We showcase a new imaging technique with label-free capability, high chemical specificity, and subcellular resolution. Our methodology can answer a questions about metabolic dysfunction in aging and diseases such as cancer Raman imaging, coupled with two photon fluorescence and second harmonic generation, offers a non-destructive and label-free imaging technology that requires minimal sample preparation and achieves high subcellular resolution without damaging the cell sample. Several imaging modalities are operated as a part of our platform.
Individuals should follow our protocol to the best of their abilities, noting that our system is home-built. Along the way they should develop their own practice to ensure success. In two separate tubes begin by measuring and mixing 10 milligrams of DMEM powder with 4.7 milliliters of double distilled water in a 15 mL conical tube.
Thoroughly vortex and invert the tube to ensure the solution is well mixed. For both tubes, add 4.7 mL of deuterium oxide, 0.5 mL of FBS, and 0.1 mL of penicillin and streptomycin before vortexing and inverting the tube. Next, add excess phenylalanine to one tube and excess tryptophan powder to the second tube and mix again.
Add methionine and threonine at 30 and 95 mg per mL, respectively, to both tubes, followed by vortexing and inverting the tube. Afterward, filter the prepared media with a 25 millimeter syringe filter with 0.22 micrometer filters. Seal all the media contained conical tubes with Parafilm.
Store prepared media at four degrees Celsius. Maintain healer cells in a culture flask using standard DMEM supplemented with 10%FBS, 1%penicillin and streptomycin. Subculture the healer cells at a split ratio of one to 10 until reaching 80%or higher cell viability.
Before seeding the cells, submerged the sterilized Poly-D-Lysine, laminin-coated, round 12 mm diameter cover slips in 70%Ethanol. Dry the cover slips using lint free wipes, and placed the clean cover slips in the appropriate wells of the plate. Wash the cells once with PBS without magnesium and calcium ions.
Add 0.25%trypsin to dissociate the adherence cells from the flask and incubate at 37 degrees Celsius and 5%carbon dioxide for three minutes. Then add DMEM supplemented with 10%FBS, 1%penicillin, and streptomycin to the cells. Gently pipette up and down.
Collect the cells by centrifugation at 300 G for three minutes at room temperature. Next, re-suspend the cells in DMEM supplemented with 0.5%FBS and 1%penicillin and streptomycin. Count the trypan blue stain cells using a hemocytometer under a light microscope and seed at a density of two times 10 to the fifth cells per well of a 24 well plate.
Distribute the cells evenly by shaking the plate gently. Incubate at 37 degrees Celsius and 5%carbon dioxide. After eight hours, replace the old culture media with 50%deuterium oxide and excess phenylalanine or 50%deuterium oxide and excess tryptophan media.
Return the cells to the incubator at 37 degrees Celsius and 5%carbon dioxide for 36 hours. Now prepare microscope slides with imaging spaces nine mm in diameter. Place 15 microliters of PBS, supplemented with calcium and magnesium ions and rinse the cells with the same solution.
Add 0.5 mL of 4%methanol-free paraformaldehyde solution and leave the plate under the biosafety hood for 15 minutes. Aspirate the paraformaldehyde solution and rinse it with PBS, supplemented with calcium and magnesium iron twice. Add one mL of PBS, supplemented with calcium and magnesium ions to each well.
Utilize sterile tweezers to remove the cover slips from each well carefully. Invert them and place the cell containing side onto the imaging spaces in contact with the PBS. Seal the outer layer of the cover slip with the imaging spacer using transparent nail polish.
Load the biological sample onto the sample holder directly underneath the objective lens. Then click the camera icon on the software to turn on the video camera and adjust the focus plane to identify a region of interest with the 50X objective lens. Switch to the 100X objective lens.
Click the stop icon to turn off the video camera. Then select a grating of 1, 800 lines per mm and set the the acquisition range from 400 to 3, 150 centimeters inverse. Lastly, set the acquisition time to 90 seconds the accumulation to three and the binning to four for the least noise and most accurate spectrum.
Click on start to warm up the laser. Start by pressing the main switch of the control box IX3CBH to On, followed by the main switch of the touch panel controller. Then turn on the main switch of the AC adapter of the power supply for LDOBIS6 laser remote connected to the main laser combiner FV31S comb.
Also switch on the AC adapter of the LDOBIS6 laser remote power supply connected to the sub laser combiner FV31S comb. After that, press the main switches of the SI photodiode detector and lock-in amplifier on. Set the laser IR at 1031 nanometers Set the pico emerald system to at tunable wavelength between 720 to 990 nm.
Mount the sample on the oil and place a large water droplet on the microscope slide where the sample is fixed. Select scan size as 512 by 512 pixels. Acquire and save the images as an Olympus.
OIR graphic file. After setting the signal to 862.0, acquire a background image and save it. Adjust the dwell time to eight microseconds per pixel and the pixel size to 512 by 512 pixels.
Set the laser shutter power parameter to 150 milliwatts For 3D image reconstruction, tune the stimulated Raman loss to 2, 850 cm inverse. Once the desired single cells have been identified, register the laser to scan from the top focus plane to detect the top and bottom layers of those cells. Deuterium oxide, probed, stimulated Raman scattering was used to visualize the spatial distribution of CD signals on single cells.
The control cells displayed moderate CD, lipid and protein bands. However, the 15 x v and 15 x trip displayed stronger CD lipid and protein bans. Quantitative analysis indicated that excess aromatic amino acids may upregulate lipid synthesis by 10 to 17%but downregulate protein synthesis by 10%Ratio metric analysis of aromatic amino acids treated cells showed a 50%increase in flavin divided by flavin plus NADH and a 10%increase in unsaturated lipid divided by saturated lipid.
The stimulated Raman 3D lipid droplet volume projection in healer cells under control and excess aromatic amino acids conditions are shown. Quantitative analyses of 3D lipid droplets showed an increase in counts, but a reduction in size in aromatic amino acids treated cells compared to the control. Imaging parameters for Raman and two photon fluorescent imaging should be optimized from one sample to the next to ensure best imaging quality.
Individuals can couple our system with second harmonic generation to study the collagen structure, which elucidates early metabolic changes in aging and diseases, and understanding the progression of neurodegeneration or cancer. Our technology is the first of this kind to visualize lipid and protein synthesis in biological systems with high subcellular resolution. The technology can also be translated into clinic for non-invasive disease detection.
