Figure 1.
The Solvatochromatic properties of MDH.
(A) MDH contains a fluorescent dansyl moiety that displays strong solvatochromatic properties. (B) MDH absorption spectrum in water and methanol. (C) MDH emission in different solvents (H2O/methanol/isopropanol/sunflower seed oil); the MDH emission maxima shifted from 570 nm to 485 nm with decreasing solvent polarity (when excited with 405 nm).
Figure 2.
A comparison between the spectral properties of commercially available LD dyes and fluorescent proteins.
The absorbance (A) and emission spectra (B) of LD dyes in sunflower seed oil, overlaid with fluorescent proteins' respective curves in water (the TagGFP2, TagYFP, and TagRFP spectra were obtained from Evrogen). The absorbance spectrum of MDH is significantly blue-shifted compared to those of the green and red fluorescent proteins. The 420–480 nm emission from MDH in oil is also free of overlap from the green and red fluorophores.
Figure 3.
MDH staining patterns are separated from known molecular markers for autophagic structures.
HepG2 cells transiently transfected with either EGFP-LC3B (top row), mcherry-atg5 (middle row), or mEos2-GABARAPL2 (bottom row) were stained with MDH and imaged. Under normal (not shown) and starvation conditions, EGFP-LC3B, TagRFP-atg5, or mEos2-GABARAPL2 puncta did not colocalize with MDH spots (right columns; merged). Scale bars, 10 µm.
Figure 4.
(A) MDH staining patterns in HepG2 cells mimicked cytosolic LDs. Fluorescence emission between 420–480 nm resulted in a high contrast, low background staining of round spots in HepG2 (top; 405 nm excitation). In contrast, emission between 550–650 nm generated images retaining the round spots, but with diffusive background stains (bottom). (B) HepG2 cells were transiently cotransfected with TagRFP-ADRP (colored red) and EGFP-tubulin (colored green), and stained with MDH (colored blue). ADRP signals surrounded the MDH spots (Inset; magnified image of the white dotted square region), indicating that MDH patterns are indeed LDs. MDH can be combined with the use of green and red fluorophores, as evidence by the separate MDH/ADRP/tubulin images. (c) MDH specifically stained the LDs in 3T3-L1 cells before and after differentiation. Top: undifferentiated. Bottom: ten days after differentiation, showing the expected increase in average LD size. Scale bars, 10 µm.
Figure 5.
LD trafficking monitored in living cells using MDH.
HeLa cells stained with MDH (A, left) were imaged every 2 seconds for 20 frames. Single LD trajectories (A, right, showing the movement of the LD in the white dotted rectangle) were then individually analyzed. LDs stained by MDH (B, top) showed similar active transport speeds compared to those stained with BODIPY 493/503 (B, bottom) (150 velocities each, in HeLa cells). The two distributions displayed similar mean (0.11 µm/s vs. 0.12) and standard deviation (0.09 vs. 0.11).
Figure 6.
Superior MDH properties for live cell imaging.
(A) The performance of MDH for long-term imaging was benchmarked against that of NileRed and BODIPY 493/503 directly in living cells. We tracked the fluorescence from MDH, NileRed, or BODIPY 493/503 stained, large/immobile LDs in differentiated 3T3-L1 cells over time (excitation laser powers were adjusted such that the images from the three respective dyes were of the same quality; Figure S3), and found that while NileRed and BODIPY 493/503 showed substantial photobleaching, MDH emission remained stable for long periods of time. (B) The LDs of HepG2 cells stained with MDH can be imaged with high contrast through two-photon fluorescence microscopy (760 nm excitation). The white-dotted line outlines the boundary of the cell. Scale bar, 10 µm.