MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

Abstract

The proper staining of the plasma membrane (PM) is critical in bioimaging as it 16 delimits the cell. Herein, we developed MemBright: a family of six cyanine--based fluorescent 17 turn--on PM probes that emit from orange to near--infrared when reaching the PM, and 18 enable homogeneous and selective PM staining with excellent contrast in mono and two--19 photon microscopy. These probes are compatible with long--term live cell imaging and 20 immunostaining. Moreover, MemBright label neurons in a brighter manner than 21 surrounding cells allowing identification of neurons in acute brain tissue section and 22 neuromuscular--junctions without any use of transfection or transgenic animals. At last, 23MemBright were used in super--resolution imaging to unravel the dendritic spines' neck. 3D 24 multicolor dSTORM in combination with immunostaining revealed en--passant synapse 25 displaying endogenous glutamate receptors clustered at the axonal--dendritic contact site. 26MemBright probes thus constitute a universal toolkit for cell biology and neuroscience 27 biomembrane imaging with a variety of microscopy techniques. 29 30Visualizing plasma membrane is particularly important in neuroscience, because it enables 1 visualization neuronal organization and membrane trafficking involved in the synaptic 2 transmission. 3 Recent years have seen a tremendous expansion of fluorescence imaging 3 techniques and tools for cellular research. In addition to genetically encoded fluorescent 4 proteins, a number of molecular probes for monitoring cellular events have been 5 developed. 4, 5 The key challenge in cellular imaging is to stain cell compartments with high 6 specificity and persistence. Although numerous efficient molecular probes have been 7 designed to selectively stain specific cellular structures including mitochondria, 6 lipid 8 droplets, 7 endoplasmic reticulum, 8 nucleus 9 and lysosomes, 10--11 there is still a demand for 9 efficient and bright fluorescent PM markers. 12 Fluorescently labelled lectins notably wheat 10 germ agglutinin (WGA) and concanavalin A 9 are popular fluorescent membrane probes. 11Despite their efficiency and ease of use, these probes are expensive and much larger than 12 molecular probes. The small size of the latter, comparable to lipids, allows their precise 13 location in the lipid bilayer, which is of key importance for studies of the lateral lipid 14 organization of biomembranes (lipid rafts), 12, 13 FRET with membrane proteins 14 and super--15 resolution imaging. 15--16 Therefore, molecular probes are an interesting alternative to 16 protein--based membrane markers. Although highly hydrophobic fluorophores are efficient 17 markers of membrane models such as liposomes, they generally fail in staining the cell PM of 18 live cells as they tend to precipitate before reaching the membrane and to quickly cross the 19 PM to stain inner membranes. 12, 17 Recent efforts have been made to develop specific and 20 efficient PM probes for cell imaging with various fluorophores including chromone, 18--19 ...

Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome

“…Staining with the lipid probe MemBright, recently developed by Collot and colleagues (46,47), showed a heterogeneous population of round-shaped vesicles in the AB and MV fractions ( Figure 2C). Cryo-electron microscopy images of MV and sEV clearly ascertained the presence of a lipid bilayer membrane surrounding the vesicles (Figure 2D, Supplementary Figure 4).…”

BODIPY with Tuned Amphiphilicity as a Fluorogenic Plasma Membrane Probe

“…Additionally, in contrast to labelled WGA, B2-AZ showed highly specific staining of PM of glioblastoma spheroids and enabled delimiting individual cells with significantly better penetration depth. The efficacy of B-2AZ led us to adopt it in the MemBright family 18 and rename it as MemBright-488. Figure 5.…”

What is Best Strategy for Water Soluble Fluorescence Dyes?—A Case Study Using Long Fluorescence Lifetime DAOTA Dyes**

“…6,7 In bio-imaging the latter is seldom desirable, but notable exceptions include the probing of membranes and staining lipophilic compartments. 8,9 Dye aggregation may lead to unwanted changes in absorption and emission spectra, reduced fluorescence intensity and lifetime. [10][11][12][13] But even when aggregates are avoided e.g.…”