Comparing Raman and NanoSIMS for heavy water labeling of single cells

Abstract

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell resolved metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, no study has yet compared results from isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell. This knowledge gap creates uncertainty about the comparability of single cell SIP data generated independently using these techniques. In this study, we conducted a comparative analysis of 543Escherichia colicells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) at single cell resolution using correlative Raman and NanoSIMS measurements. For the first time, we were able to establish the extent of data equivalence, allowing for comparisons between the two approaches. Utilizing the dataset from this study, we examined the effectiveness of preprocessing techniques and optimal wavenumbers for analyzing Raman spectra, along with identifying the ideal masses for NanoSIMS analysis of cells incubated in the presence of2H2O. We make recommendations for approaches to analyzing and comparing data using both or either of these techniques. We anticipate that the findings presented herein will enhance the comparability of studies employing either technique and ultimately contribute to the establishment of a standardized framework within the field.ImportanceAccurate and reliable measurements of cellular properties are fundamental to understanding the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. For the first time, we study the relationship of the incorporation of a stable isotope (2H through incorporation of2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.