent of relative expression levels or ratio measurements to other fluorescentproteins
As a tool to evaluate FPs in the future, what does this approach provide above what can be learned with absorbance/spectroscopy measurements performed on purified FPs?
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omparable peripheral cellregions of D-Mannitol-treated RPE cells expressing the indicated FP nanocage fusions
In the last panel (E138D), it looks like there are two dim nanocages and the rest are brighter. What could be the source of the variation?
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Thus, each nanocage carries sixty FPs (Fig. 1b) allowing comparison of the absolute fluorescence intensitybetween individual nanocages with different FPs
this is a very clever way to benchmark FPs that ensures a direct head-to-head performance comparison. By making comparisons between individual nanocages, this technique largely avoids confounds introduced by variations in expression level and cell-to-cell variability.
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- May 2024
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arxiv.org arxiv.org
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hen γ, whichsets the cell membrane tension, is low, i.e., the cell is soft,deformations are not very costly and can occur more eas-ily.
Is it possible to measure membrane tension using Brillouin microscopy or AFM? Will there be a direct linear correlation between the measured tension (or maybe Young's modulus of the cell) and the tendency for bistability?
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Evidence suggests that cell stiffness is apotential biomarker for cell malignancy with cancer cellstending to be softer [45–48]
Are there other biophysical properties of the cell that this technique is capturing that are not captured by cell stiffness alone? It would be really interesting (and clinically useful) if the bistability property was informative of the cell's propensity to become malignant
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FIG. 3.
Is there an optimal enclosure geometry that maximizes the differences in behavior?
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Within our model, we can switch cells between showinglimit-cycle dynamics and bistability either by changingphysical parameters like cell size or tension, or by chang-ing polarity dynamics like the size of protrusion fluctu-ations.
Would it be beneficial to do an e.g. RNAi screen to determine how different genes contribute to motility?
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Further experimentalstudies will also be necessary to validate our suggestionof cell properties as predictors of cell motility.
Do you think there could be clinical applicability? e.g. if you optimized the geometry to try and maximize the difference in motility b/w likely cancerous and non-cancerous cells, could this technique be used to predict pathology where other techniques may fail?
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While nuclear dynamics could be mod-eled with another phase field [9], we neglect it here forsimplicity and claim that cell center of mass is a fair ap-proximation of cell nucleus for most of the morphologieswe observe.
If a cell nucleus (or another subcellular structure) is close to the edge of the cell, would that impact cell polarity calculation?
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- Apr 2024
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www.biorxiv.org www.biorxiv.org
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A beter strategy would be to interleave epochs of intense (> 10 mW/mm2) illumina�on withepochs of darkness. Similarly, for voltage imaging of large samples (e.g. an en�re mouse heart), theexcita�on intensi�es may be low, leading to a loss of voltage sensi�vity.
That's a very valuable insight! Do you think there would be any benefit in simultaneous 1P and 2P illumination to increase SNR?
For interleaving stimulation, is the idea to use the kinetics from Fig 2d,e to determine the min period that can be used to illuminate the same spot?
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he complex photophysics of the FRET-opsin GEVIs suggest that future protein engineering efforts shouldbe accompanied, at a minimum, by a quan�fica�on of intensity-dependent voltage sensi�vity. Aninteres�ng avenue for future explora�ons would be to determine the photocycle basis for the intensity-dependent changes in voltage sensi�vity and voltage step-response waveforms shown in Figs. 1 and 2..CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeThe copyright holder for this preprint (whichthis version posted April 2, 2024.;https://doi.org/10.1101/2024.04.01.587540doi:bioRxiv preprint
Is it conceptually possible to engineer out the S2 excited state or change its excitation wavelength?
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This observa�on shows thatstatements of FRET-opsin voltage sensi�vity are only meaningful if illumina�on intensity is specified
That's very good to know!
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Figure 2
Maybe I missed it but the "I" and "II" labels in a,b,d,e are not explained in the legend. I assume I = low-power light regime and II = high-power?
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b) Fluorescence traces (le� axis, detrended ΔF/F; right axis, F) of from 1P (top) and 2P(botom) epochs of a single recording
In the 2P panel it looks like the spike amplitude paradoxically decreases as you increase laser power -- is that due to photobleaching?
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- Mar 2024
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www.biorxiv.org www.biorxiv.org
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suppressed LC
wording: "stGtACR2-expressing neurons are suppressed in the LC"?
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To ensure a smooth transition from therestraint stress to hot plate testing, we outfitted mice with a head-fixation bracket around bilateralfiber optic implants above the LC
it would be great to see a picture of the bracket!
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Figure
Minor legibility note on panels E and F: it is difficult to distinguish the injured vs non-injured mCherry and stGtACR2 line markers, especially since there is also blue shading on the "stim"
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Altogether, these resultssuggest rescued mu opioid receptor function may be a therapeutic target for the treatment chronicpain
could be promising but challenging from regulatory point of view given the opioid crisis.
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- Jan 2024
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www.biorxiv.org www.biorxiv.org
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(b)
Is it accurate to say that the retardance and orientation of infected cells was not different from uninfected? it may still be useful to plot for completeness. it may also be useful to note what kinds of investigations this unified fluorescence/label-free system allows that would not be possible (or be very challenging) with two separate systems. Overall, beautiful, useful paper!
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Our iterative alignmen
It would be interesting to learn from a user's perspective how long the initial assembly takes and how long day-to-day routine maintenance takes
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- Dec 2023
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www.biorxiv.org www.biorxiv.org
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Prediction of protein-protein binding sites using embeddings
it would be interesting to see more details here about which PPIs are predicted well/not well by SSEmb vs other models. Is a particular type of PPI consistently missed? It would also be interesting to identify what causes false positives.
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- Nov 2023
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www.biorxiv.org www.biorxiv.org
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otentially novel regulators of mTORC1
it's very cool that previously unknown genes contributing to a pathway can be identified!
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a-b.Comparison of feature embedding methodologies based on median AUC of binary classification of KOfrom WT for each genetic perturbation.
It looks like the majority of the genes have AUC ~= 0.5; what is the interpretation of that? Does that mean that most gene KOs tested do not exhibit a phenotype distinguishable from wild-type?
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he field of view images are then cropped around thecentroids of each of the segmented nuclei and masked by the corresponding cell segmentation mask tocreate tiles with a single cell in context
are pixels that are outside the mask painted with zeros on all channels?
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- Oct 2023
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www.biorxiv.org www.biorxiv.org
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Fig. 2
It would be interesting to know what is the limit of detection? i.e. the smallest voltage fluctuation that could be reliably detected?
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cell types
Another interesting cell type could be insulin-secreting beta cells. A similar ratiometric sensor was developed and tested in those cells in 2017: Schifferer, Martina, Dmytro A. Yushchenko, Frank Stein, Andrey Bolbat, and Carsten Schultz. “A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells.” Cell Chemical Biology 24, no. 4 (April 20, 2017): 525-531.e4. https://doi.org/10.1016/j.chembiol.2017.03.001.
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Fig. 5. Fluorescence–fluctuation correlation analysis uncovers strong electrical coupling among HEK293T and A375cells and weak coupling among MCF7 cells
This is a very cool result and it would be very interesting to see actual videos of the electrical coupling in action. e.g. would it be possible to see millisecond-scale membrane activity actually spreading via the gap junctions? Similar to what was done in Fig 3c of https://doi.org/10.1038/nmeth.3000?
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Fig. 5. Fluorescence–fluctuation correlation analysis uncovers strong electrical coupling among HEK293T and A375cells and weak coupling among MCF7 cells
This is a very cool result and it would be very interesting to see actual videos of the electrical coupling in action. e.g. would it be possible to see millisecond-scale membrane activity actually spreading via the gap junctions? Similar to what was done in Fig 3c of https://doi.org/10.1038/nmeth.3000?
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cell types
Another interesting cell type could be insulin-secreting beta cells. A similar ratiometric sensor was developed and tested in those cells in 2017: Schifferer, Martina, Dmytro A. Yushchenko, Frank Stein, Andrey Bolbat, and Carsten Schultz. “A Ratiometric Sensor for Imaging Insulin Secretion in Single β Cells.” Cell Chemical Biology 24, no. 4 (April 20, 2017): 525-531.e4. https://doi.org/10.1016/j.chembiol.2017.03.001.
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Fig. 2
It would be interesting to know what is the limit of detection? i.e. the smallest voltage fluctuation that could be reliably detected?
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www.biorxiv.org www.biorxiv.org
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It is the centre of mass that is used as the x/y targetcoordinates for the pipettes, so as to patch the centre of thecell, while the z-coordinate comes from the plane at whichthe cell is most in focus.
Do you have situations when the cell moves out of the way and you have to take the pipette out and try again?
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After each trial, pipettes are automatically cleaned in anenzymatic solution and then returned to their initial positions,allowing for reuse,
was wondering how often you guys encounter clogging from debris in the pipette after cleaning? we saw this happen a lot especially if the pipette was close to vertical position
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After each trial, pipettes are automatically cleaned in anenzymatic solution and then returned to their initial positions,allowing for reuse,
was wondering how often you guys encounter clogging from debris in the pipette after cleaning? we saw this happen a lot especially if the pipette was close to vertical position
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It is the centre of mass that is used as the x/y targetcoordinates for the pipettes, so as to patch the centre of thecell, while the z-coordinate comes from the plane at whichthe cell is most in focus.
Do you have situations when the cell moves out of the way and you have to take the pipette out and try again?
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www.biorxiv.org www.biorxiv.org
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Fig. 1
Can the model run at inference time with only a single image or z stack as input? Also will the model be publicly available at some point? Great work!!
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Fig. 1
Can the model run at inference time with only a single image or z stack as input? Also will the model be publicly available at some point? Great work!!
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