INTRODUCTION
1 Both giant and tiny condensates co-exist in living cells. A Representative fluorescence images of stress granules in HeLa cells under heat shock conditions (42 °C, 0.5 h). Stress granules were labeled using the antibody against G3BP1. Scale bars, 5 μm. B Representative fluorescence images of transcription factory in HeLa cells. Transcription factories were labeled using the antibody against RNA Pol II S2 phosphorylation. Scale bars, 2 μm |
Development of LLPS characterization using super-resolution imaging methods
Applications and advantages of the protocol
Limitations of the protocol
MATERIALS AND EQUIPMENT
Biological materials
Reagents
Equipment
Buffer
Software
PROCEDURE
Step 1: Sample preparation
Step 1.1 Fixed cells with antibodies
Step 1.2 Live cells with fluorescent proteins
Step 2: Microscope setup
1 Ideal working and output laser power |
Laser | Laser working power | Measure laser power |
647 | 1000 mW | 300 mW |
561 | 1000 mW | 230 mW |
405 | MAX | 10 µW |
488 | 30 mW–Low | 130 µW |
Step 3: Image acquisition
Step 3.1: Cell selection
Step 3.2: Data acquisition
2 The sequence and exposure conditions for antibody-labeled proteins |
Laser | Imaging mode | EMCCD exposure | EMCCD exposure compensation | Frames |
647 nm | Conventional | 30 ms | 30 | 200 |
647 nm–brightfield | Conventional | 10 ms | 30 | 200 |
647 nm | STORM | 10 ms | 30 | 50,000 |
3 The sequence and exposure conditions for fluorescent protein-labeled proteins |
Laser | Imaging mode | EMCCD exposure | EMCCD exposure compensation | Frames |
488 nm | Conventional | 30 ms | 90 | 200 |
561 nm–brightfield | Conventional | 30 ms | 90 | 200 |
561 and 405 nm | STORM | 30 ms | 90 | 50,000 |
Step 4: Image analysis and quantification
2 Data processing for super-resolution images of small condensates. Each frame of the single-molecule raw image was first filtered using a wavelet filter and then localized using Gaussian fitting. This process was repeated for each frame until all the frames have been processed. After single-molecule localization and image reconstruction, drift correction using cross-correlation was applied to correct the drift during image acquiring |
Step 5: Condensate identification and segmentation
3 Condensate identification and quantification using super-resolution imaging. A Voronoï-based segmentation of condensates using SR-Tesseler. Zoomed images were displayed at the bottom for more detailed information. Every single molecule has a polygon defined by its neighboring molecules. Edges of Voronoï polygons are located equidistant from the nearest two molecules. When a new molecule is added, this bisector is cut by the bisectors computed between the old molecules and the new ones. Each new molecule was computed to plot the Voronoï diagram repeatedly until all the molecules were counted. Then a threshold relative to the average localization density was set to segment the clusters of molecules within one condensate. B Create polygons in SR-Tesseler. C Parameters used to define condensates in SR-Tesseler |
Anticipated results
4 Super-resolution imaging of transcription factory induced by optogenetics. A Schematic diagram of light-induced condensation enhancement. The largest subunit of RNA Pol II (RPB1) was fused with Cry2 to induce cluster formation. Blue light activation of Opto-RPB1 leads to rapid phase separation in living cells. B Representative wide-field and STORM images of Cry2-RPB1 before and after blue light illumination. The transcription factory was labeled using the antibody against RPB1. Scale bars, 2 μm. Zoomed scale bars, 100 nm. C, D Cluster analysis of RPB1 under the light on and light off conditions. Localization per condensate (C) and condensate number per cell (D) were shown. Data are presented as mean ± SD. Individual data points correspond to the average value for one cell. n = 20 cells for dark condition, 34 cells for light condition pooled from three independent experiments. The paired two-tailed Student’s t-test was used to compare the data (C and D). Significant differences are labeled as ****p < 0.0001. Boxplots: 25 th to 75th percentiles, median, 1.5× interquartile as whiskers (C) |
TIMING
4 Troubleshooting table |
Step | Problem | Problem reason | Solution |
Step 1.1.5 | High background | Blocking missed/failed | Increase the concentration of BSA; Use fresh BSA solution |
Step 1.1.6 | High background | Artifacts | Modify the blocking protocol and use different fluorescence excitation to check if it is stained non-specifically. |
Step 1.1.6 | Structure disruption | Over permeabilization | Modify permeabilization protocol |
Step 1.1.6 | No fluorescence | Fail in permeabilization | Modify permeabilization protocol; Use fresh permeabilization solution |
Step 1.1.8 | Low intensity | Low antibody titer | Change the secondary antibody; Modify the incubation time |
Step 1.1.8 | Low intensity | Unsuitable antibody solution conditions | Check pH of antibody solution, and make fresh |
Step 1.1.8 | High intensity | Non-specifically stained | Add the control experiments ( do not add the primary antibody) |
Step 1.2.4 | No fluorescence | Fail in transfection | Use cells in a healthy state; Use the efficient transfection reagent |
Step 2.6 | Dye not blinking | STIB spoil | Use fresh STIB |
Step 3.1 | Hard to find ideal cells | Sample drift | Verify sample drift using the calibration sample and realign the focal plane |
Step 3.1 | Hard to find ideal cells | Mismatched immersion oil | Change the immersion oil |
Step 3.1 | Hard to find ideal cells | The cells are unhealthy | Optimize the cell culture |
Step 3.2.1.2 | Surplus resides in the ON state | Insufficient irradiation intensity | Increase irradiation intensity |
Step 3.2.1.2 | Fluorophores do not recover | Only a sparse subset of fluorophores is in the ON state | Reduce the intensity of the readout laser |
Step 3.2.1.2 | Fluorophores do not recover | Fluorophores are photobleached | Optimize the activation and readout laser |
Step 3.2.1.2 | Fluorophores do not recover | STIB is unsuitable | Make fresh and use within 60 min Check pH; pH should be in the range of 6–9 |
Step 4.5 | Low resolution | Drift correction missing | Perform drift correction |