Fluorescence microscopy and live cell imaging

Fluorescence microscopy has long been employed to observe biological processes within fixed cells or tissue sections. We are now more than ever able to expand our field of knowledge using the plethora of dyes and fluorophores to stain these samples and gain higher resolution of multiple processes in multiparametric fashion. Although using these dyes on fixed samples from different intervals (ie. Fixed at different times post treatment) can reveal kinetic trends, it does not allow the resolution to observe biological processes in real time. With the advent of live cell imaging, we are now capable of viewing cellular kinetics in real time within individual cells. Here in we demonstrate the use of live cell imaging to track the movement of mitochondria within live cells, and the effect of photo-bleaching when doing live cell experiments. We also demonstrate the use of live cell imaging to follow kinetics of nuclear NFκB translocation to the nucleus in response to recombinant TNF-α.

Methods

Mitochondria movement
Protocols are outlined in the MMI 490 Fall Semester 2015 lab manual with minor changes. Briefly, HeLa cells were seeded onto glass coverslips at 20% confluence in cDMEM (10% FCS) and incubated at 37°C with 5% CO₂. Slips were transferred to 6 well plates and stained with 1:333 Pico green, 0.5µM Mitotracker red CMX ros and 0.8µM Hoescht 33258 for 1 hour. Cover slips were individually transferred to live cell imaging chambers containing 0.5mL media before sampling on a Leica SP5 scanning confocal fluorescence microscope. Images were acquired once a minute for 40 minutes and analyzed using FIJI image analysis software to obtain mean fluorescence for whole cells in each channel.

Transfection of GFP-NFκB and NFκB translocation
HeLa cells were seeded onto glass cover slips at 20% confluence overnight. 100µL 1:33 Fugene 6 HD in Opti-MEM was mixed with 1 volume 200µM GFP-NFκB encoding plasmid in Opti-MEM. This mixture was incubated at 37°C for 20 minutes before drop-wise addition to cells in 1mL Opti-MEM (3% FCS). Cells were incubated at 37C 5% CO₂ before transfer of coverslips to a live cell imaging chamber. GFP distribution was then analyzed using the Olympus Wave FX 1 spinning disk fluorescence microscope. Data obtained from Dr. Steve Ogg contains images that were captured every 12 seconds for 1 hour with TNF addition at 3 minutes into the run. Fluorescence was measured using FIJI analysis software by comparing mean pixel fluorescence of the nucleus to that of the entire cell.

Results and Discussion

Mitochondrial movement in live HeLa cells
Mitochondria are often thought of as stationary blobs within the cell, but in fact their movement can be quite pronounced. Although this is the case, the health and state of cells can impact the mitochondrial behaviour, where our time lapse videos failed to show much significant movement at all. This could be due to many factors including density, activation states, temperature or cell ‘happiness’ outside of 5% CO₂ since the SP5 is not equipped with a live cell incubation unit. Another issue we had was the predominant staining with Pico green, an intercalating dye that binds to the small groove of dsDNA (2). We believe that the addition of Pico green at high concentration before the addition of Hoescht bound all the nuclear DNA and excluded the Hoechst dye from being displayed (Figure 1) since green signal can be seen dispersed throughout the cell, but also very brightly over the nucleus. The mitrotracker red displayed great affinity for mitochondria, which was expected since it localizes to mitochondrial membrane potential.

Figure 1. Photo-bleaching of vital dyes during live cell imaging. Hela cells were stained with pico green and mitotracker red previous to image capture on an SP5 spinning disk fluorescence microscope. Images were captured once a minute for 40 minutes. Images displayed from the beginning of the run and at the end, in both green and red channels.

Figure 2. Quantification of mean fluorescence of vital dyes during live cell imaging. Mean whole cell fluorescence was measured for each channel respectively in each acquired image and compared to the starting frame as calculated by (frame (x) mean fluorescence/ start frame mean fluorescence). 

Photo-bleaching is the degradation of fluorophores by light sources and is concern when doing any type of fluorescent assay. It is especially important for time-lapse imaging, since bleaching can prevent capture of further time points. Hence it is important to consider longer image capture times on longer assays to minimize exposure to high energy light and thus prevent extensive photo-bleaching. In this assay, one image was taken per minute in 3 channels for 40 minutes. Even at this setting, both detectable fluorophores (Mitotracker Red and Pico green) displayed significant levels of photo-bleaching with visually detectable differences from beginning to end (Figure 1). When quantified using FIJI (is just ImageJ), end mean fluorescence for individual cells was seen to drop by ~30% and 50% from start mean fluorescence for Pico green and Mitotracker red respectively (Figure 2). If more images had been taken during the same time frame, the images may have been dimmer or non-existent due to higher light exposures. This is especially problematic since bleaching is known to be exponential, and a lack of signal prevents any further data from being extracted from the assay.

NFkB translocation in TNFα stimulated HeLa cells
NFκB is a critical transcription factor involved in stimulating inflammatory reactions. Many studies observe the percent of cells that have translocated after specific treatments, etc., but that percentage is just a snap shot of what is happening in a cell. Using live cell imaging, we are able to show the kinetics of how fast HeLa cells translocate NFκB to the nucleus in response to TNFα stimulation. In data obtained from Dr. Steve Ogg, we observed translocation in all 3 cells found in one frame (Figure 3a), but not those found in another. When total cell fluorescence was compared to that of the nucleus, we found 3 cells experienced a mean nuclear intensity that increased steadily from 70-80% to 80-100% of that of the total cell (Figure 4). While each cell had slightly different kinetics, indicated by varying slopes of each line, each cell experiences filling in of the nucleus with the GFP tagged NFkB that is evident in the image. One the other hand, another set of cells did not display any translocation, with nuclear intensity actually dropping after treatment with TNF (Figure 3b). This could be due to the state of growth of the cells, the density on the cover slip, relative health, or crowding as outlined by the Pelkmans group in their 2009 Nature publication¹. They found that heterogeneity of cells within a single dish was not due to random variation, but could be predicted based on morphology and relative niche of each grouping of cells due to differential growth kinetics, activity, etc. Further, they could predict lower infectivity of certain cell subsets based on their positioning based on growth phase dependent expression of certain phospholipids that simian virus 40 (SV40) uses for entry.

                                                                                     

Figure 3. Translocation of NFkB-GFP in TNF stimulated HeLa cells. Transfected HeLa cells were imaged for GFP translocation to the nucleus for 30 minutes after stimulation with recombinant TNFa. a) Cells that exhibited translocation, cells 1-3. b) Cells that showed no translocation (cell 4). Yellow lines show representative regions of interest used to quantify fluorescence in Figure 4. c) Response to cell 4. Time lapse video of d) NFkB translocating cells e) non-translocating cells. (Apologies for the sloppy figure, Blogger doesn't seem to like embedding videos very much!)



d-e

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Figure 4. Quantification of NFkB translocation via nuclear fluorescence intensity. Individual cells indicated in Figure 3 were quantified for mean fluorescence in the nucleus compared to that of the whole cell for one frame every 2 minutes. A reading of 1 indicates a mean fluorescence of the nucleus identical to the whole cell. the red arrow indicates TNF addition at time = 3min.



Likely, this may contribute to why our personal experiments didn’t work, as we may have chosen to image cells that either fell into a growth phase that doesn’t express much TNF receptor, or that is somehow inhibited in the NFkB pathway to prevent proliferation of activated cells. Antibody staining for TNFR could be used to rule out the first possibility. Alternatively, it could have been due to the TNF becoming non-functional from too many freeze thaws, as we performed 3 separate technical replicates to try to obtain our own data. Problems with a speckle reducer, a module in the scope which smooths out light coming through the 50 fiber optic threads within the cable, prevented quality image acquisition on our first run. Additionally, a faulty temperature probe allowed the cell incubator to reach over 44 °C which likely had a detrimental effect on our cells. Though, as with all science one cannot expect results from the first run, and patience must be observed while trying to troubleshoot possible sources of error to fully optimize an assay.

1. Snijder, B. et al. Population context determines cell-to-cell variability in endocytosis and virus infection. Nature 461, 520–523 (2009).

2. Dragan, A. et al. Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding. Journal of Biophysics (2010).
doi: 10.1016/j.bpj.2010.09.012.