Intrinsic anti-Stokes emission in living HeLa cells.

Intrinsic fluorescence of biological material, also called auto-fluorescence, is a well-known phenomenon and has in recent years been used for imaging, diagnostics and cell viability studies. Here we show that in addition to commonly observed auto-fluorescence, intrinsic anti-Stokes emission can also be observed under 560 nm or 633 nm excitation. The anti-Stokes emission is shown to be spatially located on/in the mitochondria. The findings presented here show that sensitive imaging experiments e.g. single molecule experiments or two-photon excitation imaging can be compromised if intracellular anti-Stokes emission is not accounted for. On the other hand, we suggest that this anti-Stokes emission could be exploited as an additional modality for mitochondria visualization and cell viability investigation even in systems that are already labeled with commonly used fluorophores that rely on normal Stokes-based detection.

Introduction

Cell auto-fluorescence due to emission from intrinsic proteins, collagen, porphyrins, lipofuscin or adenine dinucleotides has been intensively investigated in the last decades and opened up possibilities for numerous applications like tissue, organelle or cell viability imaging [1-8]. The concentration distribution, pH, oxidation state of these molecules as well as temperature and polarity of the local environment were shown to influence fluorescence lifetime or/and quantum yield of naturally occurring fluorophores, making them useful intrinsic biomarkers in cells [4, 9–11]. Most commonly found in mitochondria, vesicles and extracellular matrix [4, 9, 12], these autofluorophores can help to shed a light on complex structures and functions of organelles without the need of introducing extrinsic fluorophores [13, 14].

In comparison to extrinsic fluorophores that are often used to specifically label various components of cells, the autofluorophores provide a direct relation between the emitter and its localization, while also ensure that neither the morphology nor the biological function are altered in the cells due to the introduction of external molecules. This advantage is utilized for cell viability studies, where changes in the mitochondria auto-fluorescence morphology are used to monitor cell stress and viability [15-17]. Additional applications are the label-free visualization of lysosomes [8, 18], chloroplasts [19] or tissue imaging [3, 7, 20]. Besides basic auto-fluorescence intensity measurements, more advanced methods based on auto-fluorescence lifetime [20, 21], polarization [22] or transient state kinetics [23] are also being used in both imaging and diagnostics.

Despite the number of advantages outlined above, the intrinsic auto-fluorescence in biological samples also has some drawbacks. First, most of the auto-fluorescence imaging techniques require blue or near-UV light at relatively high intensities for exciting the auto-fluorescent molecules. A consequence of this is the loss of excitation selectivity as a myriad of molecules absorb in this wavelength region. Additional issues are photo-toxicity and photo-bleaching.

Here, we demonstrate that intrinsic emission of cells can also be detected on the anti-Stokes side, that is detected at wavelengths shorter than the excitation light. Conceptually, our imaging modality is similar to coherent two-photon absorption microscopy [5, 24] or upconversion microscopy using lanthanide based nanoparticles [25, 26]. Advantages of our method is that cell viability imaging can be done on a conventional, sufficiently sensitive fluorescence microscope (e.g. a confocal single molecule fluorescence microscope, equipped with an avalanche photodiode as detector); it does not require the introduction of external emitters and requires significantly lower excitation intensity in comparison to coherent two-photon absorption microscopy.

Materials and methods

Cell cultures

HeLa cells (Sigma-Aldrich, 93021013, obtained directly from supplier) were grown in culture medium in 37°C, 5% CO2 and >95% humidity. We used a culture medium composed of Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12) with GlutaMaxTM) (Gibco), supplemented with 10% (V:V) Fetal bovine serum (FBS–Gibco). In most of the cases, unless indicated otherwise, the culture medium containing 0.0159g/L phenol red, which is used as pH indicator.

During the last two days before imaging, the cells were cultured on glass coverslips in the same medium, at 37°C, 5% CO2, The surfaces were in all cases preliminary coated with Poly-L-Lysine (Sigma Aldrich) by incubating it in PBS (Sigma Aldrich) at 1:20 ratio (V:V) during 20 minutes.

To image stressed cells, we used transient short-cold stress conditions (incubation at 4°C for 15 minutes), after which mammalian cells were shown to need several hours at physiological conditions to recover [27].

Mitochondrial labeling

The labeling of mitochondria with MitoTracker™ Green FM (Thermofisher) was done by incubation of the cell sample in 100 nM MitoTracker™ Green FM solution for 20 min in 37 oC, 5% CO2. After the incubation, the MitoTracker-Green FM solution was removed, and the cells were thoroughly washed (3 times with culture medium with phenol-red and 2 times with culture medium without phenol-red) before imaging the samples in culture medium without phenol-red.

Cell imaging

Series of label-free and MitoTracker-Green stained cells were imaged using a setup previously described in ref. [28]. However, a few modifications were introduced: a 30/70 mirror was used instead of a dichroic mirror. FF02-485/20-25, LL01-514-25, LL02-561-25, and LL01-633-25 band-pass filters from Semrock were used to clean-up the excitation beam. An OlympusUPlanSApo 100x 1.4 NA oil immersion objective was used. In the detection path LP02-488RE-25, LP02-514RE-25, BLP01-561R-25, BLP01-633R-25, SP01-561RU-25 and BSP01-633R-25 filters from Semrock were used to block the corresponding excitation light. For the fluorescence images 400 nm, 485 nm, 510 nm and 560 nm excitation lines were selected from a pulsed continuum laser (77 MHz, 10 μW– 150 μW) SuperK Extreme EXB-6) with a SuperK SELECT wavelength selector from NKT Photonics. For the anti-Stokes imaging, a 561 nm Cobolt Jive from Cobolt (~1.3 mW before the objective) and 633 nm line of HRRR170-1 HeNe laser (~2.3 mW before the objective) from Thorlabs were used. Emission spectra were recorded by directing the emission light towards a spectrograph from Princeton Instruments (Princeton Instruments SPEC-10:100B/LN eXcelon CCD camera, SP 2356 spectrometer, 300 grooves/mm). Recorded anti-Stokes emission spectra contained significant fraction of scattering, which was eliminated by subtracting normalized reference spectra measured from the water drop on cover slip (see SI, S1 Fig). For anti-Stokes emission images BLP01-633R-25 and FF01-578/16-25 filters from Semrock were added in the detection path to suppress the scattering.

Cell images were recorded using an avalanche photodiode from PerkinElmer (CD3226) and sample scanning piezo stage (PI 517.3CL from Physik Instrumente) at controlled room temperature of 23°C. The anti-Stokes emission of HeLa cells has been studied on ≈50 cells from 6 independently grown cell cultures throughout the course of half a year. The same signal localized in/on mitochondria has been detected in 100% of the healthy HeLa cells imaged and the anti-Stokes emission spectra recorded on 10% of them (randomly selected) were all peaked at around 590 nm.

All images were recorded as 90 x 90 μm images (256 x 256 pixels) with a scanning speed of 1ms/pixel and cropped for better visual representation after acquisition. Images were processed using Matlab and Fiji software.

Results and discussion

Fig 1A to 1D shows auto-fluorescence images of label-free HeLa cells, cultivated in standard culture medium composed of DMEM, phenol-red and 10% FBS, at various excitation wavelengths. This figure demonstrates that normal auto-fluorescence (Stokes emission) from living HeLa cells can be detected over a large range of the visible spectrum, using a variety of excitation wavelengths. The spatial distribution of the auto-fluorescence signal indicates that the intrinsic fluorophores are not evenly distributed in the cytosol but predominately located in subcellular structures, most likely in/on mitochondria, as it was reported previously [1, 4]. Interestingly, when imaging the anti-Stokes emission, where we excite the cell sample at 560 nm or 633 nm and monitor emission at shorter wavelengths, images with similar spatial distributions are obtained (Fig 1E and 1F).

Fig 1

Fluorescence images of label-free HeLa cells.

A to D) Auto-fluorescence upon excitation with 400 nm, 485 nm, 510 nm or 560 nm. E and F) Anti-Stokes emission upon excitation with 560 nm or 633 nm. The spectral detection ranges for both the auto-fluorescence and anti-Stokes emission are indicated in the images. Excitation wavelength (Ex.) long-pass filter (LP) band-pass filters (BP), short-pass filter (SP).

To confirm that both auto-fluorescence and anti-Stokes emission are originating from the mitochondria regions, HeLa cells were stained with MitoTracker-Green (excitation maximum ~490 nm, emission maximum ~510 nm), a well-established commercial fluorophore used for mitochondria identification in cells. Fig 2C shows that MitoTracker-Green fluorescence (Fig 2A) overlaps very well with the anti-Stokes emission (Fig 2B), confirming that the anti-Stokes emission originates from regions where the mitochondria are present. Differences in the composite images Fig 2C and Fig 2F correspond to movement of mitochondria in the living cells during the ~10 min time delay between the two imaging scans (auto-fluorescence and anti-Stokes, see also S2 Fig). Additionally, changing the excitation wavelength for the MitoTracker-Green stained HeLa cells to 560 nm (MitoTracker-Green does not absorb at this wavelength) yields again a good correlation between the auto-fluorescence and the anti-Stokes emission (see S3 Fig), proving that both signals originate from the same organelles.

Fig 2

A) MitoTracker-Green fluorescence, (B) anti-Stokes emission and C) composite images of MitoTracker-Green stained HeLa cells. D) Auto-fluorescence, E) anti-Stokes emission and F) composite images of label-free HeLa cells.

In order to start to understand the spectral properties of the anti-Stokes emission (Fig 2B and 2E) and to demonstrate the difference between auto-fluorescence (Fig 2D) and MitoTracker-Green emission (Fig 2A), emission spectra were recorded with a spectrometer coupled to the confocal microscope. Fig 3A shows that the fluorescence spectrum from the MitoTracker-Green stained cells was identical to the spectrum of pure MitoTracker-Green dissolved in water, with in both cases emission spectra peaking around 510 nm upon 485 nm excitation. Due to the high brightness of MitoTracker-Green, no significant contribution from the auto-fluorescence can be detected in the MitoTracker-Green stained HeLa cells. This can also be seen in Fig 3B, 3C and 3D where it is clear that auto-fluorescence in label-free HeLa cells only is observed at significantly higher excitation intensities, compared to the excitation intensities used to visualize the MitoTracker-Green. The auto-fluorescence spectrum is also clearly different from the MitoTracker-Green spectrum, with an emission maximum below 500 nm, as can be seen in Fig 3A. On the other hand, the anti-Stokes emission (ex. 633 nm) that is detected in the label-free HeLa cells has an emission maximum close to 590 nm. The two additional controls, exciting MitoTracker-Green stained cells and MitoTracker-Green alone in water, prove that the anti-Stokes emission does not originate from MitoTracker-Green and that presence of MitoTracker-Green in the HeLa cells has no significant impact on the spectral shape of the anti-Stokes emission.

Fig 3

A) Normalized auto-fluorescence, MitoTracker-Green fluorescence and anti-Stokes emission spectra of MitoTracker-Green stained and label-free HeLa cells, (B to D) Differences in fluorescence intensity (ex. 485 nm, em. LP 488 nm) of MitoTracker-Green stained and label-free cells. The respective excitation powers are indicated in the figure. All figures have the same intensity scale range.

Due to vast amount of different compounds present in living cells, it is not at all trivial to identify the compound(s) responsible for the observed anti-Stokes emission, let alone the mechanistic origin. Potential mechanisms that could explain the origin of the anti-Stokes emission are “hot band” excitation of thermally (Boltzmann) populated states [29], excited state annihilation (singlet or triplet) [30, 31], chemiluminescence [32] or consecutive photon absorption through a long lived intermediate state [33]. Coherent two-photon excitation can be ruled out as the intensity of the continuous wave laser used in our experiments was at least 1000 times lower than the peak intensity of pulsed Ti:Sapphire lasers often used for two-photon microscopy [34]. Additionally, with a potential two-photon excitation wavelength around 317 nm, one would expect a more blue shifted emission maximum, or at least some contribution from auto-fluorescence fluorophores in the blue part of the spectrum, which is not the case (Fig 3A). Due to the spatial location of the anti-Stokes emission, one could speculate that it is related to molecules localized in or on the mitochondria. However, the emission peak around 590 nm does not correspond to any of the commonly known endogenous auto-fluorescent compounds in the mitochondria [4, 9]. It would be beneficial to measure anti-Stokes emission excitation spectrum, lifetime and excitation power dependency to certainly rule out two photon absorption possibility, identify electronic transitions and narrow down on other potential mechanisms. However, practically it is extremely challenging, as emission intensity is weak and photobleaching is too fast for these demanding experiments. Furthermore, identifying the exact molecule, let alone the mechanism, responsible for this anti-Stokes emission is also challenging not only because of technical limitations, but also due to a sheer number of potential autofluorophores and their complex photophysical behavior depending on configuration, oxidation state or response to specific microenvironment. Lack of possibilities to prepare and measure reliable negative controls in live cells then demand ex vivo ‘deconstruction’ of mitochondria molecule by molecule, which is tedious and at too time consuming at this point.

Here we speculate that the compounds, capable of emitting the anti-Stokes emission, associated with the mitochondria, are intrinsic to the cells. To verify this hypothesis, the only other potential source of the anti-Stokes emission, the cell growth medium, should also be investigated. Rich in proteins, sugars and other vital cell growth components, this medium also often contains additives that absorb light in the visible range, e.g. the pH indicator phenol red. Despite its common use, little is known about interaction of phenol red with living cells. For a long time, it was assumed that this weakly fluorescent molecule does not interfere with any observable cell function, as its cellular uptake was considered minimal. However, modern quantification methods based on 125I labeled phenol red in cell culture medium showed that 0.31 picogram of phenol red can be accumulated per HeLa cell after just 2 hours of incubation in cell growth medium with ~10 μM phenol red [35]. Although S4 Fig shows that the phenol red containing medium by itself is able to give a weak emission with a slightly more red-shifted maximum around 610 nm, we do not believe that this is the origin of the anti-Stokes emission in the label-free HeLa cells. This is based on the fact that when HeLa cells were grown for 4 weeks (~30 division cycles) in phenol red-free media, similar anti-Stokes emission was observed as shown previously (see S4 Fig).

Here we propose to use this intrinsic anti-Stokes emission, from yet unknown compounds, to monitor cell stress in HeLa cells, even in fluorescently stained samples. As most of the autofluorophores are localized in mitochondria, autofluorescence is often used to evaluate cell stress/viability [36-39]. In case of extreme stress—cell death—membrane potential (including mitochondria membrane potential) is lost, membranes become permeable and previously localized molecules become free to diffuse throughout the cell volume.

This application is demonstrated in Fig 4, showing the well localized intrinsic anti-Stokes emission signal of healthy HeLa cells and its co-localization with the cell auto-fluorescence. Repetitive scans (Fig 4A–4F, approx. 6 min time between frames) of the same cell region showed minimal photobleaching and thereby confirm that the experimental conditions are not a relevant stress factor for the cells.

Fig 4

A-F) Multiple consecutive scans of healthy, label-free HeLa cells, G) auto-fluorescence, H) anti-Stokes emission and I) composite (images of stressed HeLa cells (15 minutes exposure to 4°C).

Upon stress, by incubation at 4°C for 15 minutes, the well-localized anti-Stokes emission in the healthy HeLa cells (Fig 4A–4F) becomes completely delocalized over the whole cell volume (see Fig 4H). Similar as for the healthy cells, the auto-fluorescence and anti-Stokes emission remain co-localized (see Fig 4G–4I). This opens opportunities to study cell viability, stress and mitochondria shape using anti-Stokes emission.

The possibility to exploit anti-Stokes emission to obtain information about the stress level of cells has several advantages. First of all, the signal is intrinsic and does not require the addition of any fluorophores to the live cells. Its detection is furthermore done with distinct optical conditions (in comparison to regular cell dyes) which makes it compatible with many other fluorophores used in cells. It also enables the use of one more fluorophore/modality in multi-labeled samples and thereby provides more information about each cell composing the sample.

These features are very timely as anti-Stokes emission microscopy using upconverting fluorophores is becoming an important and highly studied modality in bioimaging that can be detected with any microscope setup using sensitive avalanche photodiodes.

Conclusion

We have demonstrated that anti-Stokes emission from an intrinsic compound, associated with the mitochondria in HeLa cells, can be used as an additional channel for monitoring cell viability in both stained and unstained cells. Using red light excitation intensities, significantly lower than those used in coherent two-photon microscopy, yellow emission can be detected. The anti-Stokes emission signals overlap well with conventional auto-fluorescence from non-stained HeLa cells. Our results also demonstrate that attention must be paid to ensure that intrinsic anti-Stokes emission is accounted for, especially when using other anti-Stokes imaging modalities like upconversion emission from lanthanide based nanoparticles [40], optically-activated delayed fluorescence [33] or coherent two-photon absorption microscopy [24, 41].

Example of high intensity scattering from cover slip (black line) being subtracted from anti-Stokes emission spectrum of unstained HeLa cells.

(TIF)

Click here for additional data file.

Two consecutive anti-Stokes emission scans (A and B) and their composite (C) (measured on non-stained HeLa cells, ~10 min delay between the scans), showing differences between the images arising due to cell movement.

(TIF)

Click here for additional data file.

Auto-fluorescence (A), anti-Stokes emission (B) and composite (C) images of MitoTracker-Green stained HeLa cells. Note that at 560 nm MitoTracker-Green does not absorb and we are monitoring the intrinsic auto-fluorescence.

(TIF)

Click here for additional data file.

Normalized anti-Stokes emission spectra (ex. 633 nm) of various samples compared to Stokes fluorescence (ex. 560 nm, orange spectrum) of phenol red (A). Cells here were grown in phenol red containing medium and exchanged to phenol red-free medium before imaging. Label-free HeLa cells, cultivated in phenol red-free medium for 4 weeks (B).

(TIF)

Click here for additional data file.

(ZIP)

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21 Oct 2019

PONE-D-19-25352

Intrinsic anti-Stokes emission in living HeLa cells

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Reviewer #1: The paper is coherent and describes an advancement in imaging techniques. The following minor grammar corrections are suggested:

- 31: "Concentration, distribution" -> "The concentration distribution"

- 38: "autofluorophores do not" -> "autofuorophores

Reviewer #2: The manuscript titled ‘Intrinsic anti-Stokes emission in living HeLa cells’ presents an observation of anti-stokes yellow emission from the mitochondria of HeLa cells. While this was an interesting observation to start with, unfortunately the authors overlooked existing literature and failed to verify the possible source and possible underlying mechanism of this emission. The manuscript is well written and data is presented concisely.

1) First of all, it should be mentioned that auto-fluorescence from mitochondria is well known and extensively studied. Mitochondria is the powerhouse of a cell and host an enormous amount of electron carriers. In literature, most of the auto-fluorescence has been assigned to electron carriers like NAD, NADP, FAD etc which shuttles between various emissive states depending on their oxidation status. As the author conclusively show that the emission originates in mitochondria, it’s advisable that the authors discuss their findings in this context throughout the paper.

2) Redox related anti-stokes emission from cellular auto-fluorescence has been reported by Melissa C. Skala and co-workers (PNAS 2007) and followed by many other groups. The 2-photon auto-fluorescence imaging is emerging as a powerful tool for optical redox imaging of cancer cells to predict malignancy. It should be noted that FAD emission occurs in the yellow region after exciting with a red laser. The fluorescence lifetime can be measured to differentiate between bound and un-bound states of these electron carriers. It’s recommended that the author to consider these literature for the best of their interest.

3) The authors argue that 590nm emission does not correspond to any know molecules but cite a very old reference (ref 9) which actually says cellular auto-fluorescence in the 500-600 nm spectral region is mostly associated with flavins. Although it’s agreeable that the exact maxima at 590nm is not well known and FAD emission is more blue-shifted from 590nm, but care must be taken to rule out flavins. Flavins are present in diverse form of molecules and oxidation states, especially in mitochondria and fluorescence is extremely sensitive to microenvironment. How many cells did the authors measure to confirm that 590nm emission is a ubiquitous in the mitochondria of HELA cell and there is no spectral shift from 590nm? Why did the authors choose 633nm? Where is the excitation maxima for that 590nm emission, is it 633nm? An excitation spectra would be valuable as it provides clue about the absorption and electronic states of the molecule of interest. Therefore it’s not convincing that authors rule out 2-photon excitation based on the their laser intensity.

4) The authors have randomly thrown in two possible mechanism of hot-band and dark states without any clear argument. Hot band emission should have an intense stokes component in red-region along with the anti-stokes yellow emission. A dark state emission is long-lived and should have a fluorescence lifetime an order higher in magnitude. Did the authors observe any of these?

5) The data presented in figure 4 is unclear and not explained. Are the scans shown in A-F is from the same ROI? If so, what was the time interval? Why the images looks significantly different from each other? Are the scan:A-D and scan:G-I from same ROI? Why the signal was delocalized, especially if it originates in mitochondria?

6) Note: ref 4 and ref 12 are same

In summary, the observation presented in the current manuscript is interesting. However the presented data is largely unexplained, looks preliminary and not fit for publication in its current state. Authors are encouraged to perform additional experiments to characterize the phenomena thoroughly, perform an extensive revision of their manuscript in the context of present literature, clearly present the research advancement and finally improve the scientific rigor of the data analysis and discussion for the interest of their future readers.

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Reviewer #2: No

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12 Nov 2019

Reviewer 1:

The paper is coherent and describes an advancement in imaging techniques. The following minor grammar corrections are suggested:

- 31: "Concentration, distribution" -> "The concentration distribution"

- 38: "autofluorophores do not" -> "autofuorophores

We are thankful for comments and positive feedback. We included these suggestions in a revised manuscript.

Reviewer 2:

The manuscript titled ‘Intrinsic anti-Stokes emission in living HeLa cells’ presents an observation of anti-stokes yellow emission from the mitochondria of HeLa cells. While this was an interesting observation to start with, unfortunately the authors overlooked existing literature and failed to verify the possible source and possible underlying mechanism of this emission. The manuscript is well written and data is presented concisely.

1) First of all, it should be mentioned that auto-fluorescence from mitochondria is well known and extensively studied. Mitochondria is the powerhouse of a cell and host an enormous amount of electron carriers. In literature, most of the auto-fluorescence has been assigned to electron carriers like NAD, NADP, FAD etc which shuttles between various emissive states depending on their oxidation status. As the author conclusively show that the emission originates in mitochondria, it’s advisable that the authors discuss their findings in this context throughout the paper.

We are grateful for reviewer’s comments. Stokes-side autofluorescence in multiple cell organelles, including mitochondria, is indeed well-known and studied in detail. We briefly discussed most notable examples in the first two paragraphs of the manuscript. To stress the extensive research done specifically on electron carriers in mitochondria we added more references throughout the manuscript.

However, we also want to emphasize that with this paper we are presenting completely new way of mitochondria visualization using anti-Stokes autofluorophores. We strongly believe that this fluorescence is not due to two-photon absorption (see arguments in the following comment/answer), in contrary to majority of papers published on anti-Stokes autofluorescence to this day. Therefore, we believe that more extensive review of two-photon absorption based autofluorescence microscopy could be confusing and most importantly misleading to the readers.

2) Redox related anti-stokes emission from cellular auto-fluorescence has been reported by Melissa C. Skala and co-workers (PNAS 2007) and followed by many other groups. The 2-photon auto-fluorescence imaging is emerging as a powerful tool for optical redox imaging of cancer cells to predict malignancy. It should be noted that FAD emission occurs in the yellow region after exciting with a red laser. The fluorescence lifetime can be measured to differentiate between bound and un-bound states of these electron carriers. It’s recommended that the author to consider these literature for the best of their interest.

We acknowledged reviewer’s recommendation and added the reference to the manuscript.

While two-photon absorption induced emission of FAD was indeed well studied by multiple research groups (including paper by Melissa C. Skala) we believe and want to stress that anti-Stokes emission reported here is not due to coherent (simultaneous) two-photon absorption.

First, as we argue in the manuscript (lines 172-177), the power of our continuous wave 633 nm HeNe laser line we are using for the excitation is at least three orders of magnitude lower than peak intensity of pulsed lasers commonly used in two photon absorption microscopy. Furthermore, with a potential two-photon excitation wavelength around 317 nm, one would expect a more blue shifted emission maximum, as 317 nm light is expected to efficiently excite well known and most abundant autofluorophores like NADH and FAD, with broad emission peaks around 460 and 540 nm, respectively. However, we did not observe any emission bellow 560 nm (Fig 3A in the manuscript), making two-photon absorption very unlikely hypothesis to explain the anti-Stokes emission we report.

3) The authors argue that 590nm emission does not correspond to any know molecules but cite a very old reference (ref 9) which actually says cellular auto-fluorescence in the 500-600 nm spectral region is mostly associated with flavins. Although it’s agreeable that the exact maxima at 590nm is not well known and FAD emission is more blue-shifted from 590nm, but care must be taken to rule out flavins. Flavins are present in diverse form of molecules and oxidation states, especially in mitochondria and fluorescence is extremely sensitive to microenvironment. How many cells did the authors measure to confirm that 590nm emission is a ubiquitous in the mitochondria of HELA cell and there is no spectral shift from 590nm? Why did the authors choose 633nm? Where is the excitation maxima for that 590nm emission, is it 633nm? An excitation spectra would be valuable as it provides clue about the absorption and electronic states of the molecule of interest. Therefore it’s not convincing that authors rule out 2-photon excitation based on the their laser intensity.

We performed more extensive literature review on emission spectra of various autofluorophores including more recent publications. However, to the best of our knowledge, no autofluorophores (including varying oxidation states) with the emission peak around 590 nm are reported so far.

Even though flavins are most often associated with emission around 500-600 nm, number of other intrinsic and extrinsic molecules (for example cytochromes, lipopigments or phenol red) were reported to be emissive in 550-600 nm range. Furthermore, even NADH or metalloproteins cannot be safely ruled out, as their photophysical properties were reported to be highly environment dependent. In the manuscript we put special care to rule out extrinsic fluorophores like phenol red, as its contribution can be easily verified. However, finding the exact type of molecules responsible for anti-Stokes emission reported here becomes very difficult when it comes to intrinsic fluorophores. Difficulty comes not only from the sheer number of potential autofluorophores or lack of negative controls, but also complex photophysical behavior depending on configuration, oxidation state or response to specific microenvironment. Furthermore, we believe that more than one species could be responsible for observed anti-Stokes emission as anti-Stokes emission originating in mitochondria also can be observed using 560 nm excitation (Figure 1E), making identification of specific autofluorophores even more complicated.

Therefore, for now in this paper we chose to focus on applications and potential artefacts rather than identify exact type of molecule responsible for this emission.

We did not do an elaborate statistical analysis on spectral shape and peak position of observed emission as it wasn’t the scope of our work and the overall Anti-stokes emission is dim and subject to photobleaching, limiting the quality of the emission spectra and hence the analysis of emission maxima and other spectral properties. However, we measured multiple anti-Stokes emission spectra on live, non-stained HeLa cells, focusing on different cells and their mitochondria. We observed minimal spectral variations between different mitochondria and cells.

It would certainly be beneficial to measure anti-Stokes emission excitation spectrum, lifetime and excitation power dependency to certainly rule out two photon absorption possibility, but it is practically extremely challenging. Photobleaching is the biggest problem, limiting the possibility to measure excitation spectra, lifetimes and excitation power dependencies. We added these arguments to the manuscript.

4) The authors have randomly thrown in two possible mechanism of hot-band and dark states without any clear argument. Hot band emission should have an intense stokes component in red-region along with the anti-stokes yellow emission. A dark state emission is long-lived and should have a fluorescence lifetime an order higher in magnitude. Did the authors observe any of these?

When it comes to an origin of observed anti-Stokes emission, multiple photophysical pathways could be considered, including, but not limited to “hot band” excitation of thermally (Boltzmann) populated states, excited state annihilation (singlet or triplet) or consecutive photon absorption through a long lived intermediate state. We also added chemiluminescence (line 171 in the manuscript (1)) as another plausible route.

So far we showed that it’s not likely to be due to two-photon absorption (see previous comments for the arguments). To address “hot band” absorption, we have measured emission on the Stokes side (as suggested by the reviewer) and observed weak, broad and featureless emission spectrum. Even though observed tail of emission spectrum could be a strong argument in case of a sample with single type of fluorophores, it is not conclusive in mitochondria, where multiple autofluorophores have been reported.

Other possible mechanisms could be investigated in more detail by analyzing Stokes and anti-Stokes emission lifetimes. However, as we pinpoint in the previous comment, it is not practically possible to record it due to extremely low fluorescence intensity and fast bleaching.

Since we are physically limited by the number of photons that can be recorded before bleaching, in this paper we only put an effort to show that mechanism responsible for this anti-Stokes emission is different than commonly reported 2-photon absorption.

5) The data presented in figure 4 is unclear and not explained. Are the scans shown in A-F is from the same ROI? If so, what was the time interval? Why the images looks significantly different from each other? Are the scan:A-D and scan:G-I from same ROI? Why the signal was delocalized, especially if it originates in mitochondria?

Micrographs in Figure 4 A-F show consecutive scans of live, label-free HeLa cells and represent the same ROI. As we are using APD for detection instead of camera, it takes around 6 minutes to complete the scan. Meanwhile non-fixed, live cells and their organelles were observed to move (S2 Fig. in SI) significantly. We have now specified these parameters in the revised manuscript (highlighted additions at page 10).

G-I scans correspond to different ROI than A-F, but rather show different distribution of anti-Stokes emitting molecules in the cell after cell stress was induced. Multiple reports have shown that cellular response to stress can be investigated following mitochondria dynamics – fission/fusion. As most of the autofluorophores are localized in mitochondria, autofluorescence are often used to evaluate cell stress/viability (2-5). In case of extreme stress - cell death - membrane potential (including mitochondria membrane potential) is lost, membranes become permeable and previously localized molecules become free to diffuse throughout the cell volume. To make these points clearer we have now added more discussion on cell viabilityon page 10 line 202-209.

Figure 4 demonstrates one of the main advantages of the method we are reporting here, showing that cell viability can be followed by looking at anti-Stokes emission. That not only allow investigate label-free cell viability without use of intense UV irradiation but also allows observation of cellular response to stress in stained samples. This wasn’t the case previously as Stokes autofluorescence is always buried under significantly more intense emission of external fluorophore in labeled cells.

6) Note: ref 4 and ref 12 are same

References have been corrected.

In summary, the observation presented in the current manuscript is interesting. However the presented data is largely unexplained, looks preliminary and not fit for publication in its current state. Authors are encouraged to perform additional experiments to characterize the phenomena thoroughly, perform an extensive revision of their manuscript in the context of present literature, clearly present the research advancement and finally improve the scientific rigor of the data analysis and discussion for the interest of their future readers.

We share the wish of the reviewer to fully understand the molecular and mechanistic origin of this new emission. However, we also strongly believe that this first observation of the phenomena and its potential applications merits publication, even though these academically interesting questions are not yet resolved. We hope that our reply and changes to the manuscript have satisfactory addressed the reviewers’ questions.

1. Ciscato LFML, Weiss D, Beckert R, Bastos EL, Bartoloni FH, Baader WJ. Chemiluminescence-based uphill energy conversion. New J Chem. 2011;35(4):773-775.

2. Westermann B. Bioenergetic role of mitochondrial fusion and fission. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2012;1817(10):1833-1838.

3. Youle RJ, van der Bliek AM. Mitochondrial fission, fusion, and stress. Science. 2012;337(6098):1062-1065.

4. Aubin JE. Autofluorescence of viable cultured mammalian cells. Journal of Histochemistry & Cytochemistry. 1979;27(1):36-43.

5. Dittmar R, Potier E, van Zandvoort M, Ito K. Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence. Tissue engineering Part C, Methods. 2012;18(3):198-204.

Submitted filename: Response to Reviewers.docx

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5 Dec 2019

PONE-D-19-25352R1

Intrinsic anti-Stokes emission in living HeLa cells

PLOS ONE

Dear Ms. Kacenauskaite,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The main point of concern of the paper that was raised by one of the referees is the important issue of the statistical significance of the new spectral features and that part is being brushed aside.

==============================

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Additional Editor Comments

The authors seem to not interested to address the main concern of one of the reviewers on the lack of data and statistical significance of the work in their revised submission. Concern on the confidence level on new spectral signature need to be justified before the paper may be published.

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: The authors have partly addressed the comments previously made. Although the manuscript has been improved, the study appears to have limited impact in its current state. The authors argued that the signal is too weak and photo-bleaching too high to perform statistical analysis, lifetime measurement or even to identify the excitation maximum. This argument would raise a doubt about the significance or usefulness of this newly observed anti-stokes emission in this field, where other reported emissions are considerably strong. It would be advisable to take more time to perform additional experiments to characterize the new emission, thus, making the paper more valuable for the readers.

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8 Jan 2020

Reviewer #2: The authors have partly addressed the comments previously made. Although the manuscript has been improved, the study appears to have limited impact in its current state. The authors argued that the signal is too weak and photo-bleaching too high to perform statistical analysis, lifetime measurement or even to identify the excitation maximum. This argument would raise a doubt about the significance or usefulness of this newly observed anti-stokes emission in this field, where other reported emissions are considerably strong. It would be advisable to take more time to perform additional experiments to characterize the new emission, thus, making the paper more valuable for the readers.

The main concern of reviewer #2 in this second review is the question of impact or significance of our results based on the argument that if the signal is too weak to measure fluorescence lifetime or excitation spectrum, the signal might not be significant and thus relevant to be investigated.

We disagree with this reasoning. As clearly shown in the data of the manuscript, the mitochondria signal resulting from this new anti-Stokes imaging is unambiguous, reproducible and can even be used to visualize cell stress.

Anti-Stokes emission microscopy using upconverting fluorophores can be used today by anyone with a microscope setup equipped with sensitive avalanche photodiodes and is thus becoming an important and highly studied modality in bioimaging. We believe that it is important to report intrinsic anti-Stokes emission from the HeLa cells in the range that so far was always considered to be completely “background-free”, in particular when this signal is informative by indicating the stress level of the cell.

To emphasize the significance of reported anti-Stokes emission we added the following to the manuscript:

The possibility to exploit anti-Stokes emission to obtain information about the stress level of cells has several advantages. First of all, the signal is intrinsic and does not require the addition of any fluorophores to the live cells. Its detection is furthermore done with distinct optical conditions (in comparison to regular cell dyes) which makes it compatible with many other fluorophores used in cells. It enables the use of one more fluorophore/modality in multi-labeled samples and thereby provides more information about each cell composing the sample.

These features are very timely as anti-Stokes microscopy using upconverting fluorophores is becoming an important and highly studied modality in bioimaging that can be detected with any microscope setup using sensitive avalanche photodiodes.

We share the wish of the reviewer to fully understand the molecular and mechanistic origin of this new emission and we agree that the lifetime and excitation spectra measurements, that reviewer #2 wish for, could be a good complement to our results from the fundamental point of view. However, getting these results requires very different instrumentation and emission intensities than what is required for imaging. We admit that we are not able to obtain these data as we depend on and are physically limited by the instrumentation that is available.

Indeed, to record a reliable excitation spectrum of observed anti-Stokes emission, extremely powerful whitelight laser/lamp is necessary (output power of at least 10 mW per excitation width can be estimated based on our results and experience with 633 nm line of HeNe laser). We have previously attempted to record an excitation spectra using SuperK ExtremeEXB-6 with Super K SELECT wavelength selector from NKT Photonics, but no signal was observed due to too low excitation power (less than 1mW per excitation width). SuperK Extreme EXR-20 supercontinuum laser produced by the same company (which to our knowledge is the brightest commercially available whitelight laser on market to this day), also would not be sufficient as it “only” reaches ~3-7 mW/nm. That leaves us with only a few very specialized places in the world where such experiment could be carried out. Same technical limitations, together with fast bleaching, also apply to fluorescence lifetime measurements.

Furthermore, we also do not have hope that recorded excitation spectra could actually help to identify the molecule, let alone the mechanism, responsible for this anti-Stokes emission. As it was previously also noted by the reviewer, difficulty comes not only from the sheer number of potential autofluorophores but also complex photophysical behavior depending on configuration, oxidation state or response to specific microenvironment. Lack of possibilities to prepare and measure reliable negative controls in live cells then demand ex vivo ‘deconstruction’ of mitochondria molecule by molecule, which is practically impossible. Fluorescence lifetime measurements maybe could show if triplet state formation is involved in the process, but otherwise also in practice would not be helpful to identify anti-Stokes emitting species.

Editor: The main point of concern of the paper that was raised by one of the referees is the important issue of the statistical significance of the new spectral features and that part is being brushed aside.

The authors seem to not interested to address the main concern of one of the reviewers on the lack of data and statistical significance of the work in their revised submission. Concern on the confidence level on new spectral signature need to be justified before the paper may be published.

The second point raised by both reviewer and the editor concerns statistical significance of our work. In the first review reviewer #2 specified this questions as: “How many cells did the authors measure to confirm that 590nm emission is a ubiquitous in the mitochondria of HELA cell and there is no spectral shift from 590nm”.

To ensure that observed intrinsic anti-Stokes emission is a general property of HeLa cells we imaged multiple cells from different batches under the same conditions. We have in this 2nd revision added further details on the number of cells and cell cultures investigated in the study. The following text has been added (and is highlighted in the submitted manuscript):

The anti-Stokes emission of HeLa cells has been studied on �50 cells from 6 independently grown cell cultures throughout the course of half a year. The same signal localized in/on mitochondria has been detected in 100% of the cells imaged and the anti-Stokes emission spectra recorded on 10 % of them (randomly selected) were all peaked at around 590 nm.

While this number of samples and spectra certainty is insufficient to derive any statistical significant quantitative statements e.g. of intensity of the emissions as function of cell conditions, we find it sufficient to support the qualitative scope of the paper: 1) reporting a new anti-Stokes imaging modality for mitochondria in HeLa cells and 2) drawing attention to this emission for other researchers applying imaging modalities for which this emission can interfere.

We hope that our reply and changes to the manuscript have satisfactory addressed the reviewers’ and editors concerns.

Submitted filename: Response to Reviewers.docx

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5 Feb 2020

PONE-D-19-25352R2

Intrinsic anti-Stokes emission in living HeLa cells

PLOS ONE

Dear Ms. Kacenauskaite,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In particular, address the couple of more revision of the manuscript as per the comments of the Reviewer#2.  This would greatly enhance the article and we would be happy to consider the revised version favorably.

We would appreciate receiving your revised manuscript by Mar 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Debabrata Goswami

Academic Editor

PLOS ONE

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Reviewer #2: (No Response)

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**********

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #2: The authors have improved the manuscript in this version. With the following minor revisions, it may be recommended for publication and further review may not be required.

1) It seems that the authors are physically limited by avilable facility, which often happens with scientific studies. However, if those inteded experiments can reveal imprtant clue about the emission, it needs to be discussed in the manuscript as a limitaion of the current study and possible future experiments for others in the field.

2) Why 4C, 15m was chosen for cell stress experiment may be briefly explained in the result/method section.

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Reviewer #2: No

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20 Feb 2020

Reply to editor and reviewers:

Reviewer #2: The authors have improved the manuscript in this version. With the following minor revisions, it may be recommended for publication and further review may not be required.

We are grateful for Reviewer’s#2 thorough suggestions and comments towards inproved manuscript. Reviewer comments were considered in detail and addressed bellow:

1) It seems that the authors are physically limited by avilable facility, which often happens with scientific studies. However, if those inteded experiments can reveal imprtant clue about the emission, it needs to be discussed in the manuscript as a limitaion of the current study and possible future experiments for others in the field.

We have briefly mentioned the current limitations for further spectoscopic measurements in lines 190-191 and added more details in the revised manuscript.

2) Why 4C, 15m was chosen for cell stress experiment may be briefly explained in the result/method section.

We added a short explanation on cell stress in Materials and methods section.

We hope that our reply and changes have satisfactory addressed the reviewers’ comments and manuscript can now be accepted for publication.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Mar 2020

Intrinsic anti-Stokes emission in living HeLa cells

PONE-D-19-25352R3

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4 Mar 2020

PONE-D-19-25352R3

Intrinsic anti-Stokes emission in living HeLa cells

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