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  1. Author Response

    Reviewer #1 (Public Review):

    The manuscript by Yildirim presents a method for labeling and tracking cells within organoids to enable the assessment of dynamic processes within the intact organoid. The authors use Third-harmonic generation (THG), an intrinsic signal which results from tripling the frequency of the excitation wavelength, and a modified three-photon microscope to identify and track cells within the 3D organization of cerebral organoids. Specifically, the authors focus on the ventricular zone in 35-day old organoids, when young DCX+ neurons are migrating into the cortical plate-like area of the organoid and show that THG can identify migrating cells. The authors then use a disorder model of Rett syndrome to validate the method and show that differences can be detected with their technique and, importantly, that the VZ volume is smaller and that radially migrating neurons have slower migration within RTT organoids.

    There are many strengths of the study including the use of multiple (two) isogenic pairs of control and RTT organoids, the critical comparison of the labeling method with standard markers, and the use of a relevant disease model to test the utility of the technique.

    We appreciate this constructive feedback from Reviewer 1 on both imaging system development and using it for a biologically important question.

    Reviewer #2 (Public Review):

    This paper reports an impressive technical development - Third Harmonic Generation (THG) three-photon, label-free imaging of intact cerebral organoids. This is the first paper to apply THG imaging to intact, three-dimensional organoids and offers some distinct advantages over other approaches in terms of being able to image the full depth of intact organoids. Using this approach, mutant organoids generated from Rett Syndrome patients were imaged, finding shorter migration distances, slower migration speeds, and more tortuous trajectories in these organoids. This work advances in a useful way the available imaging tools for intact, three-dimensional organoids, by allowing their full depth to be accessed. It is likely to have an impact both as a demonstration of what can be achieved through advanced bioimaging techniques and on the progress of the (recently rapidly advancing) cerebral organoids field. A caveat to the latter is that, due to the optics techniques involved, reproduction in a typical organoid/cell culture laboratory may be beyond the skill of most researchers in that field, although this could ultimately be addressed with commercialization (noting that the laser products needed are not completely "turn-key" yet).

    Strengths

    The fact that the authors were able to achieve a pulse width at the sample (in deep tissue) of 27 fs is a great technical achievement, which makes the results achieved in the paper possible. I can't emphasize enough how impressive that aspect of the paper is. As they note, pulse widths of < 30 fs have not previously been reported in such a scenario (and we would normally consider the 40-50 fs range as good going. This is a great technical achievement and is important given the apparent great sensitivity of three-photon efficiency to pulse width and shape. While the short pulse lengths are impressive, it is of interest to know how hard this will be in practice to reproduce in other laboratories. The authors might comment on how difficult it was to keep the pulse compressed to this level - was there any drift, and were adjustments needed to be made to the pulse compressor over the duration of the series of experiments?

    As well as making an impressive technical demonstration, the authors showed that it could be used to make useful measurements, showing that the system was capable of distinguishing some structural properties of mutant Rett Syndrome organoids from wild-type organoids, by means of time-lapse imaging of deep structures within the samples.

    Weaknesses

    There are some concerns about the statistical validity of the conclusions made, in particular for the analysis of the time-lapse imaging experiments. I am not convinced that the analysis made is statistically valid, due to bias effects introduced by pooling different lengths of time-lapse samples.

    We would like to thank Reviewer 2 for this constructive feedback. First of all, achieving lower pulse width values is only possible with optimizing both laser and microscope parameters. Since we have designed and implemented the custom-made microscope parts aligned with laser parameters, it is possible to achieve stable and short pulse widths in different kinds of tissues including cerebral organoids for our lab.

    Second, we agree with reviewer 2 that comparing the migration parameters of the cells which disappeared from the field of view before 12 hours and those imaged during 12 hours is not reasonable. Therefore, we removed the data from the cells which were not imaged for 12 hours and updated Figure 5 and 6.

    Reviewer #3 (Public Review):

    Yildirim et al describe a novel three-photon (3P) imaging approach which concomitantly addresses several notable roadblocks in the current state of the art when it comes to functional imaging using organoid cultures. The authors use a 3P system modified with custom laser and optics which enables label-free, deep, high-resolution, non-phototoxic, long-term imaging of intact brain organoids achieving close to 1mm penetration and imaging periods up to 96 hours. Leveraging the capacity of third harmonic generation (THG) signal to differentiate regions with distinct cellular densities, the authors demonstrate effective, label-free demarcation of ventricular zone-like regions vs regions resembling the cortical plate. Moreover, through a set of well-designed and well-powered experiments, the authors apply their system to uncover structural and functional phenotypes in organoids derived from Rett's Syndrome patient lines and corrected isogenic lines. All without the need for a fluorescent label, they describe structural changes to VZ-like regions and migration deficits in cells that emanate from these regions. The imaging of migrating cells in relation to their VZ of origin reveals an especially novel look at the radial migration of cortical neurons in organoids, something which has not been possible to assay since the VZ structures remain deep within organoids and inaccessible to co-image with migrating cells using standard approaches.

    The study is highly innovative, and looking ahead, a standardized 3P/THG imaging platform that enables deep and label-free imaging of organoids at scale, holds a lot of promise in illuminating a lot of biology which currently remains beyond reach, as well as in designing large scale, non-invasive, multi-parameter phenotyping screens using patient samples. The manuscript is well-written and the results clearly demonstrated.

    We would like to thank Reviewer 3 for their constructive feedback on how our paper addresses and resolves notable roadblocks in the current state of art of intact organoid imaging for modeling brain disorders.

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  2. Evaluation Summary:

    This manuscript will be of interest to stem cell and developmental biologists who aim to use newly emerging brain organoid models to understand the structure and function of the developing human brain. It presents a technological advance in imaging and describes an innovative method for labeling and tracking of cells within organoids to enable the assessment of dynamic processes within the intact organoid. The method is validated in a disease model and addresses a challenge in the field of human stem cell modeling of assessing cells within the 3D structure.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The manuscript by Yildrim presents a method for labeling and tracking cells within organoids to enable the assessment of dynamic processes within the intact organoid. The authors use Third-harmonic generation (THG), an intrinsic signal which results from tripling the frequency of the excitation wavelength, and a modified three-photon microscope to identify and track cells within the 3D organization of cerebral organoids. Specifically, the authors focus on the ventricular zone in 35-day old organoids, when young DCX+ neurons are migrating into the cortical plate-like area of the organoid and show that THG can identify migrating cells. The authors then use a disorder model of Rett syndrome to validate the method and show that differences can be detected with their technique and, importantly, that the VZ volume is smaller and that radially migrating neurons have slower migration within RTT organoids.

    There are many strengths of the study including the use of multiple (two) isogenic pairs of control and RTT organoids, the critical comparison of the labeling method with standard markers, and the use of a relevant disease model to test the utility of the technique.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    This paper reports an impressive technical development - Third Harmonic Generation (THG) three-photon, label-free imaging of intact cerebral organoids. This is the first paper to apply THG imaging to intact, three-dimensional organoids and offers some distinct advantages over other approaches in terms of being able to image the full depth of intact organoids. Using this approach, mutant organoids generated from Rett Syndrome patients were imaged, finding shorter migration distances, slower migration speeds, and more tortuous trajectories in these organoids. This work advances in a useful way the available imaging tools for intact, three-dimensional organoids, by allowing their full depth to be accessed. It is likely to have an impact both as a demonstration of what can be achieved through advanced bioimaging techniques and on the progress of the (recently rapidly advancing) cerebral organoids field. A caveat to the latter is that, due to the optics techniques involved, reproduction in a typical organoid/cell culture laboratory may be beyond the skill of most researchers in that field, although this could ultimately be addressed with commercialisation (noting that the laser products needed are not completely "turn-key" yet).

    Strengths
    The fact that the authors were able to achieve a pulse width at the sample (in deep tissue) of 27 fs is a great technical achievement, which makes the results achieved in the paper possible. I can't emphasize enough how impressive that aspect of the paper is. As they note, pulse widths of < 30 fs have not previously been reported in such a scenario (and we would normally consider the 40-50 fs range as good going. This is a great technical achievement and is important given the apparent great sensitivity of three-photon efficiency to pulse width and shape. While the short pulse lengths are impressive, it is of interest to know how hard this will be in practice to reproduce in other laboratories. The authors might comment on how difficult it was to keep the pulse compressed to this level - was there any drift, and were adjustments needed to be made to the pulse compressor over the duration of the series of experiments?

    As well as making an impressive technical demonstration, the authors showed that it could be used to make useful measurements, showing that the system was capable of distinguishing some structural properties of mutant Rett Syndrome organoids from wild-type organoids, by means of time-lapse imaging of deep structures within the samples.

    Weaknesses
    There are some concerns about the statistical validity of the conclusions made, in particular for the analysis of the time-lapse imaging experiments. I am not convinced that the analysis made is statistically valid, due to bias effects introduced by pooling different lengths of time-lapse samples.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    Yildirim et al describe a novel three-photon (3P) imaging approach which concomitantly addresses several notable roadblocks in the current state of the art when it comes to functional imaging using organoid cultures. The authors use a 3P system modified with custom laser and optics which enables label-free, deep, high-resolution, non-phototoxic, long-term imaging of intact brain organoids achieving close to 1mm penetration and imaging periods up to 96 hours. Leveraging the capacity of third harmonic generation (THG) signal to differentiate regions with distinct cellular densities, the authors demonstrate effective, label-free demarcation of ventricular zone-like regions vs regions resembling the cortical plate. Moreover, through a set of well-designed and well-powered experiments, the authors apply their system to uncover structural and functional phenotypes in organoids derived from Rett's Syndrome patient lines and corrected isogenic lines. All without the need for a fluorescent label, they describe structural changes to VZ-like regions and migration deficits in cells that emanate from these regions. The imaging of migrating cells in relation to their VZ of origin reveals an especially novel look at the radial migration of cortical neurons in organoids, something which has not been possible to assay since the VZ structures remain deep within organoids and inaccessible to co-image with migrating cells using standard approaches.

    The study is highly innovative, and looking ahead, a standardized 3P/THG imaging platform that enables deep and label-free imaging of organoids at scale, holds a lot of promise in illuminating a lot of biology which currently remains beyond reach, as well as in designing large scale, non-invasive, multi-parameter phenotyping screens using patient samples. The manuscript is well-written and the results clearly demonstrated.

    Read the original source
    Was this evaluation helpful?