PREreview of RaMALDI: enabling simultaneous Raman and MALDI imaging of the same tissue section

The study presents the development of RaMALDI, a novel workflow that integrates Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) on a single sample. The authors aim to overcome the limitations of individual techniques by combining their strengths to achieve highly multiplexed, multimodal tissue imaging. The proof-of-concept study demonstrates the feasibility of the RaMALDI approach and its potential for enhanced molecular specificity and spatial resolution.

This study presents a valuable piece of work to the current analysis procedures, however, the authors miss to emphasize some of their most striking findings. The following points might be useful for the authors to clarify some points within the manuscript and/or inspire further research into their area of expertise.

Major issues

• The authors structured their study well, comparing several matrices in the beginning, which is commendable. The study also highlights the compatibility of RSI with MALDI matrices, which is a significant finding and adds to the existing knowledge. The use of different samples demonstrates the versatility of the RaMALDI workflow. However, regarding the claim of being the first study to perform both techniques on the same sample, it seems that the autors were not aware of a paper by Ryabchykov et al. (2018, doi: 10.3389/fchem.2018.00257 ) where they employed the same sample for both analyses (RSI without matrix, then MSI after addition of matrix). The authors should clarify this discrepancy and acknowledge previous work in this area.

• The systematic evaluation of different matrices constitutes the most interesting question in the manuscript, and I would like to suggest to the authors to include the Raman spectra of the different matrices in the corresponding figure in the main text rather than the supplemental information. Especially since this data is the reasoning behind chosing the DAN matrix. Further, the authors indicate that the MALDI matrix appears to protect the sample, which is an important observation that enhances the practicality of the technique. This feature can be a great selling point for RSI on matrixcovered samples instead of uncoated samples and I would like to suggest to the authors a more extensive discussion of this issue in the context of the relevant literature.

• On the same topic, if the authors collected data on the effect of different matrices on biological samples, I encourage them to include this into the manuscript, since this might be highly interesting for the readership and further enhance the quality of the already interesting data set.

• The manuscript lacks a discussion on the limitations of the proposed methods. Addressing potential challenges and constraints would help the readers understand the scope and applicability of the RaMALDI workflow. A comparison to other state-of-the-art techniques commonly used in biomedical imaging, such as fluorescence imaging and histology, would provide valuable context and highlight the advantages and unique contributions of RaMALDI.

Minor issues

• The selection of homogenized liver tissue for imaging seems questionable, and the rationale behind using homogenized tissue for imaging is unclear. The choice of kidney and brain tissues, while aiming for increased complexity, could be better justified in the context of the research question. The authors claim that brain tissue displays more “molecular layers”, which is unclear for the reader without further explanation. Moreover, the manuscript would benefit from more clarity on the author’s research objectives and how these samples contribute to answering specific questions. The analysis of the sample appears incomplete, as the authors only state the presence of lipids and proteins based on RSI. A more extensive discussion on possible applications would be highly beneficial to the manuscript.

• Performing additional validation experiments on different biological tissues, with and without the matrix, would strengthen the reliability and generalizability of the findings. This would help assess the performance and accuracy of Raman spectra in the presence of the MALDI matrix across various biological samples. (although I acknowledge that this is a very work-intensive assessment and could be a stand-alone publication.

• More information on special requirements for Raman spectra processing would be helpful.

• Minor language inconsistencies, such as the use of "non-biological" and "nonbiological," should be addressed for better clarity and consistency throughout the manuscript. Additionally, attention should be given to the formatting to avoid separation of numbers and units by the use of non-breaking spaces.

Competing interests

The author declares that they have no competing interests.