Super-resolution imaging techniques operate beyond the limit set by the diffraction of light, leading to new insights in cellular biology. Among these techniques, the direct stochastic optical reconstruction microscopy (dSTORM) approach is based on the use of blinking fluorescent dyes allowing spatial resolution of about 20 nm. This post is focusing on a new type of fluorescent dye compatible with both live-cell imaging and staining of fixed cells adapted to dSTORM.
Goryo Chemical recently released HydroxyMethyl Silicon-Rhodamine (HMSiR) based stains which display spontaneous blinking in light emission.
By using this probe, cell biologists can run super-resolution imaging without thiols, oxygen scavengers or irradiation of high-power laser; all of which that were necessary for causing the blinking of fluorescent dyes in previous dSTORM setups. Therefore, the HMSiR probe enables live-cell and super-resolution imaging with low-power excitation light under physiological conditions.
Why using HMSiR fluorescent probe for super-resolution imaging techniques of both living and fixed cells ?
HMSiR displays spontaneous blinking without irradiation of high-power laser (～1 kW cm-2) which used to be necessary for dSTORM experiments (Figure 1: left – laser power: 100 W cm-2, correspondence to On/Off state in right figure). It allows us to minimize the damage of the cell by the laser irradiation used in the previous dSTORM observation.
Imaging of fixed cells
An example of HMSiR application in STORM images of microtubules in fixed HeLa cells is shown in Figure 2.
Fixed HeLa cells which were incubated for 2 hours with Mouse anti-tublin antibodies ere then stained with HMSiR labeled Goat anti-rat IgG at room temperature for 2 hours in the dark. The left picture shows an averaged image, the right one shows a super-resolution image visualized by N-STORM (Nicon; 647 nm laser: 100 W/cm2). The imaging was performed without any additives such as thiols or oxygen scavengers. 5000 images were captured and analyzed.
Live-cell imaging with super resolution under the physiological conditions
Figure 3 shows an application in live cell imaging. Velo cells, in which HaloTag®-β-tubulin was expressed, were stained by HMSiR-Halo and visualized in medium Leibovitz L15 medium containing 10%FBS after replating. Images were obtained by STORM microscopy (N-STORM：TIRFM：647 nm, 100 W/cm2, 15 ms/frame, 1000 frame). Picture 3A shows an averaged image, Figure 3B shows a super-resolution image. Figure 3C shows the measurement of localization precision at the same laser conditions like in Fig. 3A and 3B.
Antibodies were labeled with HMSiR, absorbed on the glass surface, and observed by STORM (N-STORM). Positions of the molecules were repeatedly obtained and their distribution was fitted by Gaussian. Only signals over 150 photons were analyzed. Super-resolution imaging with HMSiR did not need thiols or oxygen scavengers and was operated under physiological conditions.
Where to find HMSiR-based stains ?
Various references are available. Here is a first selection of HMSiR-based references together with respective applications:
- For living cells: HMSiR-Halo – HMSiR-Halo has a chloroalkane base that specifically attaches to HaloTag® fusion proteins (HaloTag® is a trademark of Promega Corporation).
- For fixed cells:
Goryo Chemical’s HMSiR references are available in Europe through tebu-bio. Other HMSiR labeled antibodies (e.g., Goat anti-Mouse IgG and Goat anti-Rabbit IgG and labeled IgG F(ab)’ fragments) will be available soon.
Stay tuned for more HMSiR-based probes for dSTORM applications
To be informed about the new developments related to these HMSiR stains, browse tebu-bio’s web site regularly. You can also benefit from trial offers regarding HMSiR probe by looking at Special offers on tebu-bio’s web site or by contacting me via the form below!
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Uno SN, Kamiya M, Yoshihara T, Sugawara K, Okabe K, Tarhan MC, Fujita H, Funatsu T, Okada Y, Tobita S, Urano Y., Nat Chem. 2014 (681-689).