Fluorescence to measure light intensity

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.


Decision Letter, initial version:
Dear Ludovic, Your Resource, "Fluorescence to measure light intensity", has now been seen by three reviewers.As you will see from their comments below, although the reviewers find your work of considerable potential interest, they have raised a number of concerns.We are interested in the possibility of publishing your paper in Nature Methods, but would like to consider your response to these concerns before we reach a final decision on publication.
We therefore invite you to revise your manuscript to address these concerns.We found the technical concerns to be fairly straightforward, although we think you should spend more time explaining and justifying your choice of dyes and proteins given how possibilities exist (see related comments from refs 1 and 2).
We think the most substantive changes may come in the presentation of the paper.We ask that you revise with a biologist reader in mind, paying careful attention to defining jargon, and making sure that readers come away with a clear sense of how to implement the protocols in their own lab and achieve accurate results.We think this will be aided by the addition of functioning software tools and detailed Supplementary Protocols.
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Reviewers' Comments:
Reviewer #1: Remarks to the Author: A. The article provides two detailed protocols, which exploit fluorescence to enable the retrieval of the spatial distribution of the excitation light intensity for different fluorescence methods, especially fluorescence microscopy, in different commonly used spectral regions.One protocol utilizes the monoexponential fluorescence intensity decays/rises of fluorescent actinometers absorbing and emitting in different wavelength regions.The other protocol exploits an absorbing chemodosimeter in combination with a common photostable fluorophore for the back-calculation of the spatial ditribution of the excitation light intensity.The potential for these methods for the calibration of fluorescence imaging systems is subsequently demonstrated.
B. Both methods rely on previously described chemodosimeters (literature cited in the article).This is absolutely mandatory for both methods as the intention is here to provide the community of fluorescence microscopists with available (or at least reproducible) tools for the determination of this very important parameter of fluorescence imaging systems.This approach is original and very significant, given the currently ongoing activities on improving the comparability and quality of fluorescence microscopy data driven by the international network QuaRep.C. The approach is valid and the quality of the data and the presentation are mostly very good.Here is some (not much!) place for improvement As addressed in section F. D. Generally, the use of statistics and treatment of uncertainties is well done.What could be helpful for the broad audience of fluorescence microscopists are some generalestimates of achievable measurement uncertainties like "with this approach an uncertainty of xxx% can be achieved".If I had to guess, the uncertainty is in the order of 15-20% which is fairly good given the relative ease of both methods and the tools.E. The conclusions drawn are well supported by the data and experiments done and meet the high standards regarding robustness, validity, and reliability.Suggetsed minor improvements are addressed in section F. F. Suggested improvements i.) To ease the reproducibility of the approach, I suggest to provide HPLC data for the commercial dyes to characterize their purity.This enables other users of these approaches to make sure that the dyes they have at hand are really comparable.
ii.)This approach relies on the assumption of excitation wavelength independent fluorescence quantum yields of the chemodosimeters or the dye rhodamine B. Although I believe that this critrium is met by the suggested tools, this prerequisite should be explicitly addressed.As proof the authros should provide graphs comparing the normalized fluorescence excitation spectra of the fluorophores (equaling the absorption spectra of the emissive species) and the absorption spectra (wavelength-dependent absorption factors, as the fluorescence intensity is proportional to absorption and not absorbance).This could be also included for one dye in Figure 1, panel d.iii.)The choice of rhodamine B is a bit problematic.This dye is well known for its temperaturedependent fluorescence quantum yield which could posibly cause problems here as thereby, an increase in temperature automaticalyl results in a reduction in fluorescence intensity.Therefore, this dye property should b explicitly addressed wit one or two references and it mus be checked whether and to which extent this temperature dependence can affect the intensity measurement.Dyes like rhodamine 6G and rhodamine 101 would have been a better choice here as these dyes have a temperatureindependent fluorescece quantum yield as the fluorescence quenching channel, the dialkyl amino group is bridged an sterically hindered from rotation (use of julolidine groups) G.The refernces are appropriate.
H. The MS is very well written and the abstract and conclusion sections are appropriate.What could be helpful fro the general understanding of this approach is a very brief descrition o which parameters affect a fluorescence signal from the sample and the instrument side (including one or two references, i.e., of IUPAC recommendations) and an explanation of the meaning of fluorescence excitation spectra.
Reviewer #2: Remarks to the Author: This manuscript describes two methods and several dyes/fluorescent proteins that can be used to calibrate fluorescence excitation and field uniformity in terms of irradiance.This is not an easy task and this work provides a solution with a manuscript that is very thorough and in depth.The authors present dyes that cover the entire relevant wavelengths for many different applications from the UV to the near IR.Methods are detailed and there are significant supplemental materials.They demonstrate that results are consistent with measurements using traditional methods such as power meters or spectrometers.
The manuscript is very detailed, supplemental materials give in depth information about dye synthesis, purification of Dronpa, sample preparation and dye characterization.The figures present a lot of information.The characterization of the LED light source with DDAO shown in Figure 5 is particularly impressive.The figure captions contained a lot of details but the formatting was very difficult to follow.I suggest moving some of the detail as text directly on the figure panels themselves or as tables that are easier to follow.Important sample preparation information was buried in the extensive supplemental materials section.For example, the fact that some of the dyes must be dissolved in acetonitrile and require a completely glass sample holder and many dye solutions are only stable for a short time period.
While this manuscript shows a large body of work and broad application it is very technical in nature and will not be of interest to a broad readership in its current format.A more focused manuscript specifically geared towards biological imaging with detailed easy to follow workflows and protocols would be of more broad interest.The authors mention a software tool "ploty data application" and python scripts but these are only mentioned in the discussion and are not described in detail in the manuscript.In addition, it is mentioned that end users can implement the protocols in 1 hour but clear workflows and protocols are not presented.For example, can the biologists enter key parameters about the dye used and the instrument settings that would automatically calculate irradiance from images?Having many dyes covering from UV to near IR is impressive but the fact that the samples all have to be prepared and characterized individually and many are not stable over the long term is problematic.
The manuscript uses a lot of terms that are not common to biologists making it difficult to follow.These terms should be clearly defined.The manuscript is not written with enough information to know exactly what would need to be done for each experiment.Often partial information is provided and the supplemental materials are referred to for key details.Some samples require specialized holders and diffusion of dyes lead to error and the authors claim estimates of irradiance are within an order of magnitude which is not sufficient.
Since Dronpa works so well could a single solution with multiple fluorescent proteins be used to cover the spectral range instead of using different dyes and different sample preparations?Could the proteins/dyes be immobilized on the glass slide to remove diffusion artifacts for calibration of intensity and field uniformity for experiments with thin samples such as cultured cells?In turn, the dyes/fluorescent proteins could be immobilized in a gel (as mentioned at the end of the discussion) with similar refractive index as biological tissue to mimic thicker samples and calibrate irradiance with depth.It would be good to see this kind of data in the manuscript.Finally, it is not clear if the end user has to characterize the dyes when they use them on their systems of if the characterization that was done in this manuscript can be used to gain key dye parameters?Then users would just need to prepare the dye/fluorescent protein sample, image the sample and perform analysis to calculate irradiance.
Minor Points: b and c are mixed up in the Figure 2 caption.Dyes are mentioned in paragraph two on page 3 but then defined later in paragraph three.Define each dye when it is first mentioned.
Reviewer #3: Remarks to the Author: (A) In this manuscript Jullien et al. present a set of cleverly designed and comprehensive methods for the qualitative and quantitative measurement of light using fluorescence -either directly (first protocol) or indirectly (second protocol).They show that their approach is applicable in a very wide spectral range but combining different approaches.They apply their methods to wide-field and confocal microscopyeven deep within samples -and demonstrate their methods' use for the wavelength-resolved measurement of light intensity.
(B) This topic is of very high interest for a number of communities both in the photochemistry and in the micropscopy field and falls in a very "exciting" time of recent photochemical developments over a wide spectral range which are just waiting to be applied properly for example in microscopy setups.Indeed, local (!) quantification of photons has always been an underinvestigated issue in this respect.
(C) The storyline is extremely succinct -but due the the succinctness also requires quite some background knowledge for the broader understanding of the implications.The arguments for the usefulness are very clearly stated and I support those arguments.The data appear to be of high quality to me -with few exceptions: What is the explanation for the unusual shape of the curve in Figure 1d?(D) I do not see anything to complain here (E) I do not see anything to complain here (F1) I strongly suggest that -where possible -the photoreaction is depicted in the graphics of the main manuscript.This will make it much easier to follow the different photoreaction concepts compared to only describing it with words as it is done now.Also, the structure of DDAO should be shown in the main text.
(F2) Is the fluorescence quantum yield of DDAO the same over the given spectrum?See comment about the unusual curve shape above.
(F3) Many of the underlying principles become only clear when reading the second part of the supporting information.At least the underlying theory of fitting photochemical conversions with simple exponential functions and the meaning of sigma and tau should be briefly discussed in the main text as too many colleagues in the field of photochemistry bother too little about the correct mathematical treatment.True line shapes can be much more complicated with competing chromophores, more than one reaction path and large spectral shifts in the photoprocess.Have the authors confirmed -where possible -that they are dealing with only one, clean photochemical process?(F4) Typos: p3 "can travel to much of the whole irradiated area" should probably read "can travel too much out of the whole irradieated area"?p4 "As demonstrated in ->the<-Supporting Information" Figure 1 only fits to the text if the labels "b" and "c" are exchanged (G) Nothing to complain about here.
(H) I especially liked how the relevance and applicability for each of the methods is explicitly stated along with references.The text is rather short but very clear to read.I am wondering if it might be too short for the many different target communities I can imagine to immediately understand the high value of these methods.

Author Rebuttal to Initial comments Decision Letter, first revision:
Dear Ludovic, Thank you for submitting your revised manuscript "Fluorescence to measure light intensity" (NMETH-A51899A).It has now been seen by the original referees and their comments are below.The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Methods, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
In response to reviewer 2, we ask that you organize and present the material as clearly as possible for the biologist reader (with some microscopy expertise) to implement with as much ease as possible.Please leave the more expert technical details well-organized in the supplement.
We are now performing detailed checks on your paper and will send you a checklist detailing our editorial and formatting requirements in about two weeks.Please do not upload the final materials and make any revisions until you receive this additional information from us.
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Sincerely, Rita
Rita Strack, Ph.D. Senior Editor Nature Methods Reviewer #1 (Remarks to the Author): Since this is a revision, I can only confirm that all points raised by me ad the other reviewers have been excellently considered in the revised MS and SI and have been addressed in the rebuttal in a detailed and clear way.There is not much to add as to congratulate the authors to this important work.
Reviewer #2 (Remarks to the Author): The revised manuscript is significantly improved.Thank you for all of the changes.
I still find the figures and captions difficult to follow.Having to refer to a supplemental table is not ideal.
I understand the space limitations with the current manuscript format but one of the most valuable aspects of the paper is the method of making the measurements and the software.This information is still difficult to find within very long supplemental materials.This is a excellent quality paper on an important topics that is in depth and thorough.However, it is highly technical and I do not think it will be of general interest to the broad Nature Methods readership.
Reviewer #3 (Remarks to the Author): I see that Jullien et al. have responded very well not only to my comments but also to the comments of the other reviewers.As for the answers to my comments I am fully satisfied and have no further suggestions or concerns.I also think that the comments of the other reviewers were addressed properly.I believe that this was a "healthy" revision process and am sure that the revised version will be a very fine manuscript in Nature Methods that will receive much attention.
-Reviewer #3 (Remarks to the Author): I see that Jullien et al. have responded very well not only to my comments but also to the comments of the other reviewers.As for the answers to my comments I am fully satisfied and have no further suggestions or concerns.I also think that the comments of the other reviewers were addressed properly.I believe that this was a "healthy" revision process and am sure that the revised version will be a very fine manuscript in Nature Methods that will receive much attention.
We thank the reviewer for her/his comments.

Final Decision Letter:
Dear Ludovic I am pleased to inform you that your Article, "Fluorescence to measure light intensity", has now been accepted for publication in Nature Methods.Your paper is tentatively scheduled for publication in our December print issue, and will be published online prior to that.The received and accepted dates will be March 6, 2023 and October 2, 2023.This note is intended to let you know what to expect from us over the next month or so, and to let you know where to address any further questions.
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