Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy

J. A. Steyer, Wolfhard Almers

Research output: Contribution to journalArticle

190 Citations (Scopus)

Abstract

We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 800 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ~30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ~70 nm space around a granule. The 'cage' itself moves only slowly (D = 2 x 10-12 cm2/c). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

Original languageEnglish (US)
Pages (from-to)2262-2271
Number of pages10
JournalBiophysical Journal
Volume76
Issue number4
StatePublished - 1999

Fingerprint

Chromaffin Cells
Secretory Vesicles
Fluorescence Microscopy
Exocytosis
Cell Membrane
Cytosol
Uncertainty
Glass

ASJC Scopus subject areas

  • Biophysics

Cite this

Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. / Steyer, J. A.; Almers, Wolfhard.

In: Biophysical Journal, Vol. 76, No. 4, 1999, p. 2262-2271.

Research output: Contribution to journalArticle

@article{e07dfb3c313b4c43bb62a7095e2acfd4,
title = "Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy",
abstract = "We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 800 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ~30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ~70 nm space around a granule. The 'cage' itself moves only slowly (D = 2 x 10-12 cm2/c). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.",
author = "Steyer, {J. A.} and Wolfhard Almers",
year = "1999",
language = "English (US)",
volume = "76",
pages = "2262--2271",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "4",

}

TY - JOUR

T1 - Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy

AU - Steyer, J. A.

AU - Almers, Wolfhard

PY - 1999

Y1 - 1999

N2 - We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 800 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ~30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ~70 nm space around a granule. The 'cage' itself moves only slowly (D = 2 x 10-12 cm2/c). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

AB - We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 800 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ~30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ~70 nm space around a granule. The 'cage' itself moves only slowly (D = 2 x 10-12 cm2/c). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

UR - http://www.scopus.com/inward/record.url?scp=0032995380&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032995380&partnerID=8YFLogxK

M3 - Article

VL - 76

SP - 2262

EP - 2271

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 4

ER -