This method will help illustrate molecular mechanisms of a multifaceted biological phenomena. Such as mechanobiology, optical electrophysiology, and the super-resolution DNA/RNA protein imaging using automatic live-cell imaging techniques. This protocol enables automatic, multifunctional, high-throughput, self-drift correcting, and long-term image acquisition.
It is compatible with most fluorescent microscopic platforms, such as NYCOM. Anyone who tries this technique for the first time should understand the function of each step, instead of only following the protocol strictly, to ensure a successful experiment. Begin by placing the environment chamber onto the motorized stage of the inverted microscope.
Set the CO2 flow rate to 160 milliliters per minute and adjust the temperature of the chamber. Then add 40 milliliters of purified water into the bath of the chamber. Take out the glass-bottom Petri dish with cells from the incubator and place it in the environment chamber.
Turn on the confocal controller and the inverted microscope. Switch the light path to the right. Observe the cells attaching using Micro Manager.
If sufficient cells have been attached to the gel, transfer the Petri dish back to the incubator. If not, continue the cell incubation for another 30 minutes for B2B, and 60 minutes for lung cancer or PC-9 cells. Cut 2 small pieces of adhesive tape and stick them on the chamber around the circular hole.
Then apply a little adhesive glue onto the area of the tape that the Petri dish will cover. Take out the Petri dish from the incubator. Slowly place the Petri dish in the chamber and let the bottom of the dish make contact with the glue.
Press the lid of the Petri dish for 1 minute to allow the glue to make full contact with the Petri dish and solidify. Gently push the Petri dish horizontally to ensure that the glue has solidified and the Petri dish does not move. Then close the lid of the chamber.
Open IntelliJ and set a parameter T1 in line 93 of the file elements_script.java. Ensure that this value is larger than the running time of the macro and elements used for confocal imaging of one field of view. Click on the Run button to start the AMFIP IntelliJ project.
Click live and multi-deacquisition button on the main interface of Micro Manager, then switch the light path of the inverted microscope to the right for bright field imaging. Switch to the 10 times objective and open the LED light with the intensity set at 5%Click on the light path microscope objective and LED lamp button in the elements TI2 panel. Adjust the XY joystick and the knob of the Z plane to find the correct position and the in focus plane of the gel on the Petri dish.
Use the 10 times objective to find the appropriate fields of view of multiple single cells attached to the gel. Check the multiple positions XY box on the multidimensional acquisition window. Click on the edit position list button and observe the stage position list window that pops up.
Change the objective to 40 times, increase the intensity of the LED light to 15%Readjust the XY motorized stage to locate the fields of view and record the coordinates by clicking on the mark button on the stage position list window. Record 6 to 7 desired fields of view. Click on the save as button on the stage position list window to record the coordinates and input T1 as the time interval section of imaging acquisition to T1 in the time point section in the multidimensional acquisition window.
Open elements, change the light path to the right for confocal imaging and turn off the LED light. Then click on the remove interlock button and turn on the FITC laser channel for YAP imaging by checking the FITC box. After adjusting the scanning speed to 1 frame per 2 seconds, spin the knob of the Z plane to find the Z position of the attached cells quickly.
Record the lower and upper limits for the Z stack. Click on macro on the top ribbon, select macro editor under the macro dropdown menu and input the lower and upper limits for the Z stack into a macro file. Turn on the DAPI laser channel for bead imaging by checking the DAPI box to find and record the focused Z position of beads.
Go to macro editor and input the recorded values into the macro file. To set the task of moving the motorized stage using AMFIP, go to Micro Manager, click on plug-ins automation to open the graphical user interface of AMFIP. Then click on add point or remove point buttons to acquire the exact number of fields of view selected.
Input the recorded coordinates of fields of view into the coordinates panel. Define the total experiment time in the total experiment time text field. Click on the additional time configuration button and define the time interval T2 of moving the motorized stage to each FOV.
Maximize the window size of elements and drag GUI of AMFIP to the right side of the screen to avoid the GUI disturbing the automatic operations of the cursor and then click on the enter button. After the first macro finishes. click on the acquire button in the multidimensional acquisition window.
After finishing the long term imaging, stop the AMFIP task by clicking on the pause button in the automation plug-in window, and the stop button in the multidimensional acquisition window. Open elements and set Z stack imaging by clicking the top and bottom buttons in the ND acquisition window. Switch the light path to the right and open the LED light.
Slowly and carefully remove the lids of the chamber and the Petri dish while monitoring the bright field view for any drift of the field of view. Use a plastic pipette to take up 0.5 milliliters of SDS solution. Carefully hold the plastic pipette a little above the culture medium in the Petri dish and add 1 to 2 droplets of the SDS solution into the culture medium.
Once the cells and the bright field view are dissolved, close the LED light, switch the light path to the left, click on the remove interlock button. Run the Z stack imaging and save the image stack as reference_N, where N is the sequence number of each field of view. Click on the multiple positions XY button on the multidimensional acquisition window.
Then select the next field of view and click on the go-to button to move the motorized stage to the second FOV. Repeat this step for each field of view. The nuclear localization is of yes-associated protein or YAP in B2B ells increased with increasing substrate stiffness, whereas PC-9 cells showed similar YAP concentration in the nucleus and cytoplasm on substrates of varying stiffness.
The B2B cell monotonically increased the spread area over time, along with a decrease in the YAP nuclear to cytoplasmic ratio, while the PC-9 cell maintained a comparatively unchanging cell spread area, orientation, and YAP nuclear to cytoplasmic ratio throughout the 10 hour spreading process. During the 10 hour duration of the early spreading, the representative B2B cell constitutively deformed the substrate surface and applied time evolving cell traction across the whole cell area. In contrast, the representative PC-9 cell only developed displacement and traction at the 2 ends of the cell body, and its traction diminished after 7.5 hours.
The YAP nuclear to cytoplasmic ratio and dipole traction of B2B cells appeared to follow two distinct trends, suggesting that there might have been two groups of B2B cells that coexisted in the experiment. In the first group, the YAP nuclear to cytoplasmic ratio and dipole traction increased along with the enlargement of the cell spread area and reached their maxima at approximately 1, 000 square micrometers. In the second group, the YAP nuclear to cytoplasmic ratio and dipole traction increased at a slower rate with the enlargement of the cell spread area and maintained nearly constant values when the cell spread area continued to increase.
The Z stack ranges for all the laser channels need to be determined carefully to overcome the potential drift of the field of view during stage movement in long term imaging. This novel technique enables automatic, non-invasive long-term and multiposition imaging, and can help elucidate molecular biology mechanism that requires temporal and spatial tracking of cells and organelles.
