The flexible environmental chamber technique enables time-lapse as SRS imaging with transmitted signal detection on an operating microscope frame. SRS microscopy used a high numerical aperture objective and a high ended condenser, leaving only a few millimeters gap where a conventional chamber cannot fit. A flexible chamber solve this problem.
Researchers using SRS microscopy can easily adopt this technique. The flexible chamber can also be used on a dissecting or a sterile microscope to maintain the specimen under optimal physiological conditions. To begin, mark the locations of the feet of the SRS microscope frame and the motorized stage using a marker pen on the optical table.
After removing microscope frame and stage from the optical table, lay the silicone rubber sheet on the table. Cut the silicone rubber along the marks using a knife. Remove small rubber pieces, and place the square mica ceramic sheets into the same locations.
Then move the microscope frame and the stage back to the optical table and carefully align the feet onto the mica ceramic sheets along the marker lines. After realigning the SRS optical path, assemble the environmental enclosure on top of the thermal insulation foundation to cover the entire microscope frame, using five pieces of large polycarbonate sheets. Seal the edges and interfaces of the box using aluminum foil tape.
Connect the flexible duct hose to the inlet and the outlet ports of the enclosure box to allow circulated, warm airflow pumped and controlled by the heater module. Mount the machined hollow cylindrical aluminum piece 1 to the nose piece of the objective using three set screws. Next, mount the machined hollow cylindrical aluminum piece 2 to the sample holder using four screws.
Fit the sample holder with aluminum piece 2 onto the motorized stage and mount it using screws. Place the natural rubber film sleeve between the two machined aluminum pieces and mount it using rubber bands at each end. Connect the compressed CO2 cylinder to the gas mixer module using proper tubing and connectors.
Set the CO2 input pressure at 20 to 25 psi. Use the built-in CO2 sensor and a controller to ensure the air mixer module can regulate and mix 5%CO2 into the airflow. Use inline filters to clean up the airflow.
Using proper tubing and connectors, guide the mixed air to the pre-sterilized water bottle placed on the hotplate and then guide the humidified air to the flexible chamber. Set the hotplate at 37 degrees Celsius. Bubble the airflow in the warm water to increase the humidity of the airflow.
Disconnect the machine aluminum piece 2 from the sample holder by removing the screws. Wipe all parts of the flexible chamber with 70%ethanol, including the nose piece, the sample holder, and water dipping objective. Decontaminate the entire enclosure system using a UV lamp placed in the enclosure for 20 to 30 minutes.
Then load the cell culture dish. Remove the cover of the dish and immobilize it using the clamps. Lower the objective into the cell culture media for coarse focusing.
Lower the aluminum piece 2 to enclose the flexible chamber and mount it to the sample holder using screws. Then insert the sensor. Set the air supply with 5%CO2 and 19%O2 for normal cell culture with an airflow rate of 200 cubic centimeters per minute and temperature to 37 degrees Celsius.
Tune the laser wavelength to 805 nanometers to target the 2, 854 centimeter inverse Raman shift, which is attributed to the vibration of CH2 chemical bonds. Use low laser power to reduce photo damage to the cells. Adjust and focus the objective to achieve good SRS imaging of the cells using the focus button on the main controls panel of the software.
To perform rapid focusing, set the pixel number to be 512 by 512 pixels with a pixel dwell time of 4.8 microseconds on the configuration panel. After achieving good focusing, set the imaging resolution to be 2048 by 2048 pixels for a 175 micrometers squared field of view for the acquisition of high quality images. Check the save function on the main controls panel and check the SRS channel on the channels panel.
Set the interval time between two frames to be 180 seconds on the main controls channel. Set the acquisition number to 480 for time-lapse imaging over 24 hours. Start automated imaging acquisition using the loop function on the main controls panel.
The performance of the flexible chamber system for time-lapse SRS imaging was evaluated. The temperature inside the microscope environmental enclosure reached the expected 37 degrees Celsius and did not significantly affect room temperature. The temperature in the flexible chamber reached 37 degrees Celsius in 1.5 hours and was stably maintained for at least 24 hours.
The relative humidity in the flexible chamber reached 85%in one hour and could be maintained for at least 24 hours. The time-lapse SRS imaging of live SKOV3 cells showed the rapid and active movement of intracellular lipid droplets with a temporal resolution of three minutes. By the end of the 24 hour imaging session, the cells still showed normal morphology and density, indicating that the cells were healthy.
Next, SKOV3 cells treated with oleic acid were imaged and lipid droplet accumulation was tracked over 10 hours. The lipid droplet amounts were quantified using the lipid droplet to cell body area ratio and total SRS intensity of the lipid droplets. The results indicate that the amount of lipid droplets kept increasing over 10 hours.
Simultaneous forward SRS imaging of lipid droplets and backward two photon fluorescence imaging of lysosomes labeled with a fluorescence dye DND-189 was also demonstrated. A very low degree of colocalization of lipid droplets and lysosomes was observed. When attempting this protocol, keep in mind that focal drift is a common issue in live cell imaging.
Refocusing may be needed after about two hours of time-lapse imaging. After its development, time-lapse SRS imaging has been used for various studies to track label-free molecules or labeled molecules with Raman text in live cells.
