The scope of our research is to develop and apply OPTIR-FISH to identify individual cells and analyze their metabolism at the single cell level. We aim to understand how individual cells interact within a complex environment and understand their physiology and roles. Analyzing metabolism at the individual cell level, particularly within the native environments, could provide invaluable insights into revealing the heterogeneous complex activities in biological systems.
However, the concurrent task of differentiating cellular identity and elucidating metabolism at the single cell level remains challenging. Our advanced vibrational imaging platform, optical photothermal infrared offers the study of single cell metabolism. At the same time, it is naturally highly compatible with the well established fluorescence in situ hybridization for the identification of microbial species.
Therefore, straightforward integration offers metabolism and identification at the same imaging resolution simultaneously. To begin, take bacterial cells fixed in para formaldehyde, centrifuge the cells at 14, 000 G for 10 minutes, then remove the supernatant. Re-suspend the pellet in 100 microliters of 96%analytical grade ethanol and incubate for one minute at room temperature.
Repeat the centrifugation for five minutes. Then remove the ethanol and air dry the cell pellet. Now hybridize the cells in a solution containing fluorescently labeled oligonucleotide for three hours at 46 degrees Celsius.
Immediately after hybridization, centrifuge the samples in a rotor preheated at 46 degrees Celsius for 15 minutes at the maximum allowed temperature. Then wash the samples in a buffer with suitable stringency for 15 minutes at 48 degrees Celsius. After centrifuging the sample once again, wash the cells with 500 microliters of ice cold PBS.
Re-suspend them in 20 microliters of PBS and store them at four degrees Celsius until further use. To begin, take standardized calcium fluoride slides. Immerse them in 70%ethanol for 15 minutes, and rinse them two times with deionized water.
Transfer the clean slides into 0.1%poly-L-lysine solution and incubate them overnight at four degrees Celsius. Then rinse the slides with deionized water and let them dry. Next, thaw the prepared cell samples at room temperature while keeping them protected from light.
To avoid excessive salt crystals from PBS during sample drying, wash the thawed cells twice with deionized water. Centrifuge at 14, 000 G for 10 minutes and remove the supernatant. Reintroduce the original volume of deionized water to the cells and gently vortex them.
After washing, centrifuge the cells at 14, 000 G for 10 minutes to concentrate the bacterial cell samples and remove the supernatant. Gently vortex the remaining solution for approximately 30 seconds and apply one to two microliters of this solution onto the prepared calcium fluoride slide.
