Spinning disk TIRF microscopy can be a useful tool for studying the role of protein during cytoskeletal network formation. This technique allowed three-dimensional imaging of fast cellular approaches that occur in the cell and at the same the precise localization of fluorescence molecule that are placed at the plasma membrane. This method can provide insight to research areas like microbiology, cell biology, and all those branches where it's important to study the interaction between the plasma membrane and the cellular environment.
This method can be extended to those live imaging experiments where it's important to localize the structures in the plasma membrane and where you also want to maintain a high resolution of the remaining cellular volume. The crucial things one has to consider are the choice of the proper cell line to set up the TIRF illumination and the other imaging parameters and to find the focus of the transfected cells. The visual demonstration of this protocol would certainly help to avoid problems with the handling of the cells during image acquisition.
Two days prior to the experiment, seed 3, 000 HELA or NIH 3T3 cells in two milliliters of full growth medium into each well of a six-well cell culture plate. The next day, prepare the transfection reagents. First, dilute one microgram of RFP Livact and one microgram of YFP Vinculin in a total of 200 microliters of reduced serum medium.
Vortex the transfection reagent briefly. Then add four microliters of the mixture to 200 microliters of DNA. Vortex the reagents again and then incubate the transfection mix for 15 to 20 minutes at room temperature.
After incubation, add the entire transfection mix drop-wise directly to the cells. Mix the wells by shaking the plate and then place back into the incubator. On the day of the experiment, prepare the sample for live imaging by first coating a 35 millimeter glass bottom dish with a 10 microgram per milliliter solution of Fibronectin in PBS.
Leave the solution on the glass surface for 30 minutes at room temperature then remove it and let the dish air dry. Next, dilute a 0.1 micron diameter multi-fluorescent bead solution to a density of 1.8 billion particles per milliliter in distilled water. Add the solution to the Fibronectin-coated glass surface for 30 to 60 seconds.
Then remove the solution and let the dish air dry. Precoating of the dishes with fluorescent beads is only necessary for two reasons, first if you want to find the TIRF plane before seeding the cells or to acquire a two-colored bead image for image registration. Now prepare an anti-oxidizing growth medium by adding 0.1 molar ascorbic acid solution at a final concentration of 0.1 millimolar in growth medium.
Prewarm the media by placing it in a 37 degree Celsius water bath. Next, wash the cells with two milliliters of PBS and add 250 microliters of Trypsin EDTA to each well of the plate. Incubate the cells at 37 degrees Celsius and wait two to three minutes until the cells are fully detached.
Resuspend the cells carefully in one milliliter of prewarmed anti-oxidizing growth medium and add them to an additional four milliliters of the medium in a 15 milliliter cell culture tube. Place the cell suspension with a slightly opened lid in an incubator set at 37 degrees Celsius buffered with 5%carbon dioxide. To begin, start the environmental control of the microscope to achieve stable cell culture conditions.
Then place the glass bottom dish containing one milliliter of prewarmed anti-oxidizing growth medium into the sample holder of the preheated microscope. Next, in the imaging software, set up the acquisition settings at the microscope. Set the acquisition time interval to 30 seconds and the duration to between 60 and 90 minutes.
Also, activate the autofocusing function of the hardware-based autofocus for every time point by setting the value to one. Now adjust the camera exposure to 200 milliseconds, the gain level to 500, and the laser power to 20%for every channel. High gain levels, low exposure time, and low laser power are recommended to reduce phototoxicity.
Next, set the Z-stack for the spinning disk channels to 10 microns with 0.4 micron spacing, then set the bottom offset to zero so that the lowest plane will be the focus position of the hardware autofocus and deactivate the Z-stacks for the TIRF channels. Finally, activate the multi-point function stage positions. This will allow for up to three positions to be recorded over a 30-second interval.
Now find the fluorescent beads with epifluorescent illumination, activate one TIRF channel and set the illumination angle to a value that denotes TIRF illumination. Next, activate the autofocus by pushing the button at the microscope panel and adjust the focus with the offset wheel. Once in focus, acquire a two-colored data set using TIRF 488 and TIRF 561 for subsequent bead-based image registration.
Remove the cells from the incubator and mix the cell suspension by inverting the closed tube two to three times. Then apply one milliliter of the cells to the imaging dish. Quickly find double transfected cells with low level epifluorescent illumination.
Once found, center the cells in the live camera preview using bright-field illumination and mark the position. Then find an additional one to two points of interest, save them to the positions list and begin data acquisition by clicking on the sequence button. In the beginning, the cells can easily detach due to the stage movement so it's better to set four to five positions and later on discard one or two.
In order to generate a registration-free hyperstack in Fiji, first download and save the provided file SD-TIRF_helper_jove. ijm in the Fiji subfolder macros. Run the macro by clicking on the menu command starting with plugins then macros and finally run.
If the color channels need registration correction, select the option and create a new bead-based registration reference known as a landmark file. Next, import the data with the bioformats importer and choose hyperstack as a viewing option. Load the image data set, select the spinning disk series in the first step and the TIRF series in the second step.
At this point, Fiji will display the data sorted by channel and stage position. Apply the registration correction to the respective channels by loading the predetermined landmark file. Then select the desired color lookup table for every spinning disk and TIRF channel and merge them into a single multidimensional hyperstack.
During the processing of the TIRF images, a number of zed planes is added to the bottom plane and this number matches with the number of the planes in the spinning disk data sets. That's very important for the visual representation of the final hyperstack. Using the setup described in this video, YFP and RFP-expressing cells were selected and their adhesion process observed during a 60-minute timelapse.
As expected, cells were round shaped and only weakly adherent at the beginning with membrane protrusions sensing the environment and making contact with the substrate. Cell matrix contact strengthened quickly upon formation of so called nascent adhesions. Over the course of time, adhesion complexes enlarged and matured to focal adhesions.
These elongated structures were predominantly apparent at the periphery of the cell. This resulted from forces that were exerted by actomyosin fibers which induced the strengthening of cell matrix adhesions as well as the bundling of actin fibers. The cell also flattened as a result of the actin network formation.
The acquisition speed is dependent on the following parameters, the time interval, the exposure time, the number of zed planes and the number of positions. So it is very important to optimize these parameters for high acquisition rates. The implementation of the newest spinning disk technology will turn spinning disk TIRF microscopy into new super resolution microscopy technique that will allow imaging at high temporal rates.
Spinning disk microscopy is a very powerful technique for studying processes that occur between the cell interior and the cell membrane. We have been shown that we can follow the dynamic of vesicle by acquiring very large image stacks within few seconds. Collimated laser light is hazardous for the eyes.
Never look directly into the beam and use the safety precautions of the instrument to avoid direct light exposure.
