In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

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To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

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Golshani, P., Portera-Cailliau, C. In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice. J. Vis. Exp. (13), e681, doi:10.3791/681 (2008).


To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.


  1. Prepare the fluorescent calcium indicator solution. We use acetoxymethyl ester dyes, or AM dyes for short. These are dyes that can be taken up by cells when injected extracellularly. We typically use Oregon-Green BAPTA-1 AM or Fluo-4 AM at a concentration of 1 mM. AM dyes can be obtained from Invitrogen in 50 microgram vials. We first prepare a 20% solution of Pluronic F-127 in DMSO. We warm the solution prior to use every day until its clear. We dissolve the calcium indicator the 20% Pluronic F-127/DMSO for 15-20 minutes to a concentration of 10 mM. We then dilute the solution to 1mM in a solution containing, in mM: 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. In addition, 100 µM Sulforhodamine 101 is usually included to label astrocytes and visualize the pipette during the injection. We filter the solution prior to injection with a 0.49 micron centrifugal filter (Stosiek et al., 2003; Garaschuk et al., 2006).

  2. Implant a cranial window over the area to be imaged. We have described the general approach in JoVE, but the following modifications are important for calcium dye injection:

    Make a smaller craniotomy of 2 mm in diameter over the cortical area of interest, taking extreme care not to damage the underlying dura. Secure a coverslip in place over the craniotomy, but only partially cover the craniotomy, leaving a small gap between the edge of the craniotomy and the glass coverslip to allow room for a pipette to enter the cortex obliquely. Prior to injection, make a shallow well with dental cement around the craniotomy. The well should extend as rostral in direction and be shallow enough to allow insertion of the micropipette for injection.

  3. Transfer the mouse transferred to the stage of the microscope and immobilize the head, as shown in our video on blood flow imaging.

  4. Pull glass microelectrode pipettes to a tip diameter yielding 2-4 Mega Ohms in resistance, using a pipette puller. Load the calcium indicator dye mix onto the glass pipette using a microsyringe. Using a micromanipulator, gently lower the micropipette onto the surface of the brain using a 4X objective, which is then finely positioned under a 40X objective. Choose a suitable location for injection, such that there are few or no overlying blood vessels to obscure the imaging. Using two-photon microscopy, the tip of the pipette is visualized and advanced slowly into the cortex, to a depth of 200 micrometers below the dura. Then, pressure inject the dye mixture at 10 PSI for 1 minute using a Picospritzer. We usually deliver two to four injections of the AM calcium dye mix, separated in space by approximately 200-300 microns. This bulk loading approach of slightly overlapping injections ensures that an area as large as 600 x 600 microns is adequately stained for calcium imaging. Begin imaging 1 hour after the injection of the AM dye mixture, to allow for the dye to diffuse and be taken up by neurons.

  5. Perform in-vivo two-photon imaging with a custom made two-photon microscope using a Chameleon Ti-Sapphire Laser (tuned to 800-880 nm) and Cambridge Technology galvanometer mirrors. We aquire images using ScanImage Software developed in Karel Svoboda's laboratory (Pologruto et al., 2003). Laser Power is typically well below 70 mW at the sample. Whole field images are acquired either using a 20X (0.95 NA) or 40X (0.8 NA) Olympus objectives, at 1.95-15.63 frames per second (512 x 256 pixels to 256 x 64 pixels). Line scans are acquired at 500 Hz. During imaging, animals are kept under light isoflurane anesthesia (0.6-0.9%), and are also kept at 37°C using a Harvard Apparatus Temperature Control Device. Care is taken to keep the respiratory rate of animals as close to 100 breaths/minute as possible. This level of anesthesia use is the lowest level that immobilizes the animal, but still permits spontaneous activity to be recorded.

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The advantage of this method over electrophysiological recordings is that it is less invasive and allows the recordings of activity in dense neuronal neuronal networks. When doing this procedure it s important to remember to take extreme care when performing the craniotomy not to damage the dura. Even a small amount of subdural bleeding with greatly obscure the imaging.

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We would like to acknowledge Olga Garaschuk for helpful discussions on optimizing the bulk loading of calcium indicators.


Name Type Company Catalog Number Comments
Oregon Green BAPTA1-AM Reagent Invitrogen O-6807
Fluo 4- AM Reagent Invitrogen F-14201
Sulforhodamine 101 Reagent Invitrogen S-359
Pluronic F-127 in DMSO Reagent Invitrogen P-3000MP
Centrifugal Filter (0.45 micron) Reagent EMD Millipore UFC3 0HV 0S



  1. Garaschuk, O., Milos, R. I., Konnerth, A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc. 1, (2006).
  2. Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. In-vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA. 100, 7319-7324 (2003).
  3. Pologruto, T. A., Sabatini, B. L., Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes.   Biomed Eng Online 2:13. Biomed Eng Online. 2, (2003).



  1. Dear Dr. Golshani,     Do you close the craniotomy site completely with the coverslip and secure it after the injection fo the indicator?  Or do you image with the half open craniotomy hole?  Also during craniotomy, do you keep the animal under deeper anesthesia unlike during imaging? Thanks James T. Russell

    Posted by: Anonymous
    July 15, 2008 - 11:27 AM
  2. Hi James,   I image with a ²/3 closed craniotomy.  The craniotomy is performed under isoflurane anesthesia (breath-rate around 60-80) with all withdrawl reflexes completely suppressed.  We image animals under light isoflurane anesthesia (with the breath-rate close to 100 bpm).

    Posted by: Anonymous
    August 2, 2008 - 8:30 PM
  3. Any trick to go trough the dura with the pipette ? (as opposed to just push the dura with the pipette) Thanks

    Posted by: Anonymous
    July 17, 2008 - 11:56 AM
  4. We position the pipette at 30 degrees to the dura, and then slowly advance it into the brain.  We have had no problems with the pipette breaking or with penetration of the dura.  We are imaging mice.  The situation may be different with rats or other animals.   Peyman

    Posted by: Anonymous
    August 2, 2008 - 8:32 PM
  5. Dear Dr. Golshani,
    Thanks for your share.In my lab,we use P-97 Micropipette Puller.Could you tell me the Parameters for pulling the Micropipette in your experiment?

    Posted by: Anonymous
    November 4, 2009 - 7:44 AM
  6. Dear Dr. Golshani,
    I am wondering if you used the recording chamber, just as in Garaschuk's paper.


    Posted by: Anonymous
    March 12, 2010 - 3:19 AM
  7. No, the cortex buffer solution is stationary above the brain. Garaschuk uses a perfusion method, much like slice recordings.

    Posted by: Anonymous
    August 13, 2010 - 12:22 PM
  8. Hi Dr. Golshani,
    thanks for the great video. I have a question about using fluo-4--I find it much trickier than using OGB-1. It seems not to dissolve as well--when I filter it, there seems to be a large amount of the dye filtered out, even if I've vortexed and sonicated for half an hour, leaving the resulting solution very pale. Is this normal, or there any trick to using fluo-4 (warming the solution? sonicating for longer?) as opposed to OGB-1?
    thanks very kindly,

    Posted by: Kelly C.
    November 10, 2010 - 6:47 PM
  9. I treated Fluo-4 just like OGB and was able to stain a smaller proportion of cells. Cells appear dimmer but flash brighter with each spike.

    Posted by: Anonymous
    November 10, 2010 - 8:08 PM
  10. Thanks--maybe I am just using the wrong filter material. Can you recommend the model of filter you use for your solution? Thanks!

    Posted by: Kelly C.
    November 12, 2010 - 2:28 PM
  11. The filter is mentioned above: 0.45 micron centrifugal filter.

    Posted by: Anonymous
    November 17, 2010 - 10:42 AM
  12. Right sorry, I meant, what manufacturer/model number? The filter material itself might play a role in affecting the final dye concentration--for example if the material has an affinity for esters or something of that nature.

    Posted by: Kelly C.
    November 18, 2010 - 4:42 PM
  13. Hi Dr. Golshani and Portera-Cailliau,

    Thanks for the excellent video! We mean to begin performing experiments with a similar method and I have a quick question. Are you able to perform the experiments chronically, or must it be acute because the entire cortex is not covered and presumably the imaging window becomes occluded?


    Posted by: William F.
    December 10, 2012 - 4:17 PM

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