Live‐cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

Abstract

Analysis of intracellular cholesterol transport by fluorescence microscopy requires suitable fluorescent analogues of cholesterol. Most existing cholesterol analogues contain lipophilic dyes which can compromise the sterol properties in membranes. An alternative strategy is to introduce additional double bonds into the sterol ring system resulting in intrinsic fluorescence, while at the same time keeping the cholesterol-like properties of the analogues. Existing polyene sterols, such as dehydroergosterol (DHE) or cholestatrienol (CTL), however, contain only three double bonds and suffer from low brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system. We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes.

Structural design of intrinsically fluorescent oxysterols

“…predict an overweight of the keto-form [31], and the same might be expected for 25-OH-3a. However, due to the much reduced 2PA for the keto-form compared to the enol-form only the enol-form will be visualized in two-photon microscopy.…”

“…Still, due to the different membrane properties of the enol-and keto-forms it is necessary to lock the probe in the enol-form in order to prevent the probe from inverting in the membrane. A locked enol form may be acquired by acetylation of the 3α-OH group, as will be described in future work [31], but the membrane effects and orientation of such a probe should be further investigated.…”

“…Probe 25-OH-3a is inspired by our previous work on cholesterol probes [29,30]. Here, we also consider the keto form of this probe because we found that probe 3a can undergo keto-enol tautomerization [31], and this is probably also the case for 25-OH-3a. The chemical structures and numbering of the oxysterols treated in this study are given in Figure 1.…”

Image segmentation and separation of spectrally similar dyes in fluorescence microscopy by dynamic mode decomposition of photobleaching kinetics

“…Here, a concern is often that prolonged fluorescence imaging of dynamic processes in living cells can lead to photo destruction of fluorophores [40]. Therefore, it is important to determine photobleaching characteristics of fluorescent dyes under various conditions, either for optimizing probe design or for optimizing imaging conditions [14, 20, 41, 42]. For example, sensitive single molecule imaging and super-resolution microscopy critically depends on development of bright and photostable fluorophores, and here, DMD of dye photobleaching in cells can be very useful for designing improved organic fluorophores [43].…”

The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations

“…Following these computational studies, the C4P Chol analogue was ultimately synthesized and used for imaging in human fibroblasts [ 155 ]. However, upon synthesis, the fluorescent enol form undergoes tautomeric equilibration with the triene keto form ( Figure 13 b), and from electronic structural calculations of free energies of the two forms in vacuum, the authors estimated the keto:enol ratio to be ~6 × 10 3 at 25 °C.…”