smFISH is known for measuring the absolute number as sub cellular location of mRNAs. Our two color sMFISH protocol has the added capability of measuring the kinetics of transcription and mRNA degradation. Using this method, many samples can be processed simultaneously without extra time or effort.
For example, four time course experiments yielding 48 samples can be processed for imaging in eight hours. Our protocol is broadly applicable for many genes and bacterial species. To prepare cover slips and glass slides for the experiment.
Use forceps to place cover slips and slides in a Coplin jar containing 100%ethanol and place the jar in a water bath ultrasonic cater for 15 to 20 minutes. At the end of the sonication wash the slides and cover slips three to four times with ultra pure water before refilling the jar with 70%ethanol and repeating the sonication. When all the sonication steps are done, dry the cover slips and slides with nitrogen gas.
Place the cover slips into an empty 1000 microliter pipette tip box and place the slides into a clean slide box. Then, following the holes of the pipette tip box use a hydrophobic marker to draw circles onto the cover slips and apply a 20 microliter drop of 0.1%Poly l lysine to each of these wells. To set up a time course experiment, add 750 microliters from a 20 milliliter exponentially growing E.coli culture to a 1.5 milliliter culture tube marked time zero.
And immediately invert the tube. Induce lac Z expression with 0.2 to one millimolar IPTG and start a timer. After checking the timer, collect the cell culture at each consecutive experimental time point as just demonstrated.
At the appropriate time point during the experiment, add five millimolar ONPF to the flask to repress lac Z expression and continue to sample the cultures to track the mRNA degradation. At the end of the time course sample acquisition, incubate the samples at room temperature for 15 minutes followed by incubation on ice for 30 minutes. At the end of the incubation centrifuge the samples to remove the fixative and use a pipette to remove the supernatant.
Re-suspend the bacteria in one milliliter of DEPC PBS and wash the cells two more times in one milliliter of fresh DEPC PBS per wash. After the last wash, re-suspend the cells in 30 microliters of fresh DEPC PBS. For permeabilization of the cells.
First add the sample from each time point to individual hydrophobic wells on one of the ethanol sterilized glass cover slips. And allow the cells to adhere to the cover slip with a 10 to 30 minute incubation at room temperature. At the end of the incubation, add 15 microliters of 70%ethanol to each well.
After four minutes, aspirate the ethanol from each well so that the wells are completely dry. After washing add 30 microliters of pre hybridization solution to each well of permeabilized cells, and incubate the cells for 30 minutes in a 37 degrees Celsius oven. Replace the pre hybridization solution with approximately 30 microliters of probe hybridization solution per well.
And cover the chamber with aluminum foil for a two hour incubation in the 37 degrees Celsius oven. At the end of the incubation use a multi-channel pipette to rinse the wells three to five times with 30 microliters of wash solution per well per wash. After the last wash, incubate the cells for 15 to 30 minutes at 37 degrees Celsius before washing the wells five times with 30 microliters of fresh DEPC PBS per wash.
After the last wash, aspirate all of the liquid from the cover slip. And add four microliters of fresh DEPC PBS to each well. Use forceps to carefully place the cover slip onto an ethanol sterilized glass slide sample side down, and use silicone dental gum to seal the edges of the cover slip.
To locate an area of interest select the live mode of phase contrast imaging and maneuver the stage joystick to change the field of view within a well. Located an area in which the cell density is optimal and adjust the Z focus such that the phase contrast cell images are in focus. Then acquire a C five image with a four second exposure, a C three image with a two-second exposure and a phase contrast image with a 0.2 second exposure for approximately 10 different areas per well.
Alternatively, an NDI acquisition in which C five, C three and phase contrast images are acquired in series can be run. When all of the wells have been imaged, open an appropriate cell segmentation tool, and unclick stack. Load the phase contrast images of interest and select independent frames and click compute phase profile as zero.
To begin the segmentation process during which the cells will be identified and their contours will be calculated. Load the parameters provided in the GitHub folder and click all frames. At the end of the processing select contour to visualize the mesh.
Next, under the spot detection tab, uncheck stack, check meshes and click rub to begin the spot identification and quantification based on the two D Gaussian fitting. Select the folder containing the fluorescent images and the mesh file that was just calculated from the cell detection procedure and indicate the file name to which the spot detection results will be saved. Then use the image analysis workflow provided on GitHub to analyze the fluorescence images.
In this full field of view approximately 500 E.coli cells at a good density for cell segmentation can be observed. The morphology of the cells in the phase contrast images should remain comparable to that of live cells for segmentation purposes. If the cells are over permeabilized, their morphology will become unsuitable for segmentation.
The distribution of lac Z mRNA spot intensities before induction does not fit well with a normal or Poisson distribution due to the presence of spots with high intensities. When the expression of lac Z is induced the signal of five prime lac Z mRNA increases before the three prime lac Z mRNA signal increases. If the expression of lac Z is repressed both the five and three prime lac Z mRNA signals decrease.
To obtain the rate of transcription elongation, the rise of the five and three prime signals are fit with lines. While the rate of mRNA degradation is obtained by fitting the decay region with an exponential function. Because single molecule fish is a single cell technique, cell to cell variabilities in transcription can also be analyzed.
In addition, analysis of the colocalization of five prime and three prime lac Z mRNA allows the density of the RNA polymerases on the lac Z gene to be quantified. As samples from different time points are handled simultaneously during this procedure. It is important to keep the sample separated while they're in the tubes and on the cover slip.
Using this single molecule single cell microscopy technique. Researchers can now explore how transcription and mRNA degradation kinetics are related to the absolute numbers or subcellular locations of mRNAs.
