The overall goal of this procedure is to demonstrate the acquisition, detection and analysis of local IP three mediated calcium signals in intact mammalian cells using camera-based imaging. This is accomplished by first culturing the cells onto the glass bottom cover dishes and loading them with the cell membrane permeable forms of the fluorescent calcium indicator. CAL five 20 and caged IP three.
The second step is to position the cover glass onto the stage of an inverted microscope and illuminate the sample with 488 nanometer light to excite the calcium sensitive fluorescent dye Cal five 20. Next local calcium signals are induced by exposing the cells to a brief pulse of UV light. This photo liberates IP three from a caged precursor such that it can now bind to the acetol trisphosphate receptor and mediate calcium release from the endoplasmic reticulum.
The final step is to import the image stack into the custom written algorithm to initiate the automated analysis of the recorded data. Ultimately, the algorithm rapidly localizes sites of calcium release with sub pixel resolution, allows user review of data and outputs time sequences of fluorescent ratio signals together with amplitude and kinetic data in an Excel compatible format. This approach allows for the imaging of hundreds of channels or events simultaneously, but yields vastly more data than traditional approaches and the massive amount of data generated render manual processing, visual identification and analysis.
Impractical demonstrating this procedure will be Jeff Locke, a postdoc from my lab. To begin this procedure, prepare the loading buffer by diluting the stock solutions of membrane permeant CAL five 20 and CI IP three to a final concentration of five micromolar and one micromolar respectively in calcium HBSS. Next, aspirate the culture media and rinse the cells with calcium HBS S3 times.
Then remove calcium HBSS and incubate the cells in loading buffer for 60 minutes at room temperature in the dark. Subsequently, remove the loading buffer and rinse the cells with calcium HBS S3 times to facilitate the study of local calcium signals by blocking calcium waves. Incubate the cells with five micromolar EGTA am in calcium HBSS in the dark for 30 to 60 minutes at room temperature.
Following this incubation, rinse the cells three times with calcium HBSS. Afterward, incubate the cells in calcium HBSS for 30 minutes to allow for D esterification of loaded reagents immediately prior to imaging. Replace calcium HBSS with fresh calcium HBSS in order to remove any dye that might have leaked into the bath during the final incubation.
In this procedure, place a small drop of immersion oil onto the 100 XAPO to turf objective. Then mount the imaging dish on the stage of the inverted microscope. Bring the cells into focus using transmitted light.
Next, illuminate the cells with light from a blue laser. In order to excite CAL five 20, collect the emitted fluorescence using a high speed E-M-C-C-D camera. Using the E-M-C-C-D software.
Reduce the imaging field from the full 512 by 512 pixels in order to collect data at the necessary temporal resolution to capture local calcium events. Configure the software to automatically record a few seconds of baseline activity, then deliver a UV flash to the cells to evoke a desired frequency of local calcium events and continue recording for another 10 to 30 seconds. At the end, save the files as image stacks for offline analysis.
In this procedure, convert the image files to the multiplane TIFF file format. Locate the folder containing the custom written analysis algorithm and double click run exe. Then select the TIFF file to be analyzed.
Choose the analysis parameters in the open dialogue box. Determine the black level to be offset from the image stack by either moving the cursor to a part of the field of view that does not contain a cell or by manually entering a black level using the set black level prompt. Then the algorithm will run four windows on the screen appear following the analysis by the software.
C is a monochrome image of resting fluorescence from the cells being analyzed. White squares superimposed on this image are event locations determined as the OID positions of two dimensional Gaussian functions fitted to the events. Use the forward or backward cursor key to cycle through the locations of puff activity on window C.Upon doing so, the activity at each site presented as background subtracted Gaussian smooth fluorescence ratio changes, updates in window D.Next, click on a red highlighted event to update window E that displays the temporal evolution of the event.
Then click on window F, which displays the spatial profile of the event averaged over its time course. Together with the spatial profile of the fitted Gaussian function, manually review the events that have been identified so that the artifacts can be rejected from analysis. Delete such events by right clicking the red highlighted events.
Export the data by selecting save to Excel or export data on a per cell basis by drawing around the cell of interest and selecting save cell. Shown here is a monochrome image of resting Cal five 20 fluorescence onto which the locations of local IP three mediated calcium signals have been superimposed and are indicated by the white circles. These are the corresponding traces that illustrate the puffs evoked at three different sites.
In response to photo liberation of IP three from its caged precursor, red highlighted events are the puffs identified to have originated from that particular site. The superimposed traces of the representative calcium puffs are aligned and shown on an expanded scale, and this figure shows the monochrome turf footprint image of the resting cal five 20 fluorescence in SH SY five Y cells loaded with CI IP three and EGTA white circles denote the puff site origins. The traces illustrate puffs evoked at three different sites, which correspond to the white circles in the image.
The superimposed traces of representative calcium puffs are aligned and shown on an expanded scale. After watching this video, you should have a good understanding of how to image local calcium signals in intact mammalian cells, and also use our algorithm to automate analysis of recorded data. The high throughput nature of the algorithm for automating identification and analysis of local calcium events is highly useful for imaging calcium channelopathies in disease states via single cell models such as Alzheimer's disease and autism spectrum disorder.
