Review Reports - Improving Speed and Efficiency of DESI Imaging with the Xevo MRT Mass Spectrometer for Analyte Mapping

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a robust and timely evaluation of the Xevo MRT mass spectrometer coupled with DESI-MSI, successfully addressing the critical bottleneck of balancing high spatial resolution with analytical throughput. By demonstrating stable 100 Hz acquisition speeds, implementing an Enhanced Duty Cycle (EDC) mode for significant signal amplification, and leveraging up to 100,000 FWHM mass resolution to separate isomeric lipids, the study provides a comprehensive technical benchmark for the field.

It is an excellent engineering innovation that significantly pushes the performance envelope of DESI-MSI, offering a powerful new tool for the community, even if it lacks a fundamentally new scientific mechanism.

-The positioning of the manuscript needs further clarification. The current version reads more like an instrument performance demonstration or an application note. I strongly recommend that the authors emphasize the methodological innovations and reduce the promotional language regarding the product.

-The section on materials and methods was written too simply. Critical parameters for DESI-MSI (e.g., spray angle, spray distance, acquisition step size, m/z extraction window, and normalization methods) should be clearly described. Furthermore, details regarding reproducibility must be supplemented. The authors should specify the number of tissue samples, the number of tissue sections, the replicates for API spotting, and the criteria for ROI selection to enhance the reliability of the results.

-The terminology for animal tissues needs unification. Terms such as 'rat brain', 'murine brain', and 'mouse brain' are used interchangeably. Please clarify if different species were used and explicitly state the origin in the Methods section.

-The discussion on the EDC effect should be more objective. While the authors demonstrate that EDC enhances signals, they should also address potential impacts on the non-target mass range to avoid presenting only the advantages.

-There is an inconsistency in the API data. While the Abstract and Results mention five APIs, only four are listed in the Methods section.

Author Response

Comment 1: The positioning of the manuscript needs further clarification. The current version reads more like an instrument performance demonstration or an application note. I strongly recommend that the authors emphasize the methodological innovations and reduce the promotional language regarding the product.

Response 1:

This manuscript was written with the intention of being a technology advancement demonstration not a methodology or application piece, it therefore requires a description of the improvements to the instrumentation bringing about those advancements. A methodology paper is already available for the low flow capabilites this can be found as reference #4

Comment 2: The section on materials and methods was written too simply. Critical parameters for DESI-MSI (e.g., spray angle, spray distance, acquisition step size, m/z extraction window, and normalization methods) should be clearly described. Furthermore, details regarding reproducibility must be supplemented. The authors should specify the number of tissue samples, the number of tissue sections, the replicates for API spotting, and the criteria for ROI selection to enhance the reliability of the results.

Response 2:

The sprayer positioning was optimised as per Towers et al reference #4 and this has now been highlighted. Typically, the DESI settings were optimised on an experiment by experiment basis, and will vary for any laboratory wishing to reproduce the results, the ranges used have been added to the text for additional clarity. Line 121

Each image and acquisition can be considered a technical replicate in and of itself, as multiple regions and lines of acquisition can be isolated and treated as individual samples. When performing multivariate analysis utilising imaging data it is commonplace to draw several small square or circle regions of interest of identical pixel numbers as technical replicates for the statistical analysis. In order to prevent bias regions of interest are drawn at random within each area of acquisition and incorporate sufficient pixels to provide an average value. The API analysis XIC is generated from a single line pass through the API spots and was chosen at random ensuring only that each spot was included within the line.

Comment 3: The terminology for animal tissues needs unification. Terms such as 'rat brain', 'murine brain', and 'mouse brain' are used interchangeably. Please clarify if different species were used and explicitly state the origin in the Methods section.

Response 3: The reference to mouse tissue was an error, apologies, only rat brain tissue has been imaged within this manuscript, the text has been corrected accordingly.

Comments 4: The discussion on the EDC effect should be more objective. While the authors demonstrate that EDC enhances signals, they should also address potential impacts on the non-target mass range to avoid presenting only the advantages.

Response 4: A note on the effect of EDC on the rest of the mass range has been added Line 87 and further discussed within the conclusion line 361, I believe we now present both the advantages and disadvantages of EDC.

Comment 5: There is an inconsistency in the API data. While the Abstract and Results mention five APIs, only four are listed in the Methods section.

Response 5: Thank you, we have added the other API to the methods section line 109

Reviewer 2 Report

Comments and Suggestions for Authors

In mass spectrometry, desorption electrospray ionization (DESI) is a direct ionization technique used to visualize the spatial distribution of analytes, allowing the chemical analysis of samples under atmospheric conditions. This soft ionization technique produces little fragmentation and can be applied in various fields, including the food and pharmaceutical industries, pharmaceuticals, environmental monitoring, and biotechnology. The experimental strategies reported in this research are focused on increasing the speed and efficiency of this analytical technique.

The use of DESI to study secondary metabolites in situ, specifically analyzing their spatial and temporal distribution and, in this case, its enhancement, makes the subject of this manuscript interesting.

The Results section provides a general description of the experiments, and the data obtained are presented in a logical order, supported by tables and figures.

However, despite the positive aspects, some modifications are required for a better presentation of the manuscript, as reported below.

The title should be completed to make it clearer. For example: Improving Speed and Efficiency of DESI Analysis with the Xevo MRT Mass Spectrometer for Analyte Mapping.

Abstract, line 8: Please add "Desorption Electrospray Ionization (DESI)..."

The Introduction section should provide background information on the research topic, state the research question or hypotheses, and outline the importance and purpose of the study. This section is somewhat lacking and should be revised as suggested.

Additionally, please add an opening sentence. For example: Desorption Electrospray Ionization (DESI) imaging is a mass spectrometry technique that allows mapping the spatial distribution of molecules and lipids directly on surfaces or tissue sections, without complex preliminary preparations.

In the Materials and Methods section, please provide more details on the reagents, instruments, and samples used.

The Conclusions section should be expanded. Authors should summarize the main results, contextualize them within the context of current knowledge, highlight the impact of the study and any limitations, and propose possible future developments.

Please supplement the list of abbreviations used in the text.

Author Response

Comments 1: The title should be completed to make it clearer. For example: Improving Speed and Efficiency of DESI Analysis with the Xevo MRT Mass Spectrometer for Analyte Mapping.

Response 1: Thank you for the suggestion, the title has been changed to:

"Improving Speed and Efficiency of DESI Imaging with the Xevo MRT Mass Spectrometer for Analyte Mapping"

Comment 2: Abstract, line 8: Please add "Desorption Electrospray Ionization (DESI)..."

Response 2: Thankyou this has been updated.

Comment 3: The Introduction section should provide background information on the research topic, state the research question or hypotheses, and outline the importance and purpose of the study. This section is somewhat lacking and should be revised as suggested.

Response 3: Thankyou we have made extensive changes to the Introduction I hope the introduction now meets you expectations.

Comment 4:

Comment 5: In the Materials and Methods section, please provide more details on the reagents, instruments, and samples used.

Response 5: Thankyou, the methods section has been updated to include more detail.

Comment 6: The Conclusions section should be expanded. Authors should summarize the main results, contextualize them within the context of current knowledge, highlight the impact of the study and any limitations, and propose possible future developments.

Response 6:

The conclusions have been expanded thankyou for the recommendation.

Comments 7: Please supplement the list of abbreviations used in the text.

Response 7: Thank you the abbreviations list has been supplemented extensively

Reviewer 3 Report

Comments and Suggestions for Authors

The authors present the development of the Xevo MRT platform to improve DESI-MS imaging capabilities, aiming to enhance imaging speed, spatial resolution, and analytical sensitivity. The study is timely and potentially important for advancing high-resolution mass spectrometry imaging applications. However, several issues related to methodological clarity, quantitative validation, figure interpretation, and terminology consistency should be addressed before the manuscript can be considered for publication.

1. Quantitative description of performance improvements

The abstract currently describes the system performance using qualitative statements such as “data quality is excellent” and “signal intensity remains application appropriate” are insufficiently quantitative. The authors should explicitly report, improvements in spatial resolution, imaging speed, sensitivity, signal-to-noise performance, and acquisition time comparisons relative to previous systems. For example: was higher spatial resolution achieved? were lipid localization patterns better resolved? and was imaging performed at substantially higher throughput? More quantitative performance metrics are needed throughout the manuscript.

2. Introduction structure and accessibility

The latter portion of the Introduction describing technical aspects of the instrument is overly detailed for a general readership. The manuscript would be improved by moving detailed instrument descriptions to the Methods section and using the final Introduction paragraph to summarize the key findings, technical significance, and practical advantages of the platform. Also 1-2 more sentences at the initial of the introduction section about the application of DESI would be more readily friendly to readers not familiar with DESI.

3. Need for quantitative validation of imaging quality

A major concern is that several conclusions rely primarily on visual inspection of images rather than quantitative comparison. For example, in Figure 1, the manuscript appears to claim that imaging at 100 Hz provides image quality comparable to 50 Hz. However, this is evaluated largely qualitatively. The authors should provide quantitative image quality metrics, statistical comparison of signal intensity or spatial fidelity under reproducibility measurements. Visual assessment alone is insufficient to support these claims.

4. Potential overstatement of sensitivity claims

The manuscript claims that API concentrations as low as 2.5 pg/mm² are detectable. However the evidence supporting this claim is difficult to interpret visually. Also some blue signal regions appear outside the ROI. The authors should provide clearer quantitative justifications, define detection thresholds explicitly, report non-detectable cases where applicable and discuss possible background or off-target signal contributions. If sensitivity cannot be robustly demonstrated, the claims should be moderated.

Figure and Data Presentation

5. Figure 1 requires clearer explanation

Figure 1 is difficult to interpret without substantial prior knowledge. The caption should clearly explain what the color scales represent, whether the lipid species correspond to different phosphatidylserine (PS) species and how spatial distributions should be interpreted. Additionally, the comparison between 50 Hz and 100 Hz imaging should be quantitatively supported.

6. Figure 2 annotation clarity

The numerical values shown in the upper-right corners of the right panels are not explained and should be clearly defined.

7. Figures 3 and 4

The right panels of Figures 3 and 4 are all low resolution and difficult to read because of lacking clear axis labels and legends. Additionally, the definitions of “SN” and “S/N” should be explicitly provided.

Terminology and Consistency

8. Definition of abbreviations

Abbreviations such as: DESI, and EDC should be defined only at the first occurrence.

9. Consistency of lipid nomenclature

Phosphatidylserine (PS) species are described inconsistently PS 40:7, PS 36:2 & PS 38:5 and PS(18:0_22:6). A consistent naming convention should be used throughout.

10. Clarification of EDC terminology

“Enhanced duty cycle (EDC)” appears to be defined more than once. In addition, the phrase “semi-targeted (EDC) acquisition modes” is unclear and should be clarified. Is EDC itself considered a semi-targeted acquisition mode?

11. Clarification of system sensitivity

The meaning of “system sensitivity” should be more explicitly defined. For example, does this refer to the minimum detectable API concentration within tissue by DESI imaging? This should be stated clearly.

Author Response

Comment 1:

Response 1:

The conclusions have been added to better stress the improvements in speed vs the previous generation solution line 354, and the conclusions have generally be expanded to to give a better overview of the improvements. Additional context has been added to the introduction line 94. Resolution calculation have been discussed for figure 2 and statistical analysis has been added for spectra from figure 1 line 181 this has been included as supplemental data 1.

Comments 2: The latter portion of the Introduction describing technical aspects of the instrument is overly detailed for a general readership. The manuscript would be improved by moving detailed instrument descriptions to the Methods section and using the final Introduction paragraph to summarize the key findings, technical significance, and practical advantages of the platform. Also 1-2 more sentences at the initial of the introduction section about the application of DESI would be more readily friendly to readers not familiar with DESI.

Response 2:

Response 3:

Resolution calculation have been discussed for figure 2 – line 196 and statistical anlayis has been added for spectra from figure 1 line 181 this has been included as supplemental data 1. The way in which the 50 Hz and 100 Hz data were assed has been described – line 170. Imaging mass spectrometry is generally regarded as qualitative and not quantitative.

Comments 4:

Response 4: It is very difficult to quantify images and typically these are used for visualisation and fold change purposes only, the detection values reported within the manuscript are based upon the extracted ion chromatograms for each of the API concentration curves. For this we have followed industry guidelines and only reported API with a signal to noise greater than 5, typically a cutoff used for quantification, however, to err on the side of caution we have used this value as a detection cutoff in this dataset. The blue seen outside of the spot locations is background signal caused by the maximum intensity scale being reduced to aide visualisation of the lower concentration standards. Each scale is different dependant upon the ionization efficiency of the API. The Moxifloxacin (B) where some background interference is seen in the ion images, does not have a signal to noise >5 at 2.5 pg/mm² and the 25 pg/mm² has a much more intense signal than the background interference on the liver tissue.

Comments 5: Figure 1 is difficult to interpret without substantial prior knowledge. The caption should clearly explain what the color scales represent, whether the lipid species correspond to different phosphatidylserine (PS) species and how spatial distributions should be interpreted. Additionally, the comparison between 50 Hz and 100 Hz imaging should be quantitatively supported.

Response 5:

Figure 1 has been updated to make it clearer BPI numbers added and scale bar label, the figure legend has been updated and additional information added to the main text body, a description of the resolution assessment has been added

Comments 6:

Figure 2 annotation clarity

The numerical values shown in the upper-right corners of the right panels are not explained and should be clearly defined.

Response 6: These have now been explained and labbled as Base peak intensity

Comments 7:The right panels of Figures 3 and 4 are all low resolution and difficult to read because of lacking clear axis labels and legends. Additionally, the definitions of “SN” and “S/N” should be explicitly provided.

Response 7: Thank you for noting this labels have been updated sn was a typographical error and has been amended, S/N has been defined in the text as signal to noise.

comment 8: Abbreviations such as: DESI, and EDC should be defined only at the first occurrence.

Response 8 : all Abbreviations have been reviewed and I believe are now correct the Abbreviation

list has been expanded.

comment 9:

Phosphatidylserine (PS) species are described inconsistently PS 40:7, PS 36:2 & PS 38:5 and PS(18:0_22:6). A consistent naming convention should be used throughout.

Response 9: Thankyou this has been corrected

comment 10: “Enhanced duty cycle (EDC)” appears to be defined more than once. In addition, the phrase “semi-targeted (EDC) acquisition modes” is unclear and should be clarified. Is EDC itself considered a semi-targeted acquisition mode?

Response 10: Thankyou this has been corrected.

Comment 11:

Response 11:

System sensitivity is multi-faceted within mass spectrometry imaging analyses and therefore not easily defined, typically it is application dependant and users will have intimate knowledge of the sensitivity per scan of their tissue or surface. Some tissues – such as brain – are relatively analyte rich (in particular lipids which ionise well by DESI), others are less analyte rich such as fruits (which are predominantly sucrose, and this molecule ionises poorly). This is similar for LCMS analyses, where some analytes ionise better than others.

We do not have an industry recognised standard for system suitability and most biological questions answered with MSI are looking at relative changes in concentration not absolute numbers. For this manuscript we opted to show sensitivity by using the API standards, as these could be prepared to a known concentration and their approximate pg/mm² calculated. We have followed industry guidelines and only reported API with a signal to noise greater than 5, typically a cutoff used for quantification, however, to err on the side of caution we have used this value as a detection cutoff in this dataset. text as such has been added to the manuscript. Line 208

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did a great job addressing my comments and I think the manuscript is ready for accept.