This confocal microscopic protocol will permit the interpretation of the ecological behavior and adaptations of microalgae from extreme environments with slow growing in the laboratory using autofluorescence of their pigments. This technique is not invasive and minimizes artifacts as chemical compounds are not used. Knowing the performance of pigments of autotrophs and corresponding growth will permit the designing of the methods of control which is the greatest interest for touristic caves and architectural monuments.
Begin preparing the inoculum of Chroothece mobilis by transferring it from the agar culture to the seawater medium or SWES liquid medium. Maintain all the cultures for two weeks with a 16 to 8 light dark photo period at 20 degrees Celsius under a low white light intensity and without shaking until the desired cell density is obtained. Use one milliliter of the exponential phase culture having five times 10 to the third cells per milliliter cell density to inoculate in a 24-well plate for different experiments.
To reproduce the monochromatic light effect, use the green filter allowing green light to pass through from 470 to 570 nanometers with a peak at the 506 nanometer wavelength and to red light adjusted using a filter between 590 and 720 nanometers and peaks at 678 nanometers. Expose the cell cultures to this light for two weeks. Turn on all the components of the inverted confocal laser scanning microscope including the laser.
Mount the cells in the SWES growth medium from each experimental well of the 24-well plate to a 35 millimeter glass bottom dish for imaging. Choose the 63X or 1.30 numerical aperture or NA glycerol immersion objective and place glycerol over the lens. Next, place the well plate on the microscope stage ensuring the specimen does not move during image acquisition.
Center the specimen in the light path and focus on the center of the cell by selecting the plane with the highest fluorescence intensity. Once done, open the image acquisition software and choose XY lambda from the dropdown list in the acquisition mode. Select the excitation line of the laser 561 nanometers, eight bits of dynamic range and 1024 by 1024 pixels.
Collect the fluorescence emission spectra in the 10 nanometer bandwidth and the lambda step size of four nanometers within the 570 to 760 nanometer range. Set the pinhole at one air unit and run the lambda scan acquisition. After repeating this process in different fields of view and under the different conditions of red and green lights, save all the data.
Once the lambda scan is acquired, click on the quantify window at the top of the software to evaluate the collected fluorescence emission spectra. Go to the open project window and select one XY lambda file. Select stacked profile analysis in the imaging software and define a region of interest of four micrometer square in the center of a cell to analyze the mean fluorescence intensity.
Export the data and CSV before repeating the process with different cells under different conditions. Next, open the CSV files to select the different fluorescence emission peaks of all the measured regions of interest by selecting the maximum fluorescence data of phycoerythrin phycocyanobilin, C.phycocyanin, allophycocyanin, and chlorophyll A respectively in the CSV file. Once done, create a new table with all the maximum fluorescence values obtained from each phycobiliprotein and chlorophyll peak and plot the data on a graph.
Significant differences in mean fluorescence intensity were observed. The mean fluorescence intensity of phycoerythrin phycocyanobilin significantly decreased when the cells were exposed to monochromatic green light. In contrast, no difference was observed in red light compared to the control.
The green light had the opposite effect on allophycocyanin and chlorophyll A in which the mean fluorescence intensity significantly increased due to the adaptation of antenna complexes due to increased quantity or improved connectivity of the antenna complexes among others. The red light produced a non-significant increase in allophycocyanin and chlorophyll fluorescence. To study the effect of different growth conditions on cells in culture, it's crucial to perform the measurements with exactly the same microscope settings.
It's possible to analyze other environmental parameters relevant to the culture of autofluorescence organisms, as well as the fluorescence signal within the visible spectrum. This technique is a very powerful approach to a study life organisms with several autofluorescence pigments.
