Measurement of [Ca2+]i with fast video imaging of fura-2
To measure the [Ca2+]i, cells are loaded with Ca2+-specific dye fura-2. This dye is available in a cell-permeant form (acetoxymethyl (AM) ester ) that readily enters the cell and becomes trapped following the cleavage of the AM group from the dye. Cells are viewed with a fluorescence microscope. The video clip (right) shows, in sequence:

  • the black and white fluorescence image of epithelial cells loaded with fura-2 as observed by a SIT camera.

  • the overlay of white lines indicating the estimated position of the cell boundaries.
  • the pseudo-coloring of the image to facilitate intensity discrimination. 
  • the resultant [Ca2+]i (indicated by scale bar at the bottom of the image) calculated by performing ratio imaging at the onset of the experiment.  

Changes in [Ca2+]i occur rapidly and it is necessary to sample images quickly if these changes are to be correlated with changes in ciliary beat frequency.  However, techniques of ratio imaging with fura-2 to quantify [Ca2+]i can be slow. Reasons include slow filter changes and image acquisition and camera lag.  To overcome this problem, we use a single wavelength approach, that allows [Ca2+]i changes  to be recorded at 30 fps. These measurements are quantified by reference images obtained by ratioing imaging when the time constraints are less.
Outline of Fast Video Imaging  Ex 310 Ex 340
Initial starting conditions obtained from 10 frames at 340/380. From this data and calibration values, the initial [Ca2+]i can be calculated from the experimental image ratios of 340/380.   The initial fluorescence values (Fo) are determined.  

10 frames

10 frames
The experimental images are collected at 30 fps at a single wavelength of 380 nm for approximately 30 seconds. During this time the changes in  calcium are proportional to the change  in fluorescence of the image, i.e. Ca ~ Fo/Ft. 

0
to
30 sec
After about 30 seconds, a reference image is obtained at 340 nm and this is used with the 380 nm image to determine the exact calcium concentration at 30 seconds.   10 frames
Images are subsequently recorded at 30 fps, and relative changes used once again to determine the  [Ca2+]i  30 secs
to
 60 secs
Another or  final set of reference images are recorded. The process can be repeated for as long as required.  10 frames
Bleaching or dye loss
The major advantage with ratio imaging is that potential artifacts such as changes in dye concentration, e.g. bleaching, are compensated for. By returning to single wavelength recordings, this potential artifact once again becomes a concern. However, the reference 340 nm images can be used to calculate the real [Ca2+]i at known time points and these can be used to calibrate the single wavelength calculations.  The methodology for this is beyond the scope of this web page - please refer to our paper Leybaert et al., (1998) 

 

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