Calcium imaging allows us to study the movement of calcium ions into and out of the cytoplasm in response to a variety of physiological stimuli. To begin calcium imaging experiments in cortical neurons, cells are loaded with the calcium indicator dye RA 2:00 AM mounted on a perfusion chamber in RA two free media and placed on a fluorescence microscope where they are hit with light at three 40 and then three 80 nanometer in wavelength, which is absorbed by the dye. 5 0 5 nanometer emissions from the die at these two wavelengths are collected and the level of intracellular calcium can be calculated from the ratio of these values.
Hi, I'm Im Mar Barreto from the laboratory of Ricardo Domish in the Department of Neurobiology at Stanford University. Today we will show you a procedure for measuring intracellular calcium elevations in neurons. So let's get started.
The cortical neurons used for this calcium imaging procedure can be grown using established techniques, but should be plated on 15 millimeter. Number one glass cover slips. Because low fluorescence imaging of live cells is best achieved with high numerical aperture objectives that require oil.
In addition, we preco the glass cover slips with poly ornithine and laminin in order to prevent the cells from detaching or moving during imaging experiments. A variety of physiological solutions including cell culture media can be used for calcium imaging experiments. However, make sure that the solutions are free of phenol red, which greatly increases the fluorescent background for cortical neurons.
We use Tyro solution, which mimics cerebral spinal fluid and we supplement it with 0.1%bovine serum albumin. To begin the cell loading step, prepare a one millimolar stock of a c oxy methyl ester, FERRA two, or RA 2:00 AM.To do this, add 50 microliters of DMSO to a 50 microgram vial of URA 2:00 AM.It is important to use dry DMSO packed under nitrogen and it is necessary to remove the DMSO with a needle by puncturing the septum to prevent hydration of the DMSO. When not in use, keep the Fira 2:00 AM solution in a dark dry place.
FIRA 2:00 AM in DMSO is stable at room temperature for 24 hours. It is also stable at negative 20 degrees for several months. Next, aliquot two milliliters of culture media into a 15 milliliter conical tube that has been warmed to 37 degrees Celsius.
Then add two microliters of fira 2:00 AM stock to generate a one micromolar fira 2:00 AM solution. Vortex the solution vigorously for a couple of seconds, transfer this loading solution to a 35 millimeter tissue culture dish using forceps. Place the cover slip with the cortical neurons into the dish.
Incubate the neurons at 37 degrees for 30 minutes in a dark incubator time the incubation precisely during the incubation. Prepare another 35 millimeter tissue culture dish containing two milliliters of tissue culture media without fira 2:00 AM.When the incubation is complete, remove the cover slip from the loading solution and place in the new dish. After immersing the cover slip in fira 2:00 AM free media mounted onto an imaging chamber.
We use the RC 20 H imaging chamber by Warner Instruments. Apply vacuum grease to both sides of the chamber. Then place the cover slip onto the chamber and gently press it into position.
Next, screw in the side clamps. Then insert the cover slip retainer ring. Once the cells are mounted and the imaging chamber is assembled, proceed with the calcium imaging step.
For calcium imaging of cells, we use an inverted Nikon eclipse TE 2000 U microscope, equipped with a xenon arc lamp. Sutter instruments, an automated stage lule and a cooled charge. Couple device CCD camera hamamatsu orca two.
The microscope is controlled by a Macintosh computer running open lab software in provision to begin imaging, calibrate the automated microscope stage to ensure that the appropriate coordinates are found. Then place a drop of oil on the objective. For imaging, we use 40, 60, or 100 time Nikon floor oil immersion objectives with an NA exceeding 1.2.
Next, load the Tyro solution into the input line. Taking care to prevent the formation of air bubbles. Then connect the chamber to the input perfusion lines and perfuse tyro solution through the chamber.
Perfuse slowly to avoid the formation of air bubbles in the chamber. Now secure the chamber on the microscope stage and install the output perfusion line, which is connected to a suction line. Apply the input solution to confirm that the profusion lines are connected.
You should hear the input solution being aspirated into the output line. After connecting the profusion lines, focus on the cells using transmitted light. Once the cells are in focus, open the fluorescent shutter and look through the eye pieces To examine the fluorescence of the cells using illumination at three 40 and three 80 nanometer wavelengths, resting cells should be dim at three 40 and bright at three 80.
To prevent phototoxicity, the intensity of the excitation light should be reduced with a neutral density filter and cells should not be illuminated with UV light for more than 10 or 15 seconds. Now, examine the cells using the camera and set the gain and exposure of the camera to generate an image that is close to saturation when illuminated at three 80 nanometers and while below saturation. When illuminated at three 40 nanometers, keep the exposure time at 200 milliseconds or shorter if possible.
Once set, do not change the camera gain or exposure. Collect an image at each wavelength and use the region of interest tool or ROI tool to measure the background intensity in a variety of locations. For each wavelength, calculate the average of the background values and then enter the background values into the appropriate locations.
In the imaging program, the background intensity will be subtracted from each pixel in the field. Next, enter the maximum and minimum ratio values or RN and R max, which were determined previously. Once the arm in and R max are set, choose the fields that you plan to image and then use the automated stage to record the location of each field of view into the software program.
We generally collect five fields of view. Now start the experiment and collect images. During an experiment, the automated stage moves to a field, collects a ratio of the images at three 40 and three 80.
Then moves on to the next field until it returns to the first field. So if you need to collect images quickly, then only collect ratio images from a single field.Note. When testing the calcium imaging system, it is often useful to use high concentrations of stimuli that will cause an intracellular calcium rise, such as 65 millimolar potassium.
Once the experiment is complete, proceed with the results analysis to analyze the results of the experiment, convert the set of ratio images into time lapse calcium measurements for individual cells or regions of interest within cells. To do this, use the ROI tool to define the areas of the ratio image in which you want to measure calcium. It is generally useful to have at least one ROI that covers the cell body of the cell.
Once the ROIs are defined, use the software to collect time-lapse ratio measurements for each ROI. In each image, import the ratio measurements into an analysis program such as Igor Pro. In order to convert the ratio measurements into intracellular calcium values, we use a set of macros written in Igor Pro to help us carry out the analysis.
This is a calcium imaging movie of ura two loaded neurons stimulated with potassium chloride. The image is pseudo colored, so that low URA two ratios which correspond to low calcium concentrations are blue and high ratios, which correspond to high calcium concentrations are yellow and red. But upon depolarization with potassium chloride, which opens voltage gated calcium channels, calcium rises in the URA 2 3 40 to three 80 excitation ratio increases causing the neurons to become yellow and red.
The calcium is then removed from the cytoplasm, which causes the cells to become blue.Again. These images were analyzed and the ratio values were converted to calcium concentrations using a simple equation and the program ego or pro, We just show you how to perform calcium imaging of cortical neurons using inferior 2:00 AM.When doing this procedure, it's important to remember to not use the shulter plastic since it's often fluorescent at ultraviolet waylands, which makes imaging difficult. Also, make sure to avoid air bubbles when perfusing the solution through the chamber.
So that's it. Thanks for watching and good luck with your experiments.
