Table 1.

Experimental goals associated with the Raman and FT-IR imaging of single microbial cells in complex biological samples.

Goal	Recommended technique	Notes	Reference
Analysis of motile cells and/or cell sorting	Raman microspectroscopy	Optical tweezers can be used to trap or move individual cells	Huang et al., 2010; Berry et al., 2015
Detection of cells on autofluorescent and opaque surfaces	Both	Autofluorescence does not interfere with IR measurements; Raman measurements possible using resonance Raman, SERS, sample photobleaching or aging	Ojeda et al., 2009; Polisetti et al., 2016
Addressing other sources of background interference	Both	Water is a strong IR absorber; using microfluidics or an ATR accessory can reduce signal interference	Kuimova et al., 2009; Birarda et al., 2016; Loutherback et al., 2016
Stable isotope probing	Both	Approaches currently better-established for Raman analyses	Wang et al., 2016
Imaging of large (cm-scale) surface areas	FT-IR microspectroscopy	FPA detectors readily available for FT-IR instruments; Raman instrumentation also available, but not as widely accessible	Kuimova et al., 2009; Kastyak-Ibrahim et al., 2012; Miller et al., 2013; Ando et al., 2016
Localization of cells in 3D space	Raman microspectroscopy	Imaging of z-stacks possible using confocal Raman measurements	Große et al., 2015; Silge et al., 2015
High-resolution (including subcellular) measurements	Both	Raman analyses (e.g., TERS) better-established; also possible using FT-IR but requires specialist equipment or access to synchrotron beamline	Mariani et al., 2010; Opilik et al., 2013; Saulou et al., 2013; Rusciano et al., 2014; Findlay et al., 2015
Label-free discrimination between individual strains or taxa	FT-IR microspectroscopy	FT-IR analyses can outperform Raman spectroscopy in terms of spectral quality and reproducibility	AlMasoud et al., 2016; Muhamadali et al., 2016

Recommendations for analytical techniques are provided for meeting each goal.