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HaloFluor™ Series SaraFluor™ Structural Imaging Series

HaloTag® SaraFluor™ 650B ligand

[Probe for super-resolution imaging combined with HaloTag ligand]

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 new imaging methods called “superresolution fluorescence microscopy” exceeding this limitation have been developed. One of them, called single molecule localization microscopy (SMLM), is widely used. Developed exclusively for SMLM, SaraFluor B series* is a fluorescent probe that exhibits spontaneous blinking under physiological conditions.

SaraFluor 650B (SF650B, HMSiR) emits deep red fluorescence with red laser excitation. Target molecules can be labeled via HaloTag.

*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-Halo.

※HaloTag® is a registered trademark of Promega Cooperation.

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
A201-01 HaloTag® SaraFluor™ 650B ligand 15 nmol
A201-02 HaloTag ®SaraFluor™ 650B ligand 15 nmol × 2

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  • Flyer

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  • Product Information

    HaloTag® SaraFluor™ 650B ligand
    Product Information

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    Properties of SaraFluor B

    Product Name Absmax (nm) FLmax (nm) ε Φ
    SaraFluor 488B (HEtetTFER) 507 530 80,000 0.76
    SaraFluor 650B (HMSiR) 654 669 100,000 0.39

    Blinking properties of SaraFluor B

    Spontaneous blinking of SaraFluor B in PBS (pH 7.4). Measured intensity 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 650B, HEtetTFER).(It is impossible to compare two vertical axes because different optical measurement systems were used.)

    Spectra of SaraFluor B

     

     

     

     

  • Images using SaraFluor 650B antibody

    HaloTag® SaraFluor™ 650B ligand
    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

    HaloTag® SaraFluor™ 650B ligand
    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/cm2 power. 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

    HaloTag® SaraFluor™ 650B ligand
    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.

     

     

     

     

FAQ

  • Q SaraFluor 650B-NHS cannot be dissolved with DMSO completely. How should I do?
    A

    SaraFluor 650B-NHS and SaraFluor 488B-NHS show relatively low solubility to water/DMSO. Mix well in DMSO and use it for labeling proteins, according to the protocol. These compounds could assemble each other in an aqueous/DMSO solution to show low fluorescence intensity. After labeling proteins, the solubility increases and shows strong fluorescence.

     

     

  • Q My question is not in this FAQ list.....
    A

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

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