On 2017 Apr 03, Dmitri Rusakov commented:

We thank the Reviewer for following up the story. Below are our point-by-point reply to his latest set of comments. This reply and the manuscript version revised accordingly appeared to satisfy the journal Editorial Board and the other reviewer(s). We appreciate that this reviewer might not have therefore received the full set of explanations shown below. We however urge him to look at the published paper and its Supplementary data as these do contain material answers to his key questions.

Reviewer #1

Q: The authors have taken a rather minimalist approach to my suggestion that the fluorescence anistropy measurements be analysed and presented in greater detail in the MS.

A: There must have been a misunderstanding. In response to the original Reviewer's comments, we have provided a full point-by-point response with extended explanations, added quantitative evidence for the two-exponential approximation, and provided a full summary Table for the FLIM characteristics over six different areas of interest. This is precisely what was requested in the original comments.

Q: In addition, in considering this revision, additional questions have arisen. I therefore give a more detailed and prescriptive list of the data that needs to be shown. The following conditions are of interest: 1) free solution 2) with extracellular dye a) measurement inside cell b) measurement over neuropil c) measurement over synapse d) measurement inside pipette 3) with intracellular dye a) measurement inside cell 4) no dye a) measurement of autofluorescence over neuropil b) measurement inside soma For each of the above conditions, please show: 1) full fluorescence time course -1 to 12 ns 2) full anisotropy time course -1 to 12 ns 3) specimen traces 4) global averages 5) fit of global average 6) timing of the light pulse should always be indicated (I assume it occurs at 1ns, but this must be made explicit)

A: We note that all the requested information is contained, in the shape of single-parameter outcomes, in the original figures and Tables. We also note that in healthy brain slices autofluorescence (two-photon excitation) is undetectable. With all due respect, we did not fully understand the grounds for requesting excessive primary material: the process of analysing anisotropy FLIM data involves automated, pixel-by-pixel data collection and curve fittings representing tens of thousands of single-pixel plots at all stages of the data processing. Presenting such data does not appear technically feasible. However, we have added some extensive primary-data examples, as requested, to illustrate:

(a) Fluorescence decay in parallel and perpendicular detectors at different viscosity values (Fig. S1a);

(b) Instrument response for the two-detector system (Fig. S1b), indicating that it has much faster dynamics than the anisotropy decay;

(c) Anisotropy decay data in slice tissue after dye washout - indicating a specific reduction of the fast (free-diffusion) rather than slow (membrane-bound) molecular component (Fig. S1c);

(d) AF350 anisotropy decay examples recorded in a free medium, intracellular compartment, extracellular space in the synapse and neuropil extracellular space (Fig. S1d).

Q: It remains a good idea to try the same measurements with a second dye.

A: AF350 is the smallest bright fluorophore which shows no lifetime dependency on physiological cellular environment. It is therefore the best candidate to explore the movement of small ions or neurotransmitter molecules such as glutamate in similar environment. We have tried other fluorophores such as AF594, which is three times heavier, more prone to photobleaching and more likely to bind to cell membranes in slices, all of which makes interpreting the data more difficult. We believe that the experimental evidence obtained in the present study has led to a self-contained set of conclusions that are of interest to the journal readership. Repeating the entire study with a different dye, or a different animal species, or a different preparation, or else, might be a good idea for future research.

Q: Why is the rise of the anisotropy not instantaneous in Fig. 1B? I couldn't find any mention of temporal filtering.

A: The rising phase is influenced by the instrument response to the femtosecond laser pulse in raw lifetime data, which remained unmodified in the presented plots. Signal deconvolution would produce instantaneous anisotropy (while increasing noise in raw data) which is not our preferred way of presenting the data. We have added this explanation to the text.

Q: Assuming that the light pulse occurs at 1ns in Fig. S1A, why is the anisotropy shown so high? I would have thought that it should have decayed over a time constant (to about 1/e) by the point where the data shown, yet it is only reduced by about 15%. Was some additional normalisation carried out that I missed? If so it should certainly be removed and the true time course shown.

A: The example in question shows a direct comparison between decay shapes in baseline conditions and after photobleaching: for illustration purposes, the graph displays a fragment of raw decay data, including the instrument response (without deconvolution). The latter at least doubles the apparent decay constant, plus the fast component has a y-offset of 0.2-0.3 due to the slow-component. These concomitants make the fast decay appear slower but this is irrelevant for the purposes of this particular raw data illustration (in contrast, Table S1 summary data are obtained with the instrument response de-convolved and removed). The text and figure legend has been expanded to explain this.

Q: In case it is not clear, I am interested in the possibility of the fast component in fact containing two components. The data do not allow me to evaluate this possibility.

A: Whilst we appreciate personal scientific interests of the Reviewer, we see no scientific reasons, in the present context, to try and find 'the possibility' of fast anisotropy decay sub-components: we simply refer to all molecular sub-populations showing distinctly fast anisotropy decay as one free-diffusion pool.

Q: Does Scientific Reports required the authors to provide access to the original data upon publication? What is the authors' position on this?

A: All original data, including tens of thousands of single-pixel FLIM data plots at different analysis stages, etc. are available from the authors on request.

On 2017 Mar 25, Boris Barbour commented:

Below I show the key parts of my first and second reviews of this paper, which reports a very interesting and powerful optical technique for probing the microscopic properties of fluid compartments in the brain. I felt that greater detail of the analysis should be shown to support fully the conclusion of slowed diffusion in the extracellular space and synaptic cleft. It will be apparent that some questions unfortunately only occurred to me upon reading the first revision. The authors only responded to some of the points raised before the paper was published without my seeing it again. In particular, no global average anisotropy time courses are shown and the timing of the excitation pulses remains rather mysterious.

First review

This MS reports an extremely interesting approach to providing quantitative information about the diffusion of small molecules in micro-compartments of brain tissue - potentially resolving the intracellular and extracellular spaces, as well as providing information about diffusion within the synaptic cleft.

The basis for the approach is to measure the relaxation of fluorescence polarisation following two-photon excitation of small compartments. If polarised exciting light is used the emitted light is also polarised, as long as the orientation of the fluorophore remains unchanged. However, as the molecule undergoes thermal reorientations, that polarisation is lost. The authors use this technique to measure the diffusion of a small molecule - alexa fluor 350.

A subsequent section of the MS reports some synaptic modelling, applying the tissue/free ratio obtained for the fluorophore to the modelled glutamate. However, the important and by far the most interesting part of the MS is the diffusion measurement. I would be happy for the MS to consist solely of an expanded and more detailed analysis of these measurements, postponing the modelling to another paper.

My main comments relate to the analysis, presentation and interpretation of these diffusion measurements. The authors report that the relaxation time for the polarisation displays two phases - a rapid phase, which is somewhat slowed in brain tissue, and a slow phase attributed to membrane binding. But the authors do not illustrate the analysis of the fast component. As this is critical, a good deal more detail should be shown.

A first issue is whether there are additional bound/retarded states other than "fixed". To examine this the authors should show the fit to the average relaxation; it is important to be able to verify that there is a single exponential fast component rather than some mixture (it may not be possible to tell with any certainty). Some examination of the robustness and precision of the fitting would also be desirable.

The authors should also characterise the variation between different measurements of the same compartments. The underlying question here is how the various decay components might vary as, for instance, the ratio of membrane to extracellular space varies across different measurement points.

I think the authors need to give more thought to the possibility that some of the slowing they observe arises from fluorophore embedded in the membrane without being immobilised. I don't see how this can be easily ruled out - certainly the two-photon resolution does not permit distinguishing the membrane and fluid phases in the neuropil. Additionally, how would the authors rule out adsorption onto some extracellular proteins?

The reason I raise these points is because I have at least a slight difficulty with the interpretation. A slowing of molecular rotation of 70% (intracellular) suggests to me that a large fraction of fluorophores, essentially 100%, must be in direct contact with some larger molecule. This seems quite extreme even in the crowded intracellular space. I have similar reservations about the synaptic cleft and extracellular space. Is at least 50% and 30% of the volume of these spaces really occupied by macromolecules? There may be somewhat reduced diffusion due to a boundary layer at the membrane or near macromolecules. Do estimates of the thickness of such a boundary layer exist (and its effect on diffusion)?

Second review

The following conditions are of interest:

1) free solution

2) with extracellular dye

a) measurement inside cell

b) measurement over neuropil

c) measurement over synapse

d) measurement inside pipette

3) with intracellular dye

a) measurement inside cell

4) no dye

a) measurement of autofluorescence over neuropil

b) measurement inside soma

For each of the above conditions, please show:

1) full fluorescence time course -1 to 12 ns

2) full anisotropy time course -1 to 12 ns

3) specimen traces

4) global averages

5) fit of global average

6) timing of the light pulse should always be indicated (I assume it occurs at 1ns, but this must be made explicit)

It remains a good idea to try the same measurements with a second dye.

Why is the rise of the anisotropy not instantaneous in Fig. 1B? I couldn't find any mention of temporal filtering.

Assuming that the light pulse occurs at 1ns in Fig. [1E], why is the anisotropy shown so high? I would have thought that it should have decayed over a time constant (to about 1/e) by the point where the data shown, yet it is only reduced by about 15%. Was some additional normalisation carried out that I missed? If so it should certainly be removed and the true time course shown.

In case it is not clear, I am interested in the possibility of the fast component in fact containing two components. The data do not allow me to evaluate this possibility.

(Note to moderators: as the copyright holder of my reviews, I am entitled to post them.)

On 2017 Mar 25, Boris Barbour commented:

Below I show the key parts of my first and second reviews of this paper, which reports a very interesting and powerful optical technique for probing the microscopic properties of fluid compartments in the brain. I felt that greater detail of the analysis should be shown to support fully the conclusion of slowed diffusion in the extracellular space and synaptic cleft. It will be apparent that some questions unfortunately only occurred to me upon reading the first revision. The authors only responded to some of the points raised before the paper was published without my seeing it again. In particular, no global average anisotropy time courses are shown and the timing of the excitation pulses remains rather mysterious.

First review

This MS reports an extremely interesting approach to providing quantitative information about the diffusion of small molecules in micro-compartments of brain tissue - potentially resolving the intracellular and extracellular spaces, as well as providing information about diffusion within the synaptic cleft.

The basis for the approach is to measure the relaxation of fluorescence polarisation following two-photon excitation of small compartments. If polarised exciting light is used the emitted light is also polarised, as long as the orientation of the fluorophore remains unchanged. However, as the molecule undergoes thermal reorientations, that polarisation is lost. The authors use this technique to measure the diffusion of a small molecule - alexa fluor 350.

A subsequent section of the MS reports some synaptic modelling, applying the tissue/free ratio obtained for the fluorophore to the modelled glutamate. However, the important and by far the most interesting part of the MS is the diffusion measurement. I would be happy for the MS to consist solely of an expanded and more detailed analysis of these measurements, postponing the modelling to another paper.

My main comments relate to the analysis, presentation and interpretation of these diffusion measurements. The authors report that the relaxation time for the polarisation displays two phases - a rapid phase, which is somewhat slowed in brain tissue, and a slow phase attributed to membrane binding. But the authors do not illustrate the analysis of the fast component. As this is critical, a good deal more detail should be shown.

A first issue is whether there are additional bound/retarded states other than "fixed". To examine this the authors should show the fit to the average relaxation; it is important to be able to verify that there is a single exponential fast component rather than some mixture (it may not be possible to tell with any certainty). Some examination of the robustness and precision of the fitting would also be desirable.

The authors should also characterise the variation between different measurements of the same compartments. The underlying question here is how the various decay components might vary as, for instance, the ratio of membrane to extracellular space varies across different measurement points.

I think the authors need to give more thought to the possibility that some of the slowing they observe arises from fluorophore embedded in the membrane without being immobilised. I don't see how this can be easily ruled out - certainly the two-photon resolution does not permit distinguishing the membrane and fluid phases in the neuropil. Additionally, how would the authors rule out adsorption onto some extracellular proteins?

The reason I raise these points is because I have at least a slight difficulty with the interpretation. A slowing of molecular rotation of 70% (intracellular) suggests to me that a large fraction of fluorophores, essentially 100%, must be in direct contact with some larger molecule. This seems quite extreme even in the crowded intracellular space. I have similar reservations about the synaptic cleft and extracellular space. Is at least 50% and 30% of the volume of these spaces really occupied by macromolecules? There may be somewhat reduced diffusion due to a boundary layer at the membrane or near macromolecules. Do estimates of the thickness of such a boundary layer exist (and its effect on diffusion)?

Second review

The following conditions are of interest:

1) free solution

2) with extracellular dye

a) measurement inside cell

b) measurement over neuropil

c) measurement over synapse

d) measurement inside pipette

3) with intracellular dye

a) measurement inside cell

4) no dye

a) measurement of autofluorescence over neuropil

b) measurement inside soma

For each of the above conditions, please show:

1) full fluorescence time course -1 to 12 ns

2) full anisotropy time course -1 to 12 ns

3) specimen traces

4) global averages

5) fit of global average

6) timing of the light pulse should always be indicated (I assume it occurs at 1ns, but this must be made explicit)

It remains a good idea to try the same measurements with a second dye.

Why is the rise of the anisotropy not instantaneous in Fig. 1B? I couldn't find any mention of temporal filtering.

Assuming that the light pulse occurs at 1ns in Fig. [1E], why is the anisotropy shown so high? I would have thought that it should have decayed over a time constant (to about 1/e) by the point where the data shown, yet it is only reduced by about 15%. Was some additional normalisation carried out that I missed? If so it should certainly be removed and the true time course shown.

In case it is not clear, I am interested in the possibility of the fast component in fact containing two components. The data do not allow me to evaluate this possibility.

(Note to moderators: as the copyright holder of my reviews, I am entitled to post them.)