Identification of optimal fluorophores for use in the Drosophila embryo

  1. Bernardo Chapa-y-Lazo
  2. Thamarailingam Athilingam
  3. Prabhat Tiwari
  4. Prachi Pathak
  5. Shaobo Zhang
  6. Sophie Theis
  7. Timothy E Saunders

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Abstract

The use of fluorescent proteins has become ubiquitous throughout the life sciences as a key method for understanding molecular, cellular and tissue scale processes. Genetically encodable fluorophores have enabled stable genetic lines to be generated in a large array of organisms. There are now suites of fluorophores available, particularly in the green and red spectra. Yet, which fluorophore works best in vivo can depend on a range of factors, both extrinsic ( e . g . pH, temperature) and intrinsic ( e . g . photobleaching, brightness). While fluorophores have been well characterised in cell culture, such measures within in vivo systems are more limited. Here, we present a quantitative screen of nine green and eight red fluorophore lines in Drosophila , with the fluorescent protein expressed from the same genomic location and imaged under identical conditions. We analyse the expression of the fluorophores in both early and late Drosophila embryos. We provide a quantitative analysis of the bleaching and folding rates. We find amongst the green fluorophores that the suitable choice – e . g ., mEGFP, mNeonGreen, mStayGold - depends on timing and imaging requirements. For the red fluorophores, mScarlet-I performed consistently well, though no particular fluorophore stood out as ideal under all conditions. These results provide a powerful database for selecting optimal fluorophores for imaging in the Drosophila embryo in green and red channels.

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    Referee #4

    Evidence, reproducibility and clarity

    Major criticisms

    The manuscript by Chapa-y-Lazo et al. is confusing. It does not provide precise information about the three photostable monomers developed by different research groups. Please read the review (ref. 17) carefully. The monomeric version analyzed in this study was developed by Ivorra-Molla et al. and should be referred to as StayGold-E138D. This variant excels in dispersibility (monomericity), photostability, and molecular brightness (the product of the molar extinction coefficient and the fluorescence quantum yield). However, when analyzed in animal cells, StayGold-E138D is practically dim, and its brightness is poor. This can be seen in Figures 2, 3, S5, and S6 of the manuscript. The maturation efficiency of the chromophore is not so good in fly embryos. On the other hand, Ando et al. independently developed a monomeric version of StayGold called mStayGold at FPbase and Addgene. Therefore, I think that the authors should acknowledge that their analysis of StayGold monomer behavior is still incomplete. Additionally, the evolution tree of StayGold shown in Figure S2 is incorrect. The side-by side comparison of the three monomeric variants of StayGold, including StayGold-E138D and mStayGold, is documented in a recent preprint. Comparison of monomeric variants of StayGold | bioRxiv

    Minor comments

    Line 84 z-stacks were acquired using a spinning disc confocal microscope. Line 100 we collected a z-stack through each embryo. Line373 We analyzed the slices from 7 µm to 20.5 µm depth. Line 390 Depth 9 µm to 21 µm was analyzed. It is not clear what "z-stack" means in these sentences.

    Significance

    Nothing in particular.

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    Referee #3

    Evidence, reproducibility and clarity

    Chapa-y-Lazo and colleagues report the detailed characterization of a number of different genetically-encoded fluorescent proteins in Drosophila embryos. The screening and selection of an appropriate fluorescent protein for imaging tasks is an important and often neglected part of experimental design, and datasets such as this one will be extremely useful in guiding decision making for other users. The manuscript is well-written and carefully controlled for different developmental stages and nicely compares the most pertinent properties of FPs such as brightness, photobleaching, and folding time. There would be a couple of additional experiments that would be nice to see but are not strictly necessary for improving the paper as-is, but might be helpful points to include in the discussion.

    Comments:

    1. All fluorophores in this study were fused to H2Av, at the same insertion site, which makes for a nice and easy comparison between lines. However, histone-binding proteins can sometimes behave unpredictably when tagged with different things and in addition it would be interesting to see if the fusion protein affects the FP properties in anyway. I.e. would sfGFP be brighter than mEmerald when bound to a CAAX sequence or some other organelle? It would be impractical for this study to re-do all the FPs, but the top two hits could be interesting and would potentially be quite interesting if there is a significant difference in behaviour between FPs when bound to different proteins/cellular compartments. Else maybe a mention in the discussion?

    2. Another way to compare the fluorophore folding time would be to selectively bleach a portion of the embryo at the same developmental stage and measure the time it takes for each FP to recover to the same intensity as the rest of the embryo. This could potentially control for any delay for developmental reasons.

    3. Some of the lines in the figure plots could be a bit thicker - purple and pink when overlapping are hard to distinguish.

    Significance

    This manuscript will be quite useful for those who are deciding between which fluorescent protein or combination to use for their live-imaging work, and additionally has created a number of useful fly strains in the process. It will hopefully also start a discussion about proper characterization and quantification of fluorescent reporters under different conditions, ideally before all the effort to generate an entirely new genetically modified animal is performed.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript Saunders and colleagues benchmark the brightness, folding speed and photostability of a variety of red (8 versions) and green fluorescent proteins (9 versions), which have been widely used for in vivo imaging. They fused each protein to histone2Av, cloned the fusion into attP constructs and inserted them in the Drosophila genome at the same genetic location. Thus, expression levels can be compared. Nuclei at embryonic cycle 14 were imaged, segmented and fluorescence was quantified. At this early stage the maturation kinetics of the fluorophore can particularly influence its fluorescence intensity.

    Additionally, stage 15-16 embryos were imaged at the dorsal side to quantify brightness. As the histone promoter is active in all cells, the fluorescence in the nuclei of all cell types can be quantified. Brightness differences between the different proteins vary a bit between both experiments, likely taking folding versus brightness into account. Generally, sfGFP, mEGFP, mEmerald as well as mStrawberry and mScarlet are the brightest. Next, developmental movies were recorded starting at gastrulation to estimate the folding rates of the different proteins. No large differences of the relative fluorescence increase over time were reported. To estimate photostability, embryos were imaged ventrally shortly before the onset of gastrulation for 2 or 4 hours with high laser intensity and the fluorescence intensity was recorded. Consistent with data in the literature, StayGold is the most photostable green protein, although it is not the brightest from the start, likely to also slower folding. From the red proteins mRFP and mCherry are good choices for long-term imaging.

    In summary, these results do not bring huge surprises but are still valuable for future choice of protein tagging for imaging. Best green proteins are mEGFP, mNeonGreen, mStayGold with differences in brightness vs stability. For red, no protein is the clear winner, mScarlet-I is good in folding and brightness but others are better for photostability.

    Major comments:

    1. Form the methods, it is not clear which promoter is used to drive expression of the histone2Av fusions. I assume this is not UAS but the histone promotor/enhancer. Please clarify.
    2. From text is not always what the purpose of the experiment is. For example, it is not mentioned that developmental movies were recorded for the data related to Figure 3 to calculate folding, while bleaching was measured in the movies related to Figure 4. In contrast to simple single time points in Figures 1 and 2.

    Minor comments:

    1. Please add time to movie 2 and rotate it such that anterior is to the left and dorsal it up.
    2. Lines 141 - 144 should refer to Figure 3D not 4D.
    3. Movies 3 and 4, please insert time.

    Significance

    Experiments are well performed and the finding are useful to guide the future choice of fluorophores in Drosophila and possibly other model organisms. Results are not very surprising, as the major finding that StayGold is photostable (but not the brightest) is not entirely new but still reassuring. It is particularly nice to have the differences confirmed by well controlled side-by-side measurements in Drosophila. This will likely guide many Drosophila researchers to tag their favourite protein with StayGold in the future.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This is an outstanding study of high practical value, which provided the systematic performance evaluation of in vivo fluorophores under the same condition in the field of Drosophila developmental biology. By site-integrating 17 green and red fluorescent proteins into the same genomic locus and evaluating their fluorescent intensity (early/late embryos), folding time and photostability on the same imaging platform, this provides a powerful database for researchers.

    Major comments:

    Q1: All fluorescent proteins are fused with histone H2Av. Will this nuclear localization expression pattern mask the performance differences of fluorescent proteins in other subcellular structures (such as cell membranes, cytoplasm, and cytoskeleton)?

    Q2: How do the authors ensure precise developmental synchrony among different embryos to avoid the influence of developmental time differences on fluorescence intensity and folding curves?

    Q3: In this study, the authors conduct a quantitative screen of nine green and eight red fluorophore lines in Drosophila. A logical and valuable extension of this work would be a systematic evaluation of newer fluorescent proteins, including promising candidates like mBaoJin, mScarlet3, and mScarlet3-H.

    Q4：The study does not discuss the potential for fluorescent proteins to interfere with biological function. Although the proteins were expressed from the identical genomic location, variations in their size, structure, or fusion design may influence the target protein's localization or activity.

    Lines 145-147 "The only profile that did not fit well to this phenomenological function was mStayGold which did not display a clear reduction in its rate of intensity increase". What is the reason that causes mStayGold to fail to fit well? Is it related to the unique structure?

    Lines 154-156 "For the red fluorophores, the intensity profiles were more varied (Fig. 3E). They could not be reduced to a single curve, unlike the green fluorophores (Fig. S7B). The phenomenological function I(t) did not fit the curves well". Compared with green fluorophores, the intensity profiles of red fluorophores vary greatly, what's the major factors drive this difference?

    Lines 206 "Fluorophores including mEGFP and mEmerald displayed a secondary peak in intensity around an hour after experiment initiation. This is consistent with a change in the rate of protein production". What is the mechanism behind the secondary peak, and why is it distinctly observed only in mEGFP and mEmerald?

    Minor comments:

    Line 143 "curve I(t) = I0 tanh (t-tin/ts) (Fig. 4D, Methods)". It's not Fig. 4D, but Fig. 3D.

    Line 145 "time is smallest for Superfolder GFP and longest for mNeonGreen (Fig. 4D)". Not Fig. 4D, but Fig. 3D.

    Line159 "mScarlet" must be replaced with "mScarlet-I".

    Significance

    The systematic performance evaluation of in vivo fluorophores under the same condition will give a comprehensive guidence when choosing fluorescent proteins in the field of Drosophila developmental biology.

  6. Version published to 10.1101/2025.09.29.678560 on bioRxiv