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Superresolution imaging using HMSiR™-labeled secondary antibodies

  • Superresolution imaging without UV laser irradiation is possible. Additions of thiols and oxygen scavengers are not required.
  • Spontaneously blinking fluorophore for single molecule localization microscopy (SMLM) suitable for STORM/PALM microscopes.

Summary

HMSiR™-labeled secondary antibodies can be used for staining fixed cells.

HMSiR™ is a fluorescent probe which shows spontaneous blinking in physiological conditions. It enables superresolution imaging without high-power laser irradiations, without additions of thiols or oxygen scavengers, which were necessary for the previous dSTORM observation.

Imaging examples

 Figure 1. Superresolution images of microtubules in HeLa cells.
 Figure 1. Superresolution images of microtubules in HeLa cells.

 

Fixed and permeabilized HeLa cells were incubated with mouse anti-α-tubulin IgG (1/300 diluted of 625901, BioLegend) at room temperature for 2 hours. After washing the antibody, cells were incubated with 10 μg/mL of HMSiR™-labeled Goat anti-mouse IgG (H&L) (A202-01) at room temperature for 2 hours. The images were obtained using NIKON N-STORM microscope.

Figure 2. Comparison of SMLM superresolution images to conventional fluorescent images.
Figure 2. Comparison of SMLM superresolution images to conventional fluorescent images.

 

Fixed and permeabilized HeLa cells were incubated with rat anti-tubulin IgG (1/300 diluted YL1/2, NB600-506, Novus Biologicals) for 2 hours. After washing the antibody, cells were incubated with 10 μg/mL of HMSiR™-labeled Goat anti-rat IgG (H&L) (A203-01) at room temperature for 2 hours. Cells were observed using NIKON N-STORM microscope. (left) Averaged image of 5000 images of HMSiR, representing conventional fluorescent images. (right) Result of SMLM image analysis from 5000 images. Analyses were performed using NIS-Elements (NIKON).

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Further information or questions on our products, please contact us, E-mail: info_itnl@goryochemical.com.

3D-STORM imaging of microtubules using NIKON N-STORM super-resolution microscope

  • Three dimensional superresolution imaging using cylindrical lens.

Overview

HMSiR™ is a fluorescent probe that shows spontaneous blinking in physiological solution conditions. It can be observed with weak illumination for the excitation, without adding thiols or oxygen scavengers. Here we show an example of 3-dimensional imaging of microtubules using a HMSiR™-labeled antibody and cylindrical lens equipped in NIKON N-STORM microscope. With the equipment, in combination with HMSiR™, you can obtain 3D images without moving the focus plane of the objective lens.

Microtubule network in HeLa cells visualized with HMSiR-labeled Goat anti-mouse IgG (H&L)

STORM - Movie

Using cylindrical lens equipped in NIKON N-STORM microscope, Z position (position along the optical axis) of HMSiR can be determined in about 50 nm resolution. Using the 3D-STORM mode of the system, 3-dimensional image can be reconstructed. The image was obtained in Common Research Facilities in The Institute of Medical Sciences, The University of Tokyo.

An example protocol for the sample preparation.

Fixation of HeLa cells
Cell fixation in a standard method.

  1. HeLa cells cultured in a glass bottom dish were fixed with 3—4 % paraformaldehyde at 37 oC for 10-20 minutes.
  2. Rinse the cells with PBS at 37 oC.
  3. Replace the solution with cold methanol (-20 oC) and incubate for 5-10 minutes to permeabilize the cells.
Staining with antibodies
  1. Blocking the dishes with BSA. Replace the solution with PBS containing 5%BSA and incubate at room temperature for 30 minutes. Then rinse the cells with PBS for 5 minutes. Repeat the rinsing for 3 times.

  2. Staining with primary antibody. Here we used mouse anti-α-tubulin antibody (1/300 diluted 625901, BioLegend). The diluted primary antibody was applied to the cells and incubated at room temperature for 2 hours. Incubation in a wet chamber to avoid drying is recommended.
    Rinse the cells with PBS for 5 minutes. Repeat the rinsing for 3 times to remove the excess primary antibody.

  3. Staining with secondary antibody. Prepare 10 μg/mL of HMSiR™-labeled Goat anti-mouse IgG (H&L) by diluting with PBS. The diluted secondary antibody was loaded to the cells and incubate at room temperature for 2 hours in the dark. Incubation in a wet chamber to avoid drying is recommended.
    Rinse the cells with PBS for 5 minutes. Repeat the rinsing for 3 times to remove the excess secondary antibody. Usage of antifade reagent or antifade mounting medium can slow photobleaching.
Superresolution imaging with 3D-STORM
Use NIKON N-STORM microscope to observe the stained cells. Turn off the 405 nm laser because HMSiR do not require switching laser irradiation. Use 647 nm laser of about 100 W/cm2 for excitation. Insert the cylindrical lens and obtain 10,000—30,000 images by following the instructions of the microscope and obtain 3D-STORM image using the software of the microscope.
Reference
S-n Uno, M. Kamiya, T. Yoshihara, K. Sugawara, K. Okabe, M. C. Tarhan, H. Fujita, T. Funatsu, Y. Okada, S. Tobita, Y. Urano (2014) Nature Chemistry  6:681—689.

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Further information or questions on our products, please contact us, E-mail: info_itnl@goryochemical.com.

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