The overall goal of the following experiment is to use the fluorescent proteins M orange two andron for dual probe optical highlighting to selectively label four individual cell populations or cellular compartments. This is achieved by first illuminating cells with low intensity 488 nanometer excitation light to switch off all the green drawn per fluorescence. As a second step, cells are exposed to high intensity 488 nanometer excitation light, which photo converts the m orange fluorescence to red.
Next cells are exposed to 800 nanometer two photon excitation in order to switch on dral fluorescence. Again, results are obtained that show it's possible to label cells in four distinct combinations based on dual probe optical highlighting with M Orange two and drawn ppa. The main advantage of this procedure over existing experiments like subcellular fractionation is that it's done on live cells.
This procedure can help answer key questions in beta cell biology, such as why only a small fraction of insulin vesicles are secreted. Although this procedure can provide insight into beta cell function, it can also be applied to other systems. For example, tracking cancer cells in vivo, A fluorescent protein droplet sample consists of an octal water emulsion with a fluorescent protein residing in the water phase.
This emulsion is sandwiched between a microscope slide and a 22 millimeter square cover glass for microscopy applications. At the beginning of this procedure, the microscope slides and cover glasses must be cleaned and coated with a hydrophobic agent. Clean the glassware by washing it for five minutes with acetone.
After washing, leave the glassware to dry by air. Next, prepare a 2%methyl trimeth oxy seline solution in acetone and coat the glassware for two minutes in the solution. After coating, remove the glassware from the solution and allow it to dry.
Then rinse the glassware with 70%ethanol from a spray bottle. After rinsing, let the glassware air dry again and remove any remaining liquid. Using pressurized air, the coated glassware can be stored for at least one month.
Once the glassware has been prepared, obtain fluorescent proteins that have been previously purified as his tagged protein from e coli. Measure the absorbent spectrum of the purified protein. Prepare a stock dilution with an optical density of approximately 0.1 in tris buffered saline solution with pH eight containing 0.1%bovine serum albumin.
Additionally, prepare 10 milliliters of a one to one mixture of one octal and tris buffered saline solution in a 15 milliliter conical tube and mix vigorously. After mixing, leave the mixture until the phase separation is complete. The top phase is the octal to make the emulsion pipette 45 microliters of one octal and five microliters of fluorescent protein into an epi orph tube.
Tap the tube a few times to start formation of the emulsion, then sonicate the tube for 30 seconds in a sonication bath. Meanwhile, get a coated microscope slide and cover glass ready following sonication. The emulsion should be completely cloudy.
Immediately pipette four microliters of the emulsion from the middle of the tube onto a coated microscope Slide and cover it with a coated cover glass. The emulsion should spread evenly between the microscope slide and the cover glass. Within minutes, the sample should be stable.
The following procedure is a general strategy for setting up a fluorescent protein photo conversion experiment on a confocal laser scanning microscope. This procedure can be applied for purified proteins as well as for live cells. In this experiment, a droplet sample containing purified m orange two protein is used as a general starting point for the photo conversion experiment.
Set up the confocal laser scanning microscope as follows. Use a 40 x 1.3 numerical aperture oil immersion objective and an image size of 512 by 512 pixels. Set pixel dwell time to six microseconds the scan, zoom to four and increase the pinhole size to match a Z resolution of three microns.
Configure two detection channels for the initial and photo converted fluorescence as well as a photo conversion channel. Observe the orange species using 561 nanometer excitation light and collect the fluorescence between 570 and 630 nanometers. Detect the photo converted red species using 633 nanometer excitation light and collect the fluorescence between 640 and 700 nanometers.
For the photo conversion channel, select the 488 nanometer laser line and collect the fluorescence between 490 and 540 nanometers.Imaging. The photo conversion channel is not strictly necessary but can be helpful. To begin the experiment, use the detector channel to image the initial fluorescence with continuous scanning to adjust the laser power and detector gain for optimal image quality.
Then activate the photo conversion channel and select the low laser power. Start imaging a time-lapse series and gradually increase the photo conversion laser until significant bleaching of the initial fluorescence is observed. Continue scanning until the initial fluorescence is approximately 75%bleached.
Next, deactivate the photo conversion channel and activate the detection channel for the photo converted fluorescence. Start imaging with a high detector gain in low laser power. Then gradually increase the laser power until the photo converted fluorescence is detected.
Once detected, adjust the laser power and detect or gain for optimal image quality. Finally, optimize the laser power used for photo conversion as well as its duration. Increasing the laser power will accelerate the rate of photo conversion.
However, too much laser power will photobleach the protein. Once the optimal photo conversion, laser power and duration have been determined, these parameters can be used to configure a standard photobleaching module or fluorescence recovery after photobleaching module. Then the photo conversion channel will no longer be required due to the Redshift spectral properties of M orange two.
It can be used in combination with the green photo switchable fluorescent protein drawn PU for dual probe optical highlighting. To begin dual probe optical highlighting Grow hela cells in glass. Bottom mat tech dishes transfect the cells with plasmids encoding the fluorescent proteins 24 hours prior to imaging using a standard LIPECTOMY 2000 transfection.
Place the live cells on a confocal laser scanning microscope and set up the microscope for M orange two photo conversion as described earlier. Then configure the microscope for DPA photo switching. DPA is excited with 488 nanometer light and its fluorescence can be imaged using the M orange two photo conversion channel.
Be sure to minimize the laser power used for imaging MPA as too much laser power will cause inactivation of mpa. Add a channel for MPA photo activation using 800 nanometer two. Photon excitation subsequently determine the laser power required for imaging photo activation and photo inactivation OFA fluorescence.
One of the most difficult aspects of this procedure is to inactivate RPA without photo converting M orange. Because both events take place at the same wavelength. In order to prevent this, it is very important to carefully optimize the photo conversion.
Laser power and duration Take caution as photo conversion of M orange two. An inactivation of PU both occur upon excitation with 488 nanometer light to inactivate PU without significant m orange two photo conversion. Use a lower laser power once the parameters for M orange two photo conversion and DPA photo switching are set.
Dual probe optical highlighting can be achieved. First inactivate DPA fluorescence in the whole field of view with low power 488 nanometer excitation. Then select a region of interest and convert m orange two with high power 488 nanometer excitation.
Finally, select a region of interest and activate DPA fluorescence using 800 nanometer two photon excitation. Fluorescent protein droplet samples are ideally suited for setting up confocal microscope photo conversion applications. When the droplet sample is prepared correctly, the droplets appear very homogeneous.
However, when the sample is prepared without coating the microscope slide in cover glass, the droplets tend to be squashed against one of the glass surfaces in samples prepared. In the absence of 0.1%BSA, the fluorescent protein tends to accumulate at the one octal water interface. Creating a halo of fluorescence photo conversion is critically dependent on the laser power and the duration of exposure.
Too much laser power or too long exposure will cause photobleaching and results in a reduction of the photo converted red fluorescence. The distinct spectral properties of M orange two and PHO permit dual probe optical highlighting prior to photo conversion cells expressing M orange two, histone H two B and DPA mito show orange fluorescence in the nucleus and green fluorescence in the mitochondria. When al fluorescence is switched off with low power 488 nanometer excitation, there is minimal photo conversion of M orange.
Two M orange two can then be photo converted to red with high power 488 nanometer excitation. Once M orange two has been photo converted, drawn puff fluorescence can be switched back on using 800 nanometer two photon excitation. With dual probe optical highlighting cells can be labeled in four distinct ways.
Using this technique, it is possible to specifically label four different cell populations. As you've seen, one of the most difficult aspects of this procedure is to inactivate MPA without photo converting m orange. Once mastered, further experiments can be performed using this system, for example, tracking multiple populations in pancreatic beta cells.
So that's it. I hope you found this presentation useful, and good luck with your experiments.
