Abstract:
Fluorescence is re-emission of light at a higher wavelength than the excitation light which could be detected using a special fluorescence microscope. Variant types of fluorophores have been established to track different molecules inside the cells. The objective of this study was to apply these unique abilities of the fluorophores to quantify and track the movements of mitochondria inside the cell with the help of a fluorescence microscope. Endothelial cells were fixed and labeled with MitoTracker red, DAPI, and AlexaFluor 488 Phalloidin. Meanwhile, cultured human cervical carcinoma Hela cells were labeled using three different dyes (MitoTracker red CMXRoS, Hoechst 33258, and Picogreen). Both fixed and live cells were viewed using OMX wide field V4 Microscopy. Images were acquired and analyzed. Using Image J software we detected the number of mitochondria and through tracking one mitochondrion at different time points, we managed to detect the nature and direction of its movement in addition to calculation of its average velocity.
Methods:
For fixed cell imaging, bovine endothelial cells were labeled with MitoTracker red CMXRoS (to detect mitochondria), AlexaFluor 488 Phalloidin (for Actin), and DAPI (for Nuclear DNA). The cells were examined using OMX wide field V4 Microscopy. Images were acquired and analyzed to obtain quantitative data. Meanwhile, the cells which were used in live cell imaging were Human cervical carcinoma Hela cells. They were cultured at ~ 20% confluence on coverslips which were added to the bottom of a tissue culture dish. On the day of the experiment, cells were at the confluence of ~ 40%. Four coverslips were added to a 6-well plate. Two mL of media containing DMEM, 10% FCS and antibiotics were added into each well. The wells were labeled from one to four. We used three different dyes for this experiment. MitoTracker red (MT red) CMX RoS accumulates specifically in mitochondria. Hoechst 33258 intercalates into Nuclear DNA. Picogreen mainly stains mitochondrial DNA. Each of three dyes has its own excitation and emission spectra (Figure 1). MT Red was prepared by diluting the stock solution (1mM) to 100µM by adding 1 µL of MT Red to 9µL DMEM. Hoechst 33258 was prepared by diluting the stock (16mM) to 1.6 mM by adding 1 µL of the Hoechst stock solution to 9µL DMEM.
Figure 1. excitation (in blue) and emission (in red) spectra of Hoechst 33258, picogreen, and Mitotracker red CMX RoS. modified from www.thermofisher.com
At time t=0 minute, 3 µl of picogreen were added to well number 1 and well number 4. Then, The plate was incubated at 37°C and 5% CO2 conditions. At time t=30 minutes, the plate was removed from the incubator and 1 µL of the MT red working solution was added to wells number two and four. The plate was placed again in the incubator. At time t=45 minutes, 1µL of the Hoechst 33258 working solution was added to wells number three and four. The plate was returned again to the incubator. At time t=60, each coverslip was added into a live cell chamber that is designed specifically for the fluorescence microscope.
We used OMX wide field V4 Microscopy to view our sample and to acquire images. In the beginning, we added immersion oil 1.514 to the lens prior to adding the chamber to the microscope. We opened the software and we lowered down the lens using “Top 2400” setting. Following that, we added the chamber containing the coverslips and adjusted the microscope lens back again to the proper position. Then, we adjusted the viewing settings. We chose a red channel for MT red, a blue channel for Hoechst and the green channel for picogreen. After that, we selected medium mode (recommended as it indicates how fast the image readout is). We tried to lower the excitation value (20 is recommended) and the exposure time to avoid damaging the cells. While using the microscope to image the cells, we had to balance between increasing the intensity to get a good resolution and decreasing the intensity to keep the cells alive. After adjusting all parameters, we took several images at different time points showing the movements of both cells and mitochondria. Meanwhile, we could not detect any nuclear DNA using Hoechst dye.
Using Image J, we adjusted the threshold which allowed us to get more localization of the mitochondria and through “analyze particles” option we managed to get a number of mitochondria and the size of each mitochondrion. On the other hand, we have used a “manual tracker plugin” to track one mitochondrion over 91 frames which were taken over the duration of six minutes. A video was created of the mitochondrion tracking which gave us an idea about the nature of movements of mitochondria inside the cell. Following that, Results from Image J were exported to an excel file to analyze the average speed of the mitochondrion.
Results:
Mitochondria, nuclear DNA, and actin filaments of fixed endothelial cells are shown clearly in Figure 2. We have detected 141 mitochondria of different sizes ranging from 0.05 to 1 µm2 (Figure 3). A video was created showing the movement of both mitochondria and nuclear DNA (Video 1). The movements recorded were in different directions (forward, backward, and lateral movements) and speed. A second video was created demonstrating the tracking of movements of one mitochondrion inside the living Hela cell (Video 2). Data were exported from the software to excel sheet to be analyzed. The maximum speed detected was 2.337 µm/s while the lowest speed detected was 0.111 µm/s. The average speed of the mitochondrion through 91 frames was 0.445 µm/s. The velocity of the mitochondrion in each frame is shown in Figure 4.
Figure 2. Bovine endothelial cells showing nuclear DNA (blue), mitochondria (red), and actin filaments (green). Cells were fixed and labeled with MitoTracker red CMXRoS, Hoechst 33258, and Picogreen dyes. The image was acquired using OMX wide field V4 Microscopy.
Figure 3. The number of mitochondria detected in the endothelial cells and their sizes.
Discussion:
Nowadays, Fluorescence microscopy possesses variant established implications on research. The capability of fluorophores to detect different molecules made it easier for researchers to accomplish new discoveries in different biological fields (Drummen, 2012). Mitochondria, nuclear DNA, and actin filaments of fixed endothelial cells were clearly shown in Figure 1. This indicates that fixed cell imaging can provide us with a detailed description of certain molecules inside the cell at the time they were fixed. For example, we managed to get the number of mitochondria and their sizes using Image J. The same may apply to all other different molecules that a researcher would be interested in. This may help scientists to visualize those molecules and the mechanisms by which these molecules interact with different stimuli when comparing them with controls. On the other hand, the videos that we collected during the live cell imaging showed clearly the behavior of mitochondria which allowed us to track them and analyze their movements. We observed that mitochondrion moves in different directions and with an average velocity of 0.445 µm/second. Previous studies used different fluorophores for localization of mitochondria in living cells (Johnson, 1980). Due to some technical issues, we acquired blurred images that could not allow us to do tracking for the rest of the mitochondria and we were forced to perform the manual tracker.
Figure 4. The velocity of the mitochondrion at different frames. Frames were taken every 4 seconds. The analysis was performed using Image J software.
Supplementary material:
Video 1. Movements of mitochondria and nuclei in Hela cells using live cell fluorescence imaging. URL:
https://www.dropbox.com/s/vbirrhntldflbct/movement%20of%20mitochondria%20and%20nuclei.avi?dl=0
Video 2. Tracking of a mitochondrion inside a living Hela cell using Image J software.
URL: https://www.dropbox.com/s/78pe02k5738cbj7/Mitochondria%20tracking.avi?dl=0
References:
1. Drummen G. Fluorescent Probes and Fluorescence (Microscopy) Techniques – Illuminating Biological and Biomedical Research. Molecules. 2012; 17(12): 14067.
2. Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proceedings of the National Academy of Sciences. 1980; 77(2): 990-994.
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