This protocol provides a new approach to classify neural stem cell activation state with a cohort of technical advantages. Using this protocol, we can achieve live cell label-free single cell resolution analysis of neural stem cell activation state. This protocol can be used to shed light into mechanisms regulating adult neurogenesis.
Autofluorescence is much dimmer than many fluorophores researchers are used to working with. Be confident in using higher laser powers to collect your data. Demonstrating the procedure will be Chris Morrow, a former graduate student from my laboratory.
Begin by configuring the confocal microscope for autofluorescence imaging. Click 405 Laser and type in the emission window under the 405 laser menu. Proceed to click TD to collect a transmitted light image and set the HV value to visualize the sample.
Adjust the laser power to 3.5%and set the gain to 75. Set the zoom to two. For the confocal scanning parameters, set the pixel dwell to 0.5 and the resolution to 2048 by 2048 pixels.
Using 60X oil immersion objective lens under brightfield, bring the cells into focus. Click Eye Port to switch to confocal scanning mode. Ensure that the light path is directed towards the scan head and confirm that no filter cube is obstructing the light path.
Click Scan, then focus on the image and click Capture to acquire images of autofluorescence in quiescent neural stem cells and active neural stem cells. Ensure to collect single plane images with the cytoplasm of cells in focus for a representative cross-section of each cell. Start imaging with low laser powers and gradually increase the power until the autofluorescence signal is detectable without causing saturation.
Analyze quiescent neural stem cells and active neural stem cells with a flow cytometer using a 130 micrometer nozzle. Apply optical conditions as per the instrument's capabilities and detect punctate autofluorescence. Collect at least 10, 000 singlet qNSCs and aNSCs in a data file.
Utilize this data to design gates for collecting an enriched population of either qNSCs or aNSCs. Then sort the singlet cells into PLO laminin-coated wells filled with aNSC medium. After FACS, allow neural stem cells to incubate for three hours.
To fix the cells, add 4%paraformaldehyde, ensuring it is sufficient to cover the bottom of the dish and incubate. After 15 minutes, permeabilize the cells with 0.25%Triton in PBS for 15 minutes at room temperature. Following this, treat the cells with an EdU staining solution at room temperature for 30 minutes.
Next, wash the samples three times for 10 minutes each with PBS. Begin by using the oculars to locate a suitable field of view and adjust the light path to the sample on the microscope to enable imaging in the dark with the multi-photon. Set up for the collection of channel one image.
Navigate to the power gain tab and adjust the gain on the PMT to 800. Click on the 2P Laser tab and select 750 nanometers for the laser wavelength and ensure the correct filter cube is used. To collect channel one image, click the Power Gain tab and set the Pockels to 30.
Then proceed to the scanning section of the screen and click Live Scan to start preview mode. Incrementally increase the Pockels to achieve a suitable field of view at low laser power. Once a suitable field of view has been identified, adjust the laser power to 3.6 milliwatts.
Then proceed to the scanning section and click single scan to capture a channel one image over 60 seconds. To collect a channel two image, click on the 2P Laser tab and select 890 nanometers for the laser. Replace the filter cube with the channel two emission cube.
Initiate the preview mode. Increase the Pockels until the power meter shows approximately seven milliwatts and the CFD counts are between 10, 000 and 100, 000. Click on single scan and capture a channel two image over a duration of 60 seconds.
Confocal autofluorescence imaging was employed to differentiate between quiescent neural stem cells and activated neural stem cells. It was found that qNSCs display a higher number of PAF than aNSCs, demonstrating the use of autofluorescence as a marker for cell state identification. Using FACS, NSC cell states were enriched based on autofluorescence.
Cells sorted from the high autofluorescence gate showed a lower proliferation rate compared to the mixed sample, while those from the low autofluorescence gate were more proliferative, confirming the enrichment of NSC activation states. Further, FLIM was utilized to assess NSC autofluorescence and discern the activation state of NSCs. Notably, qNSCs showed a higher mean lifetime of fluorescence in channel one, but a lower proportion of the fluorescence component alpha one compared to aNSCs.
A logistic regression model incorporating data from both channels yielded a near perfect predictive capacity for NSC activation states as indicated by a receiver operator characteristic curve underscoring the potential of FLIM in classifying NSC states. Following this procedure, it would be good validate results using other approaches to measure neural stem cell activation state.
