This article describes a method for imaging Protein Kinase-A (PKA) activity in cortical neurons of an anesthetized mouse during locomotion. Using two-photon fluorescence lifetime imaging microscopy (2pFLIM), researchers can observe changes in PKA activity as the mouse transitions between rest and enforced locomotion.
Secure an anesthetized mouse with a cranial window over its motor cortex onto a treadmill.
The mouse expresses a Protein Kinase-A reporter in cortical neurons.
Position the treadmill under a two-photon FLIM objective.
Add a water droplet between the objective and the cranial window.
Allow the mouse to wake up and acclimate to the treadmill.
Using bright-field illumination, locate the imaging region. Close the microscope enclosure to block external light.
Set the imaging parameters, initiate treadmill rotation to induce locomotion, and begin imaging.
Enforced locomotion activates PKA, which phosphorylates the reporter, bringing its donor and acceptor fluorophores closer.
The microscope directs an infrared laser, exciting the donor to transfer energy to the acceptor, reducing the donor’s fluorescence lifetime.
When locomotion stops, PKA and the reporter return to their inactive states, decreasing energy transfer and increasing the donor’s fluorescence lifetime.
Analyze the images to compare cortical PKA levels during rest and locomotion.
At least two weeks after cranial window installation, set the two-photon excitation laser wavelength to 960 nanometers and confirm a lack of response to toe pinch in the anesthetized experimental mouse. Place the anesthetized mouse on the motorized treadmill and mount the head plate of the mouse to the head plate holder of the treadmill setup. Clean the surface of the cranial window coverslip with 70% ethanol and place the motorized treadmill under the 2pFLIM objective.
Apply a drop of distilled water between the cranial window coverslip in the objective, and allow the mouse to wake up and become acclimated to the treadmill and microscope environment for at least 10 minutes. Navigate to the injection location under epi-illumination and document the fiducial features under brightfield to aid in imaging of the same region of interest during subsequent imaging sessions.
Switch off the epi-illumination light source. Enclose the enclosure of the 2pFLIM microscope rig. To activate the 2pFLIM microscope photomultiplier tubes, switch on the hardware command voltage control. To acquire a Z-stack 2pFLIM image, set the image size to 128 by 128 pixels. The scanning speed to 2 milliseconds per line, the field of view to 90 to 100 micrometers in the frame, averaging to three frames.
Adjust the imaging settings based on the preparation and hardware configuration. And inspect the acquired image in FLIM view. Use a decreased field of view, decreased scanning speed, increased laser power, and increased number of frames to be averaged to increase the integrated photon counts and to reduce the lifetime estimation error as needed.
Be sure to obtain enough photons per region of interest. A workable integrated photo count for a positive soma in view is about 1,000 to 10,000 photons.
When the image has been optimized, acquire baseline repeat Z-stack images at regular intervals for at least 15 minutes at 0 speed on the treadmill. Then set the treadmill rotation to approximately centimeters per second for 15 minutes while acquiring 2pFLIM images, followed by at least 20 minutes of image acquisition after switching off the treadmill rotation to assess the duration of the protein kinase A activity after the cessation of forced locomotion.
For 2pFLIM image analysis, open the images in film view and click on the single photon counting minimum and maximum range fields to enter the appropriate minimum and maximum single photon counting range values. Click the time 0 value field to enter the time 0 value, and click the lifetime luminance minimum threshold value field to enter the desired threshold value to between 5 and 30 photons.
Click the New group button and assign an experiment group name to generate a group that combines the data from each added FLIM image. Click Region of Interest in the region of interest controls module and draw a region of interest around a T-ACCR alpha-positive soma. Move the lower and upper Z limit in the Z-stack control sliders to reduce the Z-stack range and to minimize the signal contamination originating from background photons and other Z-dips, and click Plus to add the FLIM image to the group.
Click calc to calculate the mean lifetime in the lifetime estimation error for the region of interest, and open the next file in the 2pFLIM imaging series. Measure the same T-ACCR alpha-positive soma in each consecutive image over time, adjusting the region of interest around the soma as necessary. When all of the images have been analyzed, select the delta mean photon emission time delta mean photoemission T0 in the group controls module, and click the baseline number field to enter the index for the images of interest to define the images used to calculate the baseline lifetime.
Then, click plot to generate a graph containing the FLIM response to T-ACCR alpha positive activity during the experiment in the defined regions of interest to allow a comparison of the protein kinase A activity during the locomotion of the kinase across different regions of interest.
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