SaraFluor™ 650B antibodies

[Antibodies for superresolution imaging]

650-750 nm：Far red

The theoretical limit of the resolution of fluorescence microscopy imaging is about half of the light wavelength (200-400 nm). A number of imaging methods called “superresolution fluorescence microscopy” exceeding this limitation have been developed. This fluorophore series has been developed for single molecule localization microscopy (SMLM), one of the superresolution microscopy methods. The SaraFluor B series is a fluorescent probe that exhibits spontaneous blinking under physiological conditions. This ability allows for super-resolution imaging using STORM, PALM and other similar microscopy systems under physiological conditions. It does not require the addition of thiols, deoxidizing agents or exposure to strong laser radiation to induce blinking as with general fluorescent probes. These products are secondary antibodies for immunofluorescent staining.

* SaraFluor is named after an Ainu language word “sara” which means “become visible” or “spacious and bright wetland”. Probes which exhibit spontaneous blinking are marked with letter B, first letter of the word “blinking”.
** These products are the same as those sold under the name of HMSiR labeling Goat IgG (whole) anti-mouse / rat / rabbit IgG H&L.

Products

Code No.	Product Name	Size
A202-01	SaraFluor™ 650B goat anti-mouse IgG	100 μg
A203-01	SaraFluor™ 650B goat anti-rat IgG	100 μg
A204-01	SaraFluor™ 650B goat anti-rabbit IgG	100 μg

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Protocol
Flyer
A202 SDS
A203 SDS
A204 SDS

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Product Information

SaraFluor™ 650B antibodies
Product Information

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Specifications of SaraFluor 650B antibodies

Product Name Abs_max (nm) FL_max (nm) ε* Φ

SaraFluor 650B (HMSiR) 654 669 120,000 0.39

*Molar absorption coefficient after protein labeling. Differs from the molar absorption coefficient of the probe itself.

Blinking property of SaraFluor 650B antibody

Spontaneous blinking of SaraFluor B in PBS (pH 7.4). Measured luminance change in a region corresponding to one molecule from a fluorescence image obtained by total internal reflection fluorescence microscopy with 647 nm laser excitation (SaraFluor 650 B, HEtetTFER).

Spectra of SaraFluor 650B
Images using SaraFluor 650B antibody

SaraFluor™ 650B antibodies
Images using SaraFluor 650B antibody

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Images using SaraFluor 650B antibody

An enlarged image of a HeLa cell pseudopod. Cells were fixed and stained with an anti-α-tubulin antibody (DM1A, 1/4000 dilution) and SaraFluor 650B (HMSiR) labeled secondary antibody (A202-01, 30 μg/mL). Left image (Conventional fluorescence image) is produced by averaging 25,000 images taken by the Nikon super-resolution microscope system (N-STORM) using NIKON Apo TIRF 100x (NA1.49) with 647nm laser excitation; right images are super-resolution images obtained by processing 1,000 and 25,000 images with ImageJ/ThunderSTORM software (SMLM).
Three-dimentional imaging microtubules using SaraFluor B with 3D-STORM
SaraFluor™ 650B antibodies
Three-dimentional imaging microtubules using SaraFluor B with 3D-STORM

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Three-dimentional imaging microtubules using 3D-STORM

SaraFluor B is a series of fluorophorea which show spontaneous blinking in physiological buffer conditions. By using NIKON N-STORM system with cylindrical lens, SaraFluor-B-stained three-dimentional structures cab be observed in super-resolution.

Microtubule structure of a HeLa cell stained with SaraFluor 650B labeled goat anti-mouse IgG

3D structure of microtubules imaged using cylindrical lens. Resolution in Z-axis of about 50 nm was obtained. Imaged in a N-STORM microscope in The Core Laboratories, The Institute of Medical Science, The University of Tokyo.

Procedures
1. HeLa cells cultured on a glass-bottomed dish was fixed in 3% paraformaldehyde for 20 minutes at 37℃.
2. Rinsed with PBS
3. Membrane-permeabilization in cold methanol for 5 minutes at －20℃.
4. Blocking in PBS containing 5％ BSA for 30 minutes. Rinsed three times.
5. Reacted with primary antibody. Purified anti-Tubulin-α Antibody (625901, BioLegend）was diluted to 10 μg/mL with PBS, and reacted for 2 hours at room temperature. Then, rinsed with PBS for 3 times.
6. Reacted with secondary antibody. SaraFluor 650B goat anti-mouse IgG was diluted to 10 μg/mL with PBS, and reacted for 2 hours at room temperature in dark, for 2 hours. Then, rinsed with PBS for 3 times.
7. Observed using NIKON N-STORM system. Excited with 647- nm laser of 100 W/cm²power. Cylindrical lens of the system was used to obtain images and 3D-STORM analysis was performed according to manufacture’s instructions.
Effect of frame numbers and analysis algorithms on SaraFluor B series superresolution images

SaraFluor™ 650B antibodies
Effect of frame numbers and analysis algorithms on SaraFluor B series superresolution images

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Effect of frame numbers and analysis algorithms on superresolution images

SaraFluor B series is a line of fluorophores which show spontaneous blinking in physiological pH. Without a special microscope system for PALM/STORM, a microscope which can image single fluorophore can be used to get multiple images of SaraFluor B and these images can be converted to superresolution image by using ImageJ plugins.

Besides a commonly used ImageJ plugin, ThunderSTORM, TRM mode of NanoJ SRRF , a plugin for supreresolution image analysis, can be also used to analyze SaraFluor B images to obtain superresolution images. Here, we qualitatively compare the effect of image numbers using these two plugins.

Fixed HeLa cells were reacted with anti-α tubulin antibody (DM1A, 1/4000) as a primary antibody, and then reacted with SaraFluor™ 650B goat anti-mouse IgG (A202-01, 30 μg/mL) as a secondary antibody. The cells were observed with NIKON N-STORM system by TIRF illumination using 647-nm laser, 100x Apo TIRF (NA 1.49) lens . Exposure was 30 msec/frame. Images were analyzed by the each softwares.

By using ThunderSTORM, 1,000 images were not enough to image some of the microtubule structures, whereas 10,000 images seems to be enough to image most of the microtubules. NanoJ SRRF (TRM) shows more information for fewer image numbers. ThunderSTORM provided higher contrast, whereas NanoJ SRRF did not give higher contrast and higher resolution even when using >10,000 images.

References

M. Ovesný, P. Křížek, J. Borkovec, Z. Švindrych, G. M. Hagen. (2014) Bioinformatics 30:2389-2390 DOI: 10.1093/bioinformatics/btu202 (ThunderSTORM)

Nils Gustafsson, Siân Culley, George Ashdown, Dylan M. Owen, Pedro Matos Pereira, & Ricardo Henriques (2016) Nature Communications 7: 12471 DOI: 10.1038/ncomms12471 (NanoJ SRRF)

Acknowlegements

We appreciate Dr. Kentaro Kobayashi and Dr. Motosuke Tsutsumi (NIKON Imaging Center, Hokkaido Univ.) for advices on imaging and analysis.