Potential of ultraviolet wide‐field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

Abstract

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/ emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHEstained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is $1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 lm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-lm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. Microsc. Res. Tech. 74:92-108, 2011. V V C 2010 Wiley-Liss, Inc.

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

“…Alternatively, DHE can be imaged using multiphoton excitation microscopy and photon counting detectors, leading to the same results regarding membrane organization and intracellular transport of DHE (McIntosh et al, 2003;Wüstner et al, 2011).…”

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

“…From separately acquired image stacks the bleaching of BODIPY fluorescence during acquisition was assessed. In the FLIP image stacks, a pixel-wise bleach correction was performed with this information, as described previously (Wüstner et al, 2011a). B, segmentation strategy to determine fluorescence of BODIPY in the PM (left panel), the cytoplasmic pool (middle panel) and the ERC (right panel).…”

Cholesterol and nicotinic acetylcholine receptor: An intimate nanometer-scale spatial relationship spanning the billion year time-scale

“…The smallest of such extrinsic probes is nitrobenzoxadiazole ("NBD") [94], and this fluorophore adds not only a non-negligible mass to the cholesterol moiety but may also modify its location and orientation in the membrane. An alternative minimally perturbing cholesterol-like probe is dehydroergosterol [113], which inconveniently has low quantum yield, bleaches rapidly, excites in the high-energy UV region of the spectrum in conventional wide-field microscopy [148] and is thus prone to deleterious effects on cell integrity, and requires costly UV-transparent optics for fluorescence microscopy [93]. Dehydroergosterol can also be studied using 2-photon fluorescence microscopy, circumventing one of the above problems, i.e.…”

“…Dehydroergosterol can also be studied using 2-photon fluorescence microscopy, circumventing one of the above problems, i.e. excitation in the UV region of the spectrum [147,148]. Furthermore, fluorescent cholesterol probes can partition differentially in the plasma membrane as compared to the endogenous lipids [30].…”