Extended live-cell fluorescence microscopy of novel intrinsically fluorescent cholesterol analogues with improved photophysical properties

Session

Session 4 - New Technologies: Recent advances from Acquisition to Analysis

Authors

Senjuti Halder (4), Max Lehmann (3), Peter Reinholdt (4), Mohammad Bashawat (1), Holger A. Scheidt (2), Duccio di Prima (4), Jacob Kongsted (4), Peter Müller (1), Pablo Wessig (3), Daniel Wüstner (4)

Affiliations

1. Humboldt University Berlin
2. Leipzig University
3. University of Potsdam
4. University of Southern Denmark

Keywords

Fluorescence Microscopy, sterol analogs, live-cell imaging, photobleaching, Niemann–Pick disease 

Abstract text

Fluorescence microscopy is an interesting alternative to understand the sterol-driven physiological processes and heavily relies on the functional relevance of fluorescent sterol analogs.¹ This results into the development of a wide variety of luminescent cholesterol mimics to visualize the trafficking of cellular cholesterol. However, existing fluorescent analogs have their limited physico-chemical properties compared to cholesterol² and so far, none of them seems to be ideal in all aspects. Here, we introduce novel intrinsically fluorescent sterols containing four conjugated double bonds in the ring system and either a 3’-hydroxy group (1) resembling endogenous cholesterol or a 3’-keto group (2), as found in some keto sterols.³ Both sterol probes exhibit significant red-shift in their excitation and emission spectra, which amounts to about 80 nm compared to the widely used dehydroergosterol or cholestatrienol probe. Additionally, they can be easily taken up by cultured human control fibroblasts and NPC(Niemann-Pick type C)-patient derived fibroblasts cells from albumin complexes, which together with a much-improved photostability allows for their convenient live-cell imaging on conventional wide field and confocal microscopes with a DAPI/filipin filter-cube.⁴ The imaging potential of the both polyene sterol probes is further evaluated by studying their photobleaching kinetics which is analysed with the help of PixBleach (ImageJ plugin)⁵ using pixel-wise fitting of a bi-exponential decay function. Selective photodestruction of fluorescence of both 1 and 2 was also conducted in living cells to monitor fluorescence recovery after photobleaching (FRAP)⁶ of unbleached molecules into the area, which provides information on non-vesicular sterol transport kinetics and exchange dynamics between lysosomes and cytosol. Thus, our observations demonstrate that the cholesterol-mimicking probe 1 has comparable physicochemical and membrane properties as its endogenous counterpart and validate the fact that the strategy of extending the conjugated double bond in sterol ring system is worthwhile to achieve improved optical properties which offers many future applications in live-cell imaging including multiphoton, spectral and lifetime imaging on conventional microscope systems, thereby providing novel insights into cellular cholesterol trafficking under normal and disease-related conditions. 

References

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