Table 1.

Summary of the conditions tested with the DESI XS source and their effect on signal intensity, histology, and immunophenotyping

DESI XS tested parameters		Signal intensity	Effect on H&Eb	PASc staining	mIFd	mIHCe
HTLa temperature (scan rate at 10 scans/s)	150 °C	Greater signal intensity for lower molecular mass compounds (m/z 50—350 Da)	Preserved all morphological features with some coagulation and fixation of proteins	Intact basement membranes of capillary loops and tubular epithelium highlighted by PAS	Preserved epitopes	Preserved epitopes with a positive effect from the fixation mechanism on stain intensity of pan-cytokeratin and vimentin
	450 °C	Enhanced signal of higher molecular mass compounds (m/z 600—1000 Da)	Preserved all morphological features with stronger coagulation, fixation, and fragmentation of proteins	Intact basement membranes of capillary loops and tubular epithelium highlighted by PAS	Preserved epitopes, higher background fluorescence due to coagulated proteins	Preserved epitopes with stain intensity comparable to MeOH controls
Scan rate with HTL at 450 °C	20 scans/s	Overall signal intensity decreased with the increase in the scan rate but did not compromise peak shape and resolution in any of the three conditions	Preserved all morphological features with stronger coagulation and fixation of proteins	-	-	-
	30 scans/s		Lower disturbance of tissue histology with less fragmentation of coagulated proteins	-	-	-

^aHTL heated transfer line

^bH&E hematoxylin and eosin

^cPAS periodic acid–Schiff

^dmIF multiplex immunofluorescence

^emIHC multiplex immunohistochemistry