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

M. Modzel, K. A. Solanko, M. Szomek, S. K. Hansen, A. Dupont, L. J. Nåbo, J. Kongsted, D. Wüstner*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
JournalJournal of Microscopy
Volume271
Issue number1
Pages (from-to)36-48
ISSN0022-2720
DOIs
Publication statusPublished - 2018

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Polyenes
Filipin
Membranes
Fluorescence Microscopy
Microscopy
Coloring Agents
Fluorescence
Equipment and Supplies

Keywords

  • Cholesterol
  • Electronic structure calculation
  • Fibroblasts
  • Fluorescence
  • Imaging
  • Intracellular transport
  • Niemann-Pick disease type C2
  • Photobleaching
  • Synthesis

Cite this

@article{be48a9b45c684304ba11352d4685aba8,
title = "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.",
keywords = "Cholesterol, Electronic structure calculation, Fibroblasts, Fluorescence, Imaging, Intracellular transport, Niemann-Pick disease type C2, Photobleaching, Synthesis",
author = "M. Modzel and Solanko, {K. A.} and M. Szomek and Hansen, {S. K.} and A. Dupont and N{\aa}bo, {L. J.} and J. Kongsted and D. W{\"u}stner",
year = "2018",
doi = "10.1111/jmi.12691",
language = "English",
volume = "271",
pages = "36--48",
journal = "Journal of Microscopy",
issn = "0022-2720",
publisher = "Wiley-Blackwell",
number = "1",

}

Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport. / Modzel, M.; Solanko, K. A.; Szomek, M.; Hansen, S. K.; Dupont, A.; Nåbo, L. J.; Kongsted, J.; Wüstner, D.

In: Journal of Microscopy, Vol. 271, No. 1, 2018, p. 36-48.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

AU - Modzel, M.

AU - Solanko, K. A.

AU - Szomek, M.

AU - Hansen, S. K.

AU - Dupont, A.

AU - Nåbo, L. J.

AU - Kongsted, J.

AU - Wüstner, D.

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Cholesterol

KW - Electronic structure calculation

KW - Fibroblasts

KW - Fluorescence

KW - Imaging

KW - Intracellular transport

KW - Niemann-Pick disease type C2

KW - Photobleaching

KW - Synthesis

U2 - 10.1111/jmi.12691

DO - 10.1111/jmi.12691

M3 - Journal article

C2 - 29516493

AN - SCOPUS:85043390589

VL - 271

SP - 36

EP - 48

JO - Journal of Microscopy

JF - Journal of Microscopy

SN - 0022-2720

IS - 1

ER -