STORM Microscopy (Stochastic Optical Reconstruction Microscopy)   STORM is the most widely used SRM method, and it relies on the sequential activation and time-resolved localization of photoswitchable fluorophores to generate high-resolution images. To achieve a high-quality multicolor image, specific labeling is required using direct labeling, protein conjugates, or antibody staining. Ideal fluorophores for STORM should be very bright and have a high rate of photoswitching cycles. They should also exhibit minimal photobleaching in thiol-containing buffers. With appropriate dye and buffer combinations, an optimized STORM system can generate images with 5 nm resolution. Numerous publications show combinations of Molecular Probes® dyes with a variety of targeting and labeling specificities used to generate multiplex STORM images.

Antibody conjugates and labels

Molecular Probes® fluorophores have been tested in STORM applications, as conjugates of antibodies, proteins, dextrans, and other biomolecules. Use the table below to select the fluorophore with the best wavelength and rating for your application in either dSTORM or nSTORM—citations are listed for each. By following the links you can also find all of the bioconjugates of each fluorophore currently available to target your molecule of interest; the list ranges from antibodies and phalloidins to growth factors and lectins. If you don't find the conjugate you need on the list, the links will also show you reactive dye forms or optimized labeling kits to enable you to create your own bioconjugate probes.

STORM antibody conjugates and labels
Two-color STORM image of immunolabeled microtubules. Tyrosinated tubulin was stained with Alexa Fluor® 647 dye (magenta) and detyrosinated tubulin was stained with Alexa Fluor® 750 dye (green). Image courtesy of Joshua Vaughan et al., University of Washington, Seattle, WA.
  Alexa Fluor® 488 Alexa Fluor® 532 Alexa Fluor® 555 Alexa Fluor® 568
Target Label/conjugate Label/conjugate Label/conjugate Label/conjugate
STORM buffer MEA MEA MEA MEA
Bibliography Citations Citations  Citations Citations
Laser line (nm) 488 488 488 561
Standard filter set FITC TRITC TRITC RFP
Ex/Em (nm) 495/519 532/554 555/580 578/603
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  Alexa Fluor® 594 Alexa Fluor® 647 Alexa Fluor® 680 Alexa Fluor® 750
Target Label/conjugate Label/conjugate Label/conjugate Label/conjugate
STORM buffer MEA BME BME TCEP
Bibliography Citations Citations Citations Citations
Laser line (nm) 594 594/633 633 633
Standard filter set Texas Red® dye Cy®5 Cy®5.5 Cy®7
Ex/Em (nm) 590/617 650/665 679/702 749/775
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  Alexa Fluor® 405 Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647 Alexa Fluor® 750
Target Label/conjugate Label/conjugate Label/conjugate Label/conjugate Label/conjugate
STORM probe type Activator Activator Activator Reporter Reporter
Bibliography Citations Citations Citations Citations Citations
Laser line (nm) 350/405 488 488 594/633 633
Standard filter set DAPI FITC TRITC Cy®5 Cy®7
Ex/Em (nm) 401/421 495/519 555/580 650/665 749/775
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Nucleic acid stains

For direct labeling of nucleic acids, SYTO® 13 and YOYO®-1 dyes have demonstrated excellent performance in STORM applications. Both dyes exhibit green fluorescence and are imaged using FITC filters for conventional applications. One of the highest affinity nucleic acid stains, the cell-impermeant YOYO®-1 stain exhibits over a thousand-fold increase in fluorescence when bound to dsDNA. YOYO®-1 stain has proved extremely useful in the analysis of single molecules of DNA. SYTO® 13 dye is cell permeant and shows a small shift in fluorescence characteristics (excitation and emission maxima) when bound to RNA vs. DNA. Use the table below to select a DNA dye for your STORM experiment.

  YOYO®-1 SYTO® 13
Target dsDNA Nucleic acids
STORM buffer BME/MEA BME/MEA
Bibliography
Laser line (nm) 488 488
Standard filter set FITC FITC
Ex/Em (nm) 491/509 481/509
Cat. No. Y3601 S7575

Organelle probes

Organelle probes for STORMDirect labeling probes for a range of organelles have been implemented in STORM with outstanding results. For mitochondrial labeling the optimal STORM signal is obtained with MitoTracker® Red dye, although MitoTracker® Orange and MitoTracker® Deep Red both offer good performance if the Texas Red® dye channel is already in use for multiplexing another signal.

LysoTracker® Red is recommended for lysosomes and other acidic organelles, and ERTracker® Red is highly selective for the endoplasmic reticulum.

The plasma membrane can be labeled for STORM with a range of dyes offering orange- (DiI), far-red– (DiD), or near-IR– (DiR) fluorescence. All of these dyes offer good performance in STORM, but DiD offers the strongest performance if the far-red channel is available. Use the table below to select appropriate organelle probes for your STORM experiment.

  MitoTracker® Orange dye MitoTracker® Red dye MitoTracker® Deep Red dye
Target Mitochondria Mitochondria Mitochondria
STORM buffer BME/MEA BME/MEA BME/MEA
Bibliography Citations Citations Citations
Laser line (nm) 488 561 594/633
Standard filter set TRITC Texas Red® dye Cy®5
Ex/Em (nm) 554/576 581/644 644/665
Cat. No. M7510 M22425 M22426
  LysoTracker® Red dye ER-Tracker™ Red dye
Target Lysosomes ER
STORM buffer BME/MEA BME/MEA
Bibliography Citations Citations
Laser line (nm) 561 561
Standard filter set Texas Red® dye Texas Red® dye
Ex/Em (nm) 577/590 587/615
Cat. No. L7528 E34250
  DiI DiD DiR
Target Cell membrane Cell membrane Cell membrane
STORM buffer BME/MEA BME/MEA BME/MEA
Bibliography Citations Citations Citations
Laser line (nm) 488 594/633 633
Standard filter set TRITC Cy®5 Cy®5.5
Ex/Em (nm) 449/565 644/665 750/780
Cat. No. D282 D7757 D12731

Buffers and reagents

To enable effective photoswitching, fluorescent dyes require either an appropriate activator/reporter pairing (nSTORM) or an oxygen-scavenging buffer system (dSTORM). Typical buffer systems contain catalase, glucose, and glucose oxidase (commonly referred to as GLOX) in combination with a reducing agent. The two most commonly used are mercaptoethylamine (MEA) or β-mercaptoethanol (BME). Both xanthene- and cyanine-based dyes can be used in either buffer system, however xanthene-based dyes (Alexa Fluor® 488 and Alexa Fluor® 568) tend to perform better in MEA whereas cyanine-based dyes (Cy®5, Alexa Fluor® 647, and Alexa Fluor® 750) perform better in BME and TCEP, respectively. See the buffer composition and references in the table below. Use this osmotically-stabilized imaging buffer as a starting point and optimize for a particular dye using the different reducing agents.

Basic imaging buffer
Dye-specific buffers
  MEA BME TCEP
  • 50 mM TRIS, 10 mM NaCl (to pH 8)
  • GLOX (0.5 mg/mL glucose oxidase, 40 μg/mL catalase, 10% glucose)
+ MEA to10 mM + BME to 140 mM + TCEP to 10–100 mM  (need 1 mM ascorbic acid and methyl viologen)
Dempsey et al. Nat Methods 8:1027–36 Dempsey et al. Nat Methods 8:1027–36 Dempsey et al. Nat Methods 8:1027–36 Vaughan et al. 2013 J Am Chem Soc 135(4):1197–200
For Research Use Only. Not for use in diagnostic procedures.