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Dynamic Imaging of Calcium and STIM1 in the Same Cell Using Wide-field and TIRF Microscopy
Simon Walker1, Nicholas Cunniffe1, Martin Bootman1, and H. Llewelyn Roderick1,2
1The Babraham Institute, Laboratory of Molecular Signalling, Cambridge, UK 2Department of Pharmacology, University of Cambridge, Cambridge, UK
Sponsored,vendor-submitted protocol   Sponsored by Olympus America Inc.    Published in November 2009 (p.43) DOI: 10.2144/000113032 Abstract

By employing an advanced imaging system, total internal reflection fluorescence (TIRF) illumination was used to observe the intracellular distribution of STIM-1 labeled structures at the plasma membrane, near-simultaneously with intracellular calcium concentration.

By employing an advanced imaging system, total internal reflection fluorescence (TIRF) illumination was used to observe the intracellular distribution of STIM-1 labeled structures at the plasma membrane, near-simultaneously with intracellular calcium concentration.


HEK-293 Cells (passage 35-50 from Microbix corporation)

YFP-/mCherry–tagged Constructs (gifts from R.S. Lewis, Stanford University, Palo Alto, CA, USA, and S. Muallem, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA)

JetPEI (Novagen)

Fura-2 AM (Invitrogen, Carlsbad, CA, USA) diluted to 2 mM stock in DMSO/20% Pluronic (Invitrogen)

Thapsigargin (Sigma, St. Louis, MO, USA) diluted to 2 mM stock in DMSO

Methods Cell Preparation

  1. Seed HEK-293 cells onto poly-L-lysine–coated 16 mm diameter round glass coverslips and transfect with YFP-/mCherry–tagged constructs (1 µg DNA per well) using jetPEI (Polyplus transfection) 16 h prior to imaging.

  2. For calcium imaging, load cells with Fura-2 AM (2 µM) diluted in calcium-containing extracellular (imaging) buffer (121 mM NaCl, 6 mM NaHCO3, 5.4 mM KCl, 5.5 mM D-glucose, 0.8 mM MgCl2, 25 mM HEPES, 1.8 mM CaCl2, pH 7.4) for 30 min.

  3. Remove dye-containing buffer from cells, wash with imaging buffer and leave for a period of 30 min at room temperature to allow for de-esterification.

  4. Mount coverslip in an appropriate imaging chamber (we use custom made stainless steel rings fitted with a teflon retainer) and immerse cells in imaging buffer.

Equipment and Settings

  1. Olympus cellR imaging system comprising IX81 microscope, 100×1.45 NA apochromat objective, MT-20 illumination unit, 488 nm/20 mW and 561 nm/25 mW lasers, and Hamamatsu ORCA ER camera.

  2. Solent Scientific environment chamber to maintain temperature at 32°C.

  3. Exciter filters for wide-field epifluorescence images of YFP and Fura-2: 470/40 nm and 380/15 nm, respectively. 495 DCLP and 505 nm long pass emission filter used for both dyes.

  4. YFP TIRF and mCherry TIRF images generated using 488 nm and 561 nm laser excitation, respectively, a multiband dichroic mirror with edge wavelengths at 495 nm and 573 nm and a 527/21 nm, 645/24 nm dual bandpass emission filter.

  5. Exposure times: YFP wide-field, 500 ms; YFP TIRF, 300 ms; mCherry TIRF, 400 ms; Fura-2 AM, 100 ms.

  6. Camera gain set to 100 for all experiments.

Live-cell Imaging

  1. Place imaging chamber on microscope stage.

  2. Place vacuum aspirator in imaging chamber ∼5 mm from coverslip surface.

  3. Add 2 ml fresh imaging buffer (pre-heated to 32°C) to cells.

  4. Focus on cells and capture reference images.

  5. After 60 s, apply 4 ml calcium-free buffer.

  6. After a further 60 s, apply 2 µM thapsigargin diluted in calcium-free buffer and continue recording for 10 min.

Image Analysis

All image analysis can be done using Olympus cellR software.


Figure 1 compares both wide-field epifluorescence and TIRF images from STIM1-YFP-transfected HEK cells. Thapsigargin (2 µM) was added at 0 min; bar, 10µm. Acquisition of TIRF images and wide-field epifluorescence images (Figure 2) allows STIM1 translocation to be monitored concomitant with changes in intracellular calcium concentration. Figure 2A shows the sequential acquisition of STIM1-YFP (TIRF), STIM1-mCherry (TIRF) and Fura2 (wide-field epi-fluorescence) images from the same cells. A set of three images (2× TIRF, 1× wide-field) was acquired every 2 s (images shown acquired 5 min after application of thapsigargin). Figure 2B illustrates the change in intracellular calcium concentration of cells outlined in A; bar, 10 µm.


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