Fig. 4: Multidimensional tunning strategy to generate spectrally resolvable MARS probe library.
From: 9-Cyanopyronin probe palette for super-multiplexed vibrational imaging
a The probes were rationally engineered along three dimensions: core atom, number of rings, and isotopic substitutions. All probes were named according to fitted nitrile peak positions. b Fitted spectra of nitrile stretching modes of 20 MARS NHS esters. 20 peaks can be grouped into 4 sets of isotopologues and clearly resolved. c Exemplar images of immunostaining using individual MARS NHS ester probes in fixed cells. Top: Imaging α-tubulin with MARS2155 conjugated secondary antibody; Imaging fibrillarin with MARS2155 conjugated secondary antibody. Bottom: α-tubulin imaging with MARS2222 conjugated secondary antibody; Fibrillarin imaging with MARS2222 conjugated secondary antibody. Scale bars: 20 μm. Replicates = 3. d, f The fourth dimension to expand MARS library. d MARS probes developed for fixed sample imaging. e Imaging EdU or AHA labeled HeLa cells with MARS2238-Azide or MARS2184-PEG2-Alkyne through click reaction; Imaging F-actin structure with MARS2228 conjugated phalloidin; Imaging plasma membrane with MARS2238 conjugated wheat-germ-agglutinin. Scale bars: 20 μm. Replicates = 3. f MARS probes developed for live cell imaging. g Imaging mitochondria with MARS2237 in live cells; Imaging lysosome with MARS2184-Lyso in live cells; Imaging intracellular lipids or plasma membrane with C18 derivatized MARS probes. Scale bars: 20  μm. Replicates = 3. h 8-color multiplexed cell imaging with 3 fluorescent probes and 4 epr-SRS probes. Fluorescence: NucBlue for nucleus, Alexa-488-WGA for plasma membrane, and MitoTracker Orange for mitochondria. SRS: Label-free at 2945 cm−1 for proteins, MARS2238-Azide with EdU for newly synthesized DNA, MARS2210-NHS labeled antibody for α-tubulin, MARS2184-PEG2-Alkyne with AHA for nascent proteins, and MARS2155-NHS labeled antibody for fibrillarin. Scale bar: 20 μm. Replicates = 3.
