UnaG, a fluorogen-binding protein derived from Japanese freshwater eel, fluoresces after binding non-fluorescent bilirubin, an endogenous metabolite. Notably, we found that UnaG fluorescence is restored after photobleaching when exogenous bilirubin is added to the imaging buffer solution.
Introduction
Experimental Method and Materials
- Fluorescence Microscope
- Image Analysis
- Cell Culture
- Transfection
- Fixation
- Staining
- Imaging Buffer
COS7 cells, an African green monkey kidney cell line, and HEK-293T cells were maintained at 5% CO2 and 37 °C in Minimum Essential Medium (Gibco) supplemented with 10% Fetal Bovine Serum (FBS), 50 units/ mL penicillin and 50 μg/mL streptomycin. UnaG-expressing cells were fixed with 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) at room temperature for 15 min. It is then washed three times with DPBS and permeabilized with 0.4% Triton X100 at room temperature for 1 hour.
Cells were washed three times with DPBS and blocked with 2% BSA in DPBS at room temperature for 1 h. To stain mitochondria, transfected cells were incubated in 0.2 ~ 0.5 μM MitoTracker Red (Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM) for 30 seconds and then washed three times with DMEM. Imaging buffer was prepared with Phosphate Buffered Saline (PBS) supplemented with 2% glucose, oxygen scavenger (0.5 mg/mL glucose oxidase and 40 μg/mL catalase), and 500 nM bilirubin before imaging.
Results
UnaG Photophysical Properties
Short switching mechanism UnaG. Non-fluorescent apoUnaG combines with bilirubin and then becomes fluorescent holoUnaG. HoloUnaG is excited by 488 nm illumination and emits 527 nm fluorescence. During the shut-off mechanism, oxidized bilirubin is dissociated and UnaG is converted back to non-fluorescent UnaG. ON and OFF UnaG. Purified 200 mM holo-UnaG was measured with a 488 nm laser until UnaG fluorescence was quenched. After verification, fluorescence is turned back on with 488 nm laser illumination, a 20 mW 488 nm laser re-illuminates the tube until all fluorescence is turned off.
Measurement of the switching cycle of UnaG. A) Fluorescence intensity of UnaG-expressing U2-OS cell illuminated with 488 nm laser. By analyzing the fluorescence of small ROIs on the mitochondria in all acquired images, it was possible to measure the switching rate of the molecules. The switching cycle of UnaG was 25 Hz (Figure 7. A) and it was a sufficiently high cycle speed compared to other photoswitchable green fluorescent proteins [15]-[18].
However, its task could be prolonged by adding exogenous bilirubin because oxidized bilirubin changes position with bilirubin and then restores UnaG fluorescence. The fluorescence of UnaG recovered slowly and after 10 min it recovered almost the same amount of initial fluorescence. 200 nM holo-UnaG was illuminated with different 488 nm laser intensities to study the turn-off pattern (Figure 9).
With 80mW/cm2 of 488 nm laser, the fluorescence of UnaG decreased rapidly without oxygen scavenger while the fluorescence was. The fluorescence of UnaG lasted under the oxygen scavenger system, so maintaining a low oxygen concentration in the imaging buffer was important to perform long-latency STORM imaging. After photobleaching, UnaG fluorescence recovered to combine with fresh bilirubin over time.
A fluorescence intensity of the holoUnaG was dependent on 488 nm laser intensity and the fluorescence increasing trend shows linear graph. In a low oxygen atmosphere, the fluorescence of UnaG was reduced much more slowly than normal oxygen concentration solution. The fluorescence of UnaG switched on with bilirubin and the amount of fluorescence recovery depended on the bilirubin concentration.
Live Cell STORM Imaging
The thickness profile of ER tube shows STORM imaging shows 2 times better resolution than conventional imaging from 300 nm to 150 nm (Fig. We also performed STORM imaging with another construct, vimentin, which has an important role to supporting and anchoring structure in the cytosol.(Figure 12) We performed long time-lapse STORM imaging with UnaG-sec61β and UnaG-mito, expressing mitochondria membrane.
STORM imaging for the two constructs was performed using the same imaging mode, 3 mW 488 nm laser and 10 msec sCMOS camera frame rate. The first conventional level image of UnaG-sec61β and conventional image of the same ROI after 100 min of continuous STORM imaging. Super-resolution provides nanometer scale in cells and has revealed ultrastructural information about many organelles.
However, it has proven difficult to obtain super-resolution time-lapse images and the main cause is photobleaching of dyes. Therefore, we could approach obtaining ultrastructural dynamics of cells with super-resolution imaging with UnaG construct. In this study, we studied different targeted STORM imaging with UnaG and tried to find the appropriate STORM imaging condition, especially for long time-lapse imaging.
Also to perform long delayed STORM imaging, the imaging buffer circulation system must be provided. Because exogenous bilirubin will be depleted and subsequently photobleached, removal of oxidized bilirubin and replenishment of fresh bilirubin are necessary to perform long-term imaging. It is necessary to find the right image condition that satisfies UnaG and other colors to perform long STORM time-lapse imaging.
Super-resolution fluorescence imaging of organelles in living cells with photoswitchable membrane probes. Proceedings of the National Academy of Sciences, 109(35), p. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature Methods, 8(12), p. 1027-1036. Fluorescent probes for high-resolution live cell imaging. Nature Reviews Molecular Cell Biology, 9(12), pp.929-943.
Evidence for the conversion of bilirubin to dihydroxyl derivatives in the Gunn rat.Biochemical and Biophysical Research Communications,49(5), pp.1366-1375. Photoactivatable fluorescent proteins for diffraction-limited imaging and super-resolution imaging.Trends in cell biology,19(11), pp.555-565.
Two-Color STORM Imaging
Conclusion & Discussion
