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SUPER-RESOLUTION MICROSCOPY

Validated in dozens of publications for STORM, STED, SIM, 2-photon and other specialized applications
Fluorescence lifetime data for CF® Dyes available for FLIM imaging
Unique kits and pre-labeled secondary antibody conjugates with a low degree of labeling optimal for STORM
Featuring rapid and efficient Mix-n-Stain™ labeling kits, cytoskeleton probes, and probes for other cellular structures
Featuring MemBrite® Fix cell surface stains developed for STORM imaging
Includes stains for nucleus, cell membranes, organelles, ion indicators, and more

The Best Probes for Super-Resolution

Super-resolution microscopy encompasses a broad family of imaging techniques that push beyond the diffraction limit of traditional light microscopy, revealing the finer details of biological structures. These techniques rely on extremely precise control over the excitation, emission, and image acquisition of fluorescently labeled cells and tissues. Consequently, the quality and efficiency of super-resolution imaging relies heavily on the fluorescent properties of the probe. Recent advances in super-resolution microscopy have demonstrated the importance of high-performance fluorescent probes. Biotium’s CF® Dyes have met this demand with industry-leading brightness and photochemical switching properties over other commercially available fluorescent probes. Moreover, CF® Dyes have been published in dozens of studies for a wide range of super-resolution applications.

 

CF® Dye Advantages for Super-Resolution

  • Superior brightness and photochemical switching properties ideal for super-resolution imaging
  • Validated in dozens of publications for STORM, STED, SIM, 2-photon, TIRF, and more
  • Available in over 40 fluorophores across visible spectrum with several near-IR options
  • Unique dye options designed specifically for STORM

Superior Performance for STORM Imaging

Several CF® Dyes were developed and validated for optimal performance in Stochastic Optical Reconstruction Microscopy (STORM). This includes Biotium’s red-excited CF® Dyes such as CF®647, CF®660C, and CF®680 which display superior brightness and unique photoswitching properties that are ideal for STORM. However, while STORM imaging commonly relies on photoswitchable red-excited dyes, finding comparable dyes for other excitation sources to produce quality multi-color STORM images remains a challenge. In response, Biotium has developed several green-excited dyes developed for optimal performance in STORM. Specifically, green-excited CF®583R and CF®597R were developed in collaboration with UC Berkeley and STORM scientist Ke Xu, PhD for exceptional performance in multi-color STORM imaging (Figures 1 and 2). In addition, green-excited CF®568 has also demonstrated better performance than Cy3b (Figure 3). CF®594ST is Biotium’s unique version of CF®594 developed specifically for STORM. See below for a full list of validated CF® Dyes for STORM and available products.

 

Figure 1. Typical (d)STORM image of the actin cytoskeleton in a fixed COS-7 cell labeled by phalloidin-CF®583R. (d)STORM was performed under standard conditions using a standard tris-based (d)STORM imaging buffer containing 100 mM cysteamine and an oxygen scavenger. Image courtesy of Bowen Wang, Michael Xiong, and Professor Ke Xu, College of Chemistry, University of California, Berkeley.

Figure 2. Comparison of typical (d)STORM images obtained in fixed COS-7 cells for microtubules immunolabeled by different dyes. (d)STORM was performed under standard conditions using a standard tris-based (d)STORM imaging buffer containing 100 mM cysteamine and an oxygen scavenger. (a) CF®583R. (b) CF®597R. (c) Alexa Fluor® 532. (d) Cy3B. (e) CF®568. Localization distributions are further given for single CF®583R and CF®597R molecules in the sample, in the X (in-plane; top) and Z (depth; bottom) directions, respectively. Gaussian fits (red curves) give standard deviations of ~10 and ~20 nm in the two directions, respectively. Color denotes depth (Z) values. Images and figures courtesy of Bowen Wang, Michael Xiong, and Professor Ke Xu, College of Chemistry, University of California, Berkeley.
Figure 3. CF®568 (left) produces better images than Cy®3b (right) in 3-D STORM microscopy. Images courtesy of Sam Kenny and Professor Ke Xu, College of Chemistry, University of California, Berkeley

CF® Dyes Validated for STED and FLIM Imaging by STELLARIS 8 STED Confocal Microscope

CF® Dyes were validated for high-quality super-resolution imaging of microtubules by Leica’s STELLARIS 8 STED FALCON platform. See the table on the right for fluorescent lifetime data for several CF® Dyes.

Figure 4. STED imaging of microtubules in U2OS cells. Microtubules were labeled with mouse anti-tubulin (DM1a) and anti-mouse CF®640R secondary antibody. Image acquired on a STELLARIS 8 STED FALCON confocal microscope, courtesy Leica Microsystems GmbH, Germany.

CF® Dye Fluorescence Lifetime Data

Dyeτ (ns) /free acid in PBS pH 7.4, ε (ns)τ (ns) /S.Ab§
CF®405S 3.88 ± 0.05
CF®488A 4.11 ± 0.051.705
CF®568 3.66 ± 0.05 1.539
CF®594 1.746
CF®633 3.39* (in water)3*
CF®640R 2.38 ± 0.05 1.557
CF®647 1.07 ± 0.05 1.195
CF®680 1.23 ± 0.05 1.277
CF®680R 1.22 ± 0.051.6
CF®750 0.58 ± 0.050.636
CF®790 0.39 ± 0.050.54

Measurements were made on a Stellaris 8 STED FALCON microscope courtesy Leica Microsystems, Germany. § Fluorescence lifetime measurements of CF® Dye labeled anti-mouse secondary antibodies used for immunostaining microtubules in U2OS using mouse anti-tubulin (DM1a) and mounted in ProLong™ Diamond. *Lifetime data obtained via customer communication under different experimental conditions and imaging setup.

 

 

 

Don’t Just Take Our Word for It!

Check Out More Publications Validating CF® Dyes for STORM

Sign up to our e-newsletter to download our Literature Digest: CF® Dyes for STORM Super-Resolution Microscopy.

CF® Dyes Validated for Super-Resolution Microscopy

DyeAbs/Em (nm)STORMSTEDSIM2-PhotonTIRFOther ApplicationsFeatures
CF®405S411/431ExM• Brighter than Alexa Fluor® 405
CF®405M416/452ExM• More photostable than Pacific Blue™
• Excellent choice for SIM imaging
CF®488A490/516ExM• Less non-specific binding than Alexa Fluor® 488
CF®535ST535/569• Orange dye designed specifically for STORM imaging
CF®555554/5681• Brighter and more photostable than Cy®3
• Less non-specific binding than Alexa Fluor® 555
CF®568562/5841ExM• Yields much brighter conjugates than Alexa Fluor® 568
• Outperforms Cy®3b in STORM
• Pairs well with CF®647 and CF®680 for multi-color STORM – see publication
CF®583R585/6091• One of two top-performing dyes specifically designed for STORM
with green laser (also see CF®597R) – see publication
CF®594593/615• Significantly brighter than Alexa Fluor® 594 and Texas Red®
• Extremely photostable
CF®594ST593/620• Deep-red fluorescent dye designed specifically for STORM
CF®597R597/6191• Deep-red fluorescent dye designed specifically for STORM
• Top-performing dye specifically designed for STORM
with green laser (also see CF®583R) – see publication
CF®633629/650FIONA, gSHRImp, ExM, SMT• Significantly brighter than spectrally similar far-red dyes
• Far more photostable than Alexa Fluor® 647
CF®640R642/663FLImP• Offers improved brightness and photostability over ATTO 647N
and spectrally similar dyes
CF®647652/6681ExM• Spectrally similar to Cy®5 and Alexa Fluor® 647
• Pairs well with CF®568 for multi-color STORM
• The best far-red dye for demixing-based multi-color
(d)STORM imaging when paired with CF®680 – see publication
CF®660R662/682ExM, SMLM• Much brighter than Alexa Fluor® 660
• The most photostable 660 nm dye
• Validated for use with DNA-PAINT SMLM – see publication
CF®660C667/6851• Much brighter and more photostable than Alexa Fluor® 660
• Ideal for long high-intensity 3D (d)STORM image acquisitions
with minimal photobleaching – see publication
CF®680681/6981Dual-color 3D SMLM• The brightest among spectrally similar 680 nm dyes
• Pairs well with CF®568 for multi-color STORM
• The best near-IR dye for demixing-based multi-color
(d)STORM imaging when paired with CF®647 – see publication
CF®680R680/7011Single-molecule spectroscopy, SMT• The most photostable 680 nm dye
• Suitable for labeling nucleic acids and small biomolecules
CF®750755/779• Exceptionally bright and photostable near-IR dye
• Patented pegylated dye for superior performance
A check mark indicates the respective dye has been published and/or validated for the application.

1 Dye was validated in multi-color STORM experiments

FLImP: Fluorophore localization imaging with photobleaching; SIM: Structured illumination microscopy; STED: Stimulated emission depletion; STORM: Stochastical optical reconstruction microscopy; TIRF: Total internal reflection fluorescence; FIONA: Fluorescence imaging with one-nanometer accuracy; ExM: Expansion microscopy; SMT: Single-molecule tracking; SMLM: Single-molecule localization microscopy.

CF® Dye Products Useful for Super-Resolution Microscopy

Antibody Labeling Kits

Our Mix-n-Stain™ STORM CF® Dye Antibody Labeling Kits allow you to label 50 ug of your antibody with one of Biotium’s STORM CF® Dyes to produce an antibody conjugate with a low 1-2.5 DOL (degree of labeling) optimal for super-resolution STORM (Stochastic Optical Reconstruction Microscopy). Labeling takes just 30 minutes, with minimal hands-on time and no purification. Click here to learn more about Biotium’s antibody labeling kits.

  • Choice of 8 CF® Dye colors ideal for STORM
  • CF®583R and CF®597R are novel green-excited STORM dyes
  • Labeling optimized to provide low 1-2.5 DOL
  • Less than 30 seconds of hands-on time
  • 30 minutes total reaction time
  • No purification, 100% recovery

Reactive CF® Dyes

Developing a custom probe for super-resolution applications? Biotium offers CF® Dyes in a wide array of reactive chemistries to suit your research needs.

Single-Label Secondary Antibody Conjugates for STORM

Secondary antibodies with a low degree of labeling (DOL, or number of dye molecules per antibody molecule) have been reported to be optimal for STORM (Bittel et al. 2015). Biotium offers single-label secondary antibody conjugates with an average DOL of one for STORM applications.

Probes for Cytoskeleton and Other Cellular Structures

Biotium offers a wide variety of CF® Dye bioconjugates used for labeling specific cellular structures.

  • Phalloidin conjugates for F-actin labeling available in 16 CF® Dyes. CF®647 and CF®680 phalloidins ideal for STORM imaging.
  • Con A, WGA, and PNA lectin CF® Dye conjugates for labeling glycoproteins and cellular surfaces
  • Streptavidin & biotinylated CF® Dye conjugates
  • CF® Dye nucleotides and probes for apoptosis, endocytosis, and other cellular processes also available.

Learn more about CF® Dyes for Super-Resolution Imaging:

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Membrane and Organelle Stains for Super-Resolution Imaging

MemBrite® Fix stains are validated for super-resolution and other specialized imaging applications

MemBrite® Fix stains are novel reactive fluorescent dye stains that react irreversibly with cell surface proteins, for staining that can withstand formaldehyde or alcohol fixation, and detergent permeabilization. Unlike lectins such as WGA, where surface staining varies between cell types, MemBrite® Fix dyes react widely with cell surface proteins for more uniform staining.

Several MemBrite® Fix dyes have been validated in super-resolution imaging applications or 2-photon microscopy. MemBrite® Fix-ST dyes are specialized dyes recommended for super-resolution imaging by STORM. MemBrite® Fix-ST dyes can also be used for standard microscopy applications; however, MemBrite® Fix dyes are generally more photostable than MemBrite® Fix-ST dyes. See the table on the right for a list of MemBrite® Fix dyes validated for super-resolution and other specialized imaging applications.

Figure 5. Cells were labeled with the indicated dye for 5 minutes at 37°C, then fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature, followed by permeabilization with PBS/0.1% Triton® X-100 for 10 minutes at room temperature.

MemBrite® Fix Features

  • Rapid, uniform cell surface labeling that can be fixed & permeabilized
  • Choose from 12 bright & photostable dye colors from blue to near-IR
  • MemBrite® Fix-ST designed specifically for STORM imaging
  • Several MemBrite® Fix colors validated for super-resolution and other specialized imaging applications
Catalog numberDyeSpecialized
applications
30092MemBrite® Fix 405/430SIM
30093MemBrite® Fix 488/515STED, TIRF, 2-photon microscopy
30094MemBrite® Fix 543/560N/A
30095MemBrite® Fix 568/580SIM, STORM, TIRF
30096MemBrite® Fix 594/6152-photon microscopy
30097MemBrite® Fix 640/660FLImP, SIM, TIRF
30098MemBrite® Fix 660/680N/A
30099MemBrite® Fix 680/700STORM1, Single-molecule imaging, STED,
2-photon microscopy
30101MemBrite® Fix-ST 650/665STORM
30102MemBrite® Fix-ST 667/685STORM
30103MemBrite® Fix-ST 681/698Single-molecule imaging, STORM
30104MemBrite® Fix-ST 755/777STORM
FLImP: Fluorophore localization imaging with photobleaching; SIM: Structured illumination microscopy; STED: Stimulated emission depletion; STORM: Stochastical optical reconstruction microscopy; TIRF: Total internal reflection fluorescence

1. MemBrite® Fix-ST 681/698 dye is reported to have better performance in STORM imaging than MemBrite® Fix 680/700 dye.


Lysosome and Lipid Droplet Stains Validated for Super-Resolution

LysoView™ Dyes are fluorescent stains for imaging lysosome localization and morphology in live cells. The dyes accumulate in the low pH environment of the lysosomes, resulting in highly specific lysosomal staining without the need for a wash step. LysoView™ 540 and LysoView™ 633 exhibit pH-sensitive fluorescence, further enhancing the specificity of staining. LysoView™ 488 has been validated in super-resolution imaging by SIM (see highlighted citation below), while LysoView™ 650 fluorophore is compatible with super-resolution imaging by SIM and STED.

LipidSpot™ Lipid Droplet Stains are fluorogenic neutral lipid stains that rapidly stain lipid droplets. The dyes can be used to stain lipid droplets in both live and fixed cells, with no wash step required. Cells also can be fixed and permeabilized after staining. LipidSpot™ stains show minimal background staining of cellular membranes or other organelles, unlike dyes like Nile Red. Available with green or deep-red/far-red fluorescence.

LysoView™ 488 and LipidSpot™ 488 are published for labeling lysosomes and lipid droplets in structured illumination microscopy (SIM)

Highlighted Citation: In a publication in Light: Science & Applications, Dong et al. developed a dual-mode high-speed super-resolution microscope for live cells by combining optical diffraction tomography (ODT) with structured illumination microscopy (SIM). This novel technique, deemed super-resolution fluorescence assisted diffraction computational tomography (SR-FACT), was used to acquire ~100 nm resolution images merged with 3D imaging of live cells in real time. The authors validated the technique by visualizing organelles within COS-7 cells labeled by expressed GFP fusion proteins or synthetic fluorescent probes. SIM visualization of LysoView™ 488 and LipidSpot™ 488 were shown to colocalize in the ODT channel with lysosomes and lipid droplets respectively.

Figure 6. Confirmed visualization of cellular lysosomes and lipid droplets from ODT images. The regions enclosed by the dashed yellow boxes on the left are enlarged on the right. Observation of lipid droplets in a live COS-7 cell from ODT images, as confirmed by colocalization with LipidSpot™ 488-labeled structures in the Hessian SIM channel (top row). Observation of lysosomes in a live COS-7 cell from ODT images, as confirmed by colocalization with LysoView™ 488-labeled structures in the Hessian SIM channel (bottom row). Scale bars, 5 µm (left) and 1 µm (right).
Adapted from Dong. D, et al. https://doi.org/10.1038/s41377-020-0249-4 reproduced under the Creative Commons license.

Validated Stains for Imaging Extracellular Vesicles by STORM

Characterizing exosomes and EVs by imaging remains a challenge due to their small size and the resolution limit of light microscopy. ExoBrite™ EV Membrane Stains have been validated for dSTORM by the ONI Nanoimager, allowing the study of fine morphological details and co-staining with EV biomarkers such as tetraspanin proteins CD9, CD63, and CD81.

  • ExoBrite™ 560/585 and 650/665 EV Membrane Stains are validated for dSTORM imaging of EVs
  • Understand more about vesicle morphology and structure
  • Allows antibody co-staining and localization studies with EV biomarkers
Super-resolution dSTORM images of human colorectal cancer cell line derived EVs stained for tetraspanins and with ExoBrite™ stains at 1x concentration. EVs stained with ExoBrite™ 560/585 (blue) and CF®647-labeled CD9, CD63, and CD81 antibody cocktail (purple)(left). EVs stained with ExoBrite™ 650/665 (purple), CF®568-labeled CD63 antibody (blue), and CF®488A-labeled CD9 antibody (yellow)(right). Samples were prepared using the ONI EV Profiler assay and acquired using on an ONI Nanoimager. Data was processed using a beta-release of the online ONI localization microscopy data analysis platform named CODI.

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List of Cellular Stains Validated for Super-Resolution Microscopy

LocalizationCatalog NumberProductAbs/Em (nm)STORMSTEDSIM2-PhotonTIRFOther Applications
Nucleus40046, 40047Hoechst 33342352/458 (with DNA)
40011, 40009, 40043DAPI358/461 (with DNA)
40090Oxazole Yellow Homodimer (YOYO®-1)491/509 (with DNA)
40081NucSpot® Live 488503/518Multiview confocal microscopy
40082NucSpot® Live 650655/681
40061RedDot™2665 (broad)/695 (with DNA)ExM
Membrane /Cell Surface30092MemBrite® Fix 405/430405/430
30093MemBrite® Fix 488/515488/515
30095MemBrite® Fix 568/580568/580
30096MemBrite® Fix 594/615594/615
30097MemBrite® Fix 640/660640/660FLImP
30099MemBrite® Fix 680/700680/700Single-molecule imaging
30101MemBrite® Fix-ST 650/665650/665
30102MemBrite® Fix-ST 667/685667/685
30103MemBrite® Fix-ST 681/698681/698Single-molecule imaging
30104MemBrite® Fix-ST 755/777755/777
30090CellBrite® Fix 488480/513
30107CellBrite® Steady 550562/579
30108CellBrite® Steady 650656/676
30109CellBrite® Steady 685686/708
30022CellBrite® Orange549/565
30023CellBrite® Red644/665
60017DiR748/780
70020, 70022SynaptoGreen™ C4 (FM®1-43)480/598 (in membranes)
Cytoplasm30050ViaFluor® CFSE495/515
Microtubules70063ViaFluor® 647650/675
Lysosomes70067LysoView ™ 488506/532
70059LysoView™ 650650/675
Lipid Droplets70065LipidSpot™ 488427/585
EVs & Exosomes30013ExoBrite™ 560/585562/584
Ion Indicators50033, 50034,
50033-1
Fura-2, AM Ester363/512 (no Ca2+)
335/505 (high Ca2+)
50029Fura-2, AM Ester, 1 mM Solution
50030Fura-2, Pentaammonium Salt
50031Fura-2, Pentapotassium Salt
50032Fura-2, Pentasodium Salt
Oxidative Stress1006110-Acetyl-3,7-dihydroxyphenoxazine
(Amplex® Red)
571/585 (end product)
Cell Tracing80015Lucifer Yellow CH, lithium salt428/536
80026Lucifer Yellow CH, lithium salt, 100 mM in water
80016Lucifer Yellow CH, potassium salt
A check mark indicates the respective dye is compatible for the indicated application. For more information on compatibility of our cellular stains with super-resolution applications please email techsupport@biotium.com

FLImP: Fluorophore localization imaging with photobleaching; SIM: Structured illumination microscopy; STED: Stimulated emission depletion; STORM: Stochastical optical reconstruction microscopy; TIRF: Total internal reflection fluorescence; ExM: Expansion microscopy.

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