the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 12 Feb 2024
An automated NMR platform with light-coupled cryogenic probes to detect low micromolar samples
Abstract. Recent advances in NMR fragment screening use sample illumination to boost NMR sensitivity, reduce measurement time to a few seconds, and reduce sample concentration to a few micromolars. Nevertheless, the absence of a fully automated solution to measure several hundreds of samples with photoinduced hyperpolarization limits the large-scale applicability of the method. We present a setup to couple an optical fiber with a cryogenic probe using the flow-cell accessory port. This setup is compatible with commercially available autosamplers, enabling the fully automated measurement of several hundreds of samples per day.
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RC1: 'Comment on mr-2024-3', Anonymous Referee #1, 04 Mar 2024
The comment was uploaded in the form of a supplement: https://mr.copernicus.org/preprints/mr-2024-3/mr-2024-3-RC1-supplement.pdf
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AC1: 'Reply on RC1', Felix Torres, 12 Mar 2024
Dear Referee,
Thank you for your positive appreciation of our work and your corrections and comments.
Before resubmission of the manuscript with appropriate corrections, we would like to confirm that:
We will correct the Figure 1 caption for "Lateral axial distance." thank you for noticing the typo.
We will reword the sentence line 108, change the Figure number line 137, define the STD abbreviation line 154, correct "spheric" to "spherical" line 174.
We will replace "KPO4" for "potassium phosphate buffer" with the pH indicated. This will avoid confusion, as the exact protonation state depends on the pH, and stating an exact state (K3PO4, K2HPO4, or KH2PO4) would not be appropriate.
We have experienced issues with the reference manager. We corrected the reference to August Beer, and we will verify and eventually correct the references manually.
The title of the supplementary will be changed, considering the suggestions of Referee 2.
We will reword the Figure S2 caption.
We will include the high-resolution picture of Figure S2.
Figure S3 will be reworked. The purpose of the figure is to show the different light paths when changing the position of the optic fiber. Then, it is possible to see when the light beam is collimated, divergent, or convergent.
As mentioned above, we will provide the appropriate corrections to the manuscript and resubmit in required delays.
Best regards
Felix Torres
Citation: https://doi.org/10.5194/mr-2024-3-AC1
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AC1: 'Reply on RC1', Felix Torres, 12 Mar 2024
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RC2: 'Comment on mr-2024-3', Alexander P. Golovanov, 06 Mar 2024
The manuscript by Wüster et al titled “An automated NMR platform with light-coupled cryogenic probes to detect low micromolar samples” describes an approach to how sample illumination using optical fiber can be embedded in a cryoprobe using an existing inside channel normally used for flow-cell accessory. The other end of the optical fiber can be coupled, as usual, to a laser or a laser diode. Light enters through the bottom of the NMR sample tube via an effective lens formed by the tube bottom, and if the optical density of the sample is not very high, the light then penetrates the sample and illuminates the NMR detection area. The main benefit of this approach is that it leaves the top of the magnet bore completely opened so that the sample lift and automatic sample changers can operate as usual, meaning that full sample automation present nowadays on modern NMR spectrometers can be fully operational, and be applied to samples under illuminated conditions. Overall it is a very clever way to introduce the illumination to the bottom of the sample from within the probehead itself, yet without any probehead modification, apart from routing the optical fiber through the existing channel. Such positioning of the fiber does not noticeably compromise shimming, signal lineshape, or other characteristics of the probe, as the authors demonstrate in their manuscript. From this viewpoint, the submitted paper will be undoubtedly useful for the community that aims to couple sample illumination with NMR spectroscopy, and moreover, will likely trigger follow-on studies where this approach will be further modified, optimized and improved. However, before accepting this manuscript for formal publication a few things should be done to make sure that this paper lays a robust foundation for this approach and can be used as a guide by others.
1) it will be perhaps interesting for the community if there is a short discussion included of whether all the Cryoprobes, or only specific types, have this port for the flow-probe accessory, and some discussion of usability of 5 mm vs 3 mm cryoprobes and sample tubes. The exact type of cryoprobe used in the study is not mentioned, unfortunately, only the spectrometer used. It is not clear if this was 5mm cryoprobe, or 3mm cryoprobe.
2) the title of the paper has somewhat imprecise wording at the very beginning if one considers what data is actually included in the manuscript itself. The title implies that “an automated NMR platform” was presented in the paper, but automation as a platform was not demonstrated here, just the illumination approach. We suggest modifying the title slightly (along the lines that the approach "allows automation") so it reflects more precisely the actual data included in the paper. (As another Review already noted, in the Supplemental the title is actually very different.) In the Discussion/Conclusions it is of course fair to say that this approach would be fully compatible with automation, no doubt about that.
3) the paper presents results of simulations and ray tracing, which is good for displaying that light will go inside the sample and be largely confined within the sample and the sample tube. However, the most interesting things, like the effects of light absorbance in the sample and light scattering are not described in enough detail. What will change in light distribution if 5 mm tube is used instead of 3 mm tube? (There may be more 5 mm cryoprobes around than 3mm cryoprobes, I suspect, and 5 mm tubes are common). It is mentioned in the Conclusion (line 193) that “we built setup that illuminated 3mm and 5mm NMR tubes” but no data was shown in the paper regarding 5mm tubes, so either the data for 5mm tubes needs to be added, or claim “we built setup that illuminated […] 5mm tubes” modified. Presumably, in 5mm tubes there will be more unlit areas close to the tube walls towards the bottom half of the sample, where the beam enters, but how bad the situation might be is not really obvious without a simulation and/or experimentation. Moreover, ray tracing (presumably?) would not account for light absorbance and scattering within the sample and within the tube walls, but that is what ultimately defines the distribution of light in the NMR active volume, and in the sample in general. The authors do mention (lines 197-198) that it is “possible to detect power levels at different axial positions within the liquid sample using an immersed photodiode”, however, this experimental data on such measurements is not shown anywhere, and is certainly worth adding. How uniformly the sample is illuminated in reality would be of great importance to many, if this approach is to be used as a “platform”. Apart from using a photodiode for ex-situ measuring using the model benchtop rig (Fig1C), it would be really useful to measure the experimental light uniformity inside the sample in the cryoprobe (with all the sample tube surroundings present there). Presumably, this can be done using slice-selective photo-CIDNP, or slice-selective reaction with some suitable actinometer, or just using Z-position-dependent photo-CIDNP enhancement factor using imaging experiment for illuminated and dark z-profiles, and dividing one by another (like it was done for NMRtorch in Bramham and Golovanov, Comm Chem, 5, 2022; very easy if photo-CIDNP enhancements are high enough). This will quantify the real absorbed light intensity distribution along the length of the sample inside the NMR active volume inside the spectrometer. On line 95 the claim is made that “inserting the optical fiber near NMR tube yields high illumination of the sample”, however, what is considered as “high illumination” is a bit vague, it is only compared with the illumination from the top, and looks a bit worse, for the reasons outlined in the paper. Either the statement needs to be somewhat reworded, or some quantification shown.
Further discussion will be useful in the paper as to what may be the maximal sample optical density where the light penetration and light uniformity in the NMR detection region will be still satisfactory. The current arrangement (Fig1C) implies that only a small amount of light is absorbed by the sample, and the major part just passes through, reducing the illumination efficiency. It is mentioned in the paper that a simple way to improve photo-CIDNP hyperpolarization could be to use stronger laser power (line 153), but this will not improve light uniformity and may induce local heating and excessive dye bleaching at the point where the beam enters the sample, not to mention that higher laser power may be degrading the optical fiber itself. All these issues should be mentioned or discussed in the paper, or, even better, at least some of these effects measured (ideally).
Another thing – it is fairly easy to position the end of the fiber at the very bottom of the sample tube in the open desktop rig used for testing (Fig1C), however inside the cryoprobe the bottom of the sample tube is not visible, and the tube position in the spinner is normally set by an external gauge and is not really movable. Some tips or comments would be useful to advise how the recommended small optimal distance between the end of the fiber and the tube bottom can be set inside the cryoprobe where both of these things are not visible.
In addition, there are a few minor technical issues that need fixing in the final version of the paper.
Fig1 legend, for panels D,E: it is mentioned “simulation of the absorbed power (AU)”, and the graphs show “Absorption fraction” – what exactly these mean, how these were defined and calculated? Presumably, these parameters should depend on the optical density (OD) of the sample as well? What was the sample OD for these simulations? Needs clarifying, in the figure legend and/or in the Methods.
Line 137 refers to Figure 2 – presumably should refer to Figure 3 instead. Also, in the phrase “sample measured from the top and the bottom” – presumably Authors mean that the sample is illuminated from the top and the bottom? To re-word in the text, and also in the Figure 3 legend. To add the key to Figure 3 (for red/blue), and/or fix the wording in the figure legend regarding the colors.
Line 154 – Reword statement “Such concentrations are on the low standard of typical fragment screening by NMR”
Fig 3A – the Y scale shows raw signal intensity (with an unclear small initial value without any illumination), whereas the text refers to SNE values. Perhaps the SNE values should be plotted on the figure, versus illumination time, not intensities, for consistency.
Line 200 – which cryoprobe exactly was used in the study? 5mm, or 3mm, what type exactly? Add information.
As a very minor comment, it will help text readability if paragraphs in the text are separated with extra space between them and/or paragraph idents are used.
Apart from these minor issues, the paper is easy to read and easy to follow, very nice work overall.
Citation: https://doi.org/10.5194/mr-2024-3-RC2 -
AC2: 'Reply on RC2', Felix Torres, 12 Mar 2024
Dear Prof Golovanov, dear Sasha,
Thank you for your positive appreciation of the submitted manuscript, as well as your insightful comments and suggestions.
We would like to provide feedback below before resubmitting it with appropriate implementations and corrections.
1) We will describe the installation procedure as part of the supplementary data. Succinctly, the fiber is inserted from the entry of the air conduct, which is typically connected to the BCU. The connection to the BCU is maintained using a T-connector to accommodate the optic fiber and the BCU tubing. The fiber is inserted until the user feels the resistance of the NMR tube, then the optic fiber is pulled down by a few millimeters. Concerning the cryoprobes, this system is compatible with standard 5 mm CryoProbes and Prodigy probes from 300 to 900 MHz. The compatibility with 3 mm probes is unknown, but we know that CryoFit set ups are incompatible with 3 mm probes. Therefore, we do not expect our setup to be compatible with 3mm probes.
2) We will change the title as appropriate, including your suggestion and the supplementary. For your interest, we have successfully installed an optic fiber on a cryoprobe on a 600 MHz equipped with SampleJet.
3) You are correct; the illumination profile of the 5 mm NMR tubes will differ from that of the 3 mm NMR tubes. We deliberately focus on 3 mm tubes as the primary application is drug discovery and, more specifically, fragment screening. In this case, the access to sufficient quantities of recombinant protein is often limiting, and using 3 mm tubes instead of 5 mm reduces the protein quantities by a factor of 3. Nevertheless, it is true that the light is not uniformly distributed due to the absorption of the photosensitizer, which is present in the region below the measuring area determined by the rf coils. We will provide more details in the discussion on the impact of light absorption in this particular setup and adapt the statement to consider this particular point. Furthermore, we envision that the combination of illumination from the bottom part of the CryoProbes in combination with other technologies such as NMRtorch tubes might be a starting point to correct the effect of light inhomogeneity in the NMR samples while allowing high throughput automation.
Referee 2 is right: the laser power will indeed cause more bleaching, and this option would only be useful for single-scan experiments, such as screening.
As suggested above, we will provide more details about the installation procedure for the readers who might desire to install this setup.
We will correct the minor issues for the resubmitted manuscripts, including Figure 1 caption, line 134, line 154, figure 200, and Figure 3A, in which the Y scale contains a typo.
We appreciate fully that all the comments from Referee 2 (Prof. Golovanov) will contribute to significantly improving the quality of the manuscript.
Citation: https://doi.org/10.5194/mr-2024-3-AC2
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AC2: 'Reply on RC2', Felix Torres, 12 Mar 2024
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EC1: 'Comment on mr-2024-3', Daniella Goldfarb, 13 Mar 2024
Dear Dr. Torres
I am happy with your response to the reveiwers comments. Please submit a revized version.
Sincerely ,
Daniella Goldfarb
Citation: https://doi.org/10.5194/mr-2024-3-EC1 -
AC3: 'Reply on EC1', Felix Torres, 15 Mar 2024
Dear Prof. Goldfarb,
Thank you. We resubmitted a revised version of the manuscript and a detailed answer to the referees.
Best regards
Felix Torres
Citation: https://doi.org/10.5194/mr-2024-3-AC3
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AC3: 'Reply on EC1', Felix Torres, 15 Mar 2024
Status: closed
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RC1: 'Comment on mr-2024-3', Anonymous Referee #1, 04 Mar 2024
The comment was uploaded in the form of a supplement: https://mr.copernicus.org/preprints/mr-2024-3/mr-2024-3-RC1-supplement.pdf
-
AC1: 'Reply on RC1', Felix Torres, 12 Mar 2024
Dear Referee,
Thank you for your positive appreciation of our work and your corrections and comments.
Before resubmission of the manuscript with appropriate corrections, we would like to confirm that:
We will correct the Figure 1 caption for "Lateral axial distance." thank you for noticing the typo.
We will reword the sentence line 108, change the Figure number line 137, define the STD abbreviation line 154, correct "spheric" to "spherical" line 174.
We will replace "KPO4" for "potassium phosphate buffer" with the pH indicated. This will avoid confusion, as the exact protonation state depends on the pH, and stating an exact state (K3PO4, K2HPO4, or KH2PO4) would not be appropriate.
We have experienced issues with the reference manager. We corrected the reference to August Beer, and we will verify and eventually correct the references manually.
The title of the supplementary will be changed, considering the suggestions of Referee 2.
We will reword the Figure S2 caption.
We will include the high-resolution picture of Figure S2.
Figure S3 will be reworked. The purpose of the figure is to show the different light paths when changing the position of the optic fiber. Then, it is possible to see when the light beam is collimated, divergent, or convergent.
As mentioned above, we will provide the appropriate corrections to the manuscript and resubmit in required delays.
Best regards
Felix Torres
Citation: https://doi.org/10.5194/mr-2024-3-AC1
-
AC1: 'Reply on RC1', Felix Torres, 12 Mar 2024
-
RC2: 'Comment on mr-2024-3', Alexander P. Golovanov, 06 Mar 2024
The manuscript by Wüster et al titled “An automated NMR platform with light-coupled cryogenic probes to detect low micromolar samples” describes an approach to how sample illumination using optical fiber can be embedded in a cryoprobe using an existing inside channel normally used for flow-cell accessory. The other end of the optical fiber can be coupled, as usual, to a laser or a laser diode. Light enters through the bottom of the NMR sample tube via an effective lens formed by the tube bottom, and if the optical density of the sample is not very high, the light then penetrates the sample and illuminates the NMR detection area. The main benefit of this approach is that it leaves the top of the magnet bore completely opened so that the sample lift and automatic sample changers can operate as usual, meaning that full sample automation present nowadays on modern NMR spectrometers can be fully operational, and be applied to samples under illuminated conditions. Overall it is a very clever way to introduce the illumination to the bottom of the sample from within the probehead itself, yet without any probehead modification, apart from routing the optical fiber through the existing channel. Such positioning of the fiber does not noticeably compromise shimming, signal lineshape, or other characteristics of the probe, as the authors demonstrate in their manuscript. From this viewpoint, the submitted paper will be undoubtedly useful for the community that aims to couple sample illumination with NMR spectroscopy, and moreover, will likely trigger follow-on studies where this approach will be further modified, optimized and improved. However, before accepting this manuscript for formal publication a few things should be done to make sure that this paper lays a robust foundation for this approach and can be used as a guide by others.
1) it will be perhaps interesting for the community if there is a short discussion included of whether all the Cryoprobes, or only specific types, have this port for the flow-probe accessory, and some discussion of usability of 5 mm vs 3 mm cryoprobes and sample tubes. The exact type of cryoprobe used in the study is not mentioned, unfortunately, only the spectrometer used. It is not clear if this was 5mm cryoprobe, or 3mm cryoprobe.
2) the title of the paper has somewhat imprecise wording at the very beginning if one considers what data is actually included in the manuscript itself. The title implies that “an automated NMR platform” was presented in the paper, but automation as a platform was not demonstrated here, just the illumination approach. We suggest modifying the title slightly (along the lines that the approach "allows automation") so it reflects more precisely the actual data included in the paper. (As another Review already noted, in the Supplemental the title is actually very different.) In the Discussion/Conclusions it is of course fair to say that this approach would be fully compatible with automation, no doubt about that.
3) the paper presents results of simulations and ray tracing, which is good for displaying that light will go inside the sample and be largely confined within the sample and the sample tube. However, the most interesting things, like the effects of light absorbance in the sample and light scattering are not described in enough detail. What will change in light distribution if 5 mm tube is used instead of 3 mm tube? (There may be more 5 mm cryoprobes around than 3mm cryoprobes, I suspect, and 5 mm tubes are common). It is mentioned in the Conclusion (line 193) that “we built setup that illuminated 3mm and 5mm NMR tubes” but no data was shown in the paper regarding 5mm tubes, so either the data for 5mm tubes needs to be added, or claim “we built setup that illuminated […] 5mm tubes” modified. Presumably, in 5mm tubes there will be more unlit areas close to the tube walls towards the bottom half of the sample, where the beam enters, but how bad the situation might be is not really obvious without a simulation and/or experimentation. Moreover, ray tracing (presumably?) would not account for light absorbance and scattering within the sample and within the tube walls, but that is what ultimately defines the distribution of light in the NMR active volume, and in the sample in general. The authors do mention (lines 197-198) that it is “possible to detect power levels at different axial positions within the liquid sample using an immersed photodiode”, however, this experimental data on such measurements is not shown anywhere, and is certainly worth adding. How uniformly the sample is illuminated in reality would be of great importance to many, if this approach is to be used as a “platform”. Apart from using a photodiode for ex-situ measuring using the model benchtop rig (Fig1C), it would be really useful to measure the experimental light uniformity inside the sample in the cryoprobe (with all the sample tube surroundings present there). Presumably, this can be done using slice-selective photo-CIDNP, or slice-selective reaction with some suitable actinometer, or just using Z-position-dependent photo-CIDNP enhancement factor using imaging experiment for illuminated and dark z-profiles, and dividing one by another (like it was done for NMRtorch in Bramham and Golovanov, Comm Chem, 5, 2022; very easy if photo-CIDNP enhancements are high enough). This will quantify the real absorbed light intensity distribution along the length of the sample inside the NMR active volume inside the spectrometer. On line 95 the claim is made that “inserting the optical fiber near NMR tube yields high illumination of the sample”, however, what is considered as “high illumination” is a bit vague, it is only compared with the illumination from the top, and looks a bit worse, for the reasons outlined in the paper. Either the statement needs to be somewhat reworded, or some quantification shown.
Further discussion will be useful in the paper as to what may be the maximal sample optical density where the light penetration and light uniformity in the NMR detection region will be still satisfactory. The current arrangement (Fig1C) implies that only a small amount of light is absorbed by the sample, and the major part just passes through, reducing the illumination efficiency. It is mentioned in the paper that a simple way to improve photo-CIDNP hyperpolarization could be to use stronger laser power (line 153), but this will not improve light uniformity and may induce local heating and excessive dye bleaching at the point where the beam enters the sample, not to mention that higher laser power may be degrading the optical fiber itself. All these issues should be mentioned or discussed in the paper, or, even better, at least some of these effects measured (ideally).
Another thing – it is fairly easy to position the end of the fiber at the very bottom of the sample tube in the open desktop rig used for testing (Fig1C), however inside the cryoprobe the bottom of the sample tube is not visible, and the tube position in the spinner is normally set by an external gauge and is not really movable. Some tips or comments would be useful to advise how the recommended small optimal distance between the end of the fiber and the tube bottom can be set inside the cryoprobe where both of these things are not visible.
In addition, there are a few minor technical issues that need fixing in the final version of the paper.
Fig1 legend, for panels D,E: it is mentioned “simulation of the absorbed power (AU)”, and the graphs show “Absorption fraction” – what exactly these mean, how these were defined and calculated? Presumably, these parameters should depend on the optical density (OD) of the sample as well? What was the sample OD for these simulations? Needs clarifying, in the figure legend and/or in the Methods.
Line 137 refers to Figure 2 – presumably should refer to Figure 3 instead. Also, in the phrase “sample measured from the top and the bottom” – presumably Authors mean that the sample is illuminated from the top and the bottom? To re-word in the text, and also in the Figure 3 legend. To add the key to Figure 3 (for red/blue), and/or fix the wording in the figure legend regarding the colors.
Line 154 – Reword statement “Such concentrations are on the low standard of typical fragment screening by NMR”
Fig 3A – the Y scale shows raw signal intensity (with an unclear small initial value without any illumination), whereas the text refers to SNE values. Perhaps the SNE values should be plotted on the figure, versus illumination time, not intensities, for consistency.
Line 200 – which cryoprobe exactly was used in the study? 5mm, or 3mm, what type exactly? Add information.
As a very minor comment, it will help text readability if paragraphs in the text are separated with extra space between them and/or paragraph idents are used.
Apart from these minor issues, the paper is easy to read and easy to follow, very nice work overall.
Citation: https://doi.org/10.5194/mr-2024-3-RC2 -
AC2: 'Reply on RC2', Felix Torres, 12 Mar 2024
Dear Prof Golovanov, dear Sasha,
Thank you for your positive appreciation of the submitted manuscript, as well as your insightful comments and suggestions.
We would like to provide feedback below before resubmitting it with appropriate implementations and corrections.
1) We will describe the installation procedure as part of the supplementary data. Succinctly, the fiber is inserted from the entry of the air conduct, which is typically connected to the BCU. The connection to the BCU is maintained using a T-connector to accommodate the optic fiber and the BCU tubing. The fiber is inserted until the user feels the resistance of the NMR tube, then the optic fiber is pulled down by a few millimeters. Concerning the cryoprobes, this system is compatible with standard 5 mm CryoProbes and Prodigy probes from 300 to 900 MHz. The compatibility with 3 mm probes is unknown, but we know that CryoFit set ups are incompatible with 3 mm probes. Therefore, we do not expect our setup to be compatible with 3mm probes.
2) We will change the title as appropriate, including your suggestion and the supplementary. For your interest, we have successfully installed an optic fiber on a cryoprobe on a 600 MHz equipped with SampleJet.
3) You are correct; the illumination profile of the 5 mm NMR tubes will differ from that of the 3 mm NMR tubes. We deliberately focus on 3 mm tubes as the primary application is drug discovery and, more specifically, fragment screening. In this case, the access to sufficient quantities of recombinant protein is often limiting, and using 3 mm tubes instead of 5 mm reduces the protein quantities by a factor of 3. Nevertheless, it is true that the light is not uniformly distributed due to the absorption of the photosensitizer, which is present in the region below the measuring area determined by the rf coils. We will provide more details in the discussion on the impact of light absorption in this particular setup and adapt the statement to consider this particular point. Furthermore, we envision that the combination of illumination from the bottom part of the CryoProbes in combination with other technologies such as NMRtorch tubes might be a starting point to correct the effect of light inhomogeneity in the NMR samples while allowing high throughput automation.
Referee 2 is right: the laser power will indeed cause more bleaching, and this option would only be useful for single-scan experiments, such as screening.
As suggested above, we will provide more details about the installation procedure for the readers who might desire to install this setup.
We will correct the minor issues for the resubmitted manuscripts, including Figure 1 caption, line 134, line 154, figure 200, and Figure 3A, in which the Y scale contains a typo.
We appreciate fully that all the comments from Referee 2 (Prof. Golovanov) will contribute to significantly improving the quality of the manuscript.
Citation: https://doi.org/10.5194/mr-2024-3-AC2
-
AC2: 'Reply on RC2', Felix Torres, 12 Mar 2024
-
EC1: 'Comment on mr-2024-3', Daniella Goldfarb, 13 Mar 2024
Dear Dr. Torres
I am happy with your response to the reveiwers comments. Please submit a revized version.
Sincerely ,
Daniella Goldfarb
Citation: https://doi.org/10.5194/mr-2024-3-EC1 -
AC3: 'Reply on EC1', Felix Torres, 15 Mar 2024
Dear Prof. Goldfarb,
Thank you. We resubmitted a revised version of the manuscript and a detailed answer to the referees.
Best regards
Felix Torres
Citation: https://doi.org/10.5194/mr-2024-3-AC3
-
AC3: 'Reply on EC1', Felix Torres, 15 Mar 2024
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