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.
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.
CF® Dye Fluorescence Lifetime Data
| Dye | τ (ns) /free acid in PBS pH 7.4, ε (ns) | τ (ns) /S.Ab§ |
|---|---|---|
| CF®405S | 3.88 ± 0.05 | – |
| CF®488A | 4.11 ± 0.05 | 1.705 |
| 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 ± 0.05 | 1.6 |
| CF®750 | 0.58 ± 0.05 | 0.636 |
| CF®790 | 0.39 ± 0.05 | 0.54 |
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.
CF® Dyes Validated for Super-Resolution Microscopy
| Dye | Abs/Em (nm) | STORM | STED | SIM | 2-Photon | TIRF | Other Applications | Features |
|---|---|---|---|---|---|---|---|---|
| CF®405S | 411/431 | ✓ | ExM | • Brighter than Alexa Fluor® 405 | ||||
| CF®405M | 416/452 | ✓ | ✓ | ✓ | ExM | • More photostable than Pacific Blue™ • Excellent choice for SIM imaging |
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| CF®488A | 490/516 | ✓ | ✓ | ✓ | ✓ | ExM | • Less non-specific binding than Alexa Fluor® 488 | |
| CF®535ST | 535/569 | ✓ | • Orange dye designed specifically for STORM imaging | |||||
| CF®555 | 554/568 | ✓1 | • Brighter and more photostable than Cy®3 • Less non-specific binding than Alexa Fluor® 555 |
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| CF®568 | 562/584 | ✓1 | ✓ | ✓ | ✓ | ExM | • 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 |
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| CF®583R | 585/609 | ✓1 | • One of two top-performing dyes specifically designed for STORM with green laser (also see CF®597R) – see publication |
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| CF®594 | 593/615 | ✓ | ✓ | • Significantly brighter than Alexa Fluor® 594 and Texas Red® • Extremely photostable |
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| CF®594ST | 593/620 | ✓ | • Deep-red fluorescent dye designed specifically for STORM | |||||
| CF®597R | 597/619 | ✓1 | • Deep-red fluorescent dye designed specifically for STORM • Top-performing dye specifically designed for STORM with green laser (also see CF®583R) – see publication |
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| CF®633 | 629/650 | ✓ | FIONA, gSHRImp, ExM, SMT | • Significantly brighter than spectrally similar far-red dyes • Far more photostable than Alexa Fluor® 647 |
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| CF®640R | 642/663 | ✓ | ✓ | ✓ | FLImP | • Offers improved brightness and photostability over ATTO 647N and spectrally similar dyes |
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| CF®647 | 652/668 | ✓1 | ExM | • 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 |
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| CF®660R | 662/682 | ExM, SMLM | • Much brighter than Alexa Fluor® 660 • The most photostable 660 nm dye • Validated for use with DNA-PAINT SMLM – see publication |
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| CF®660C | 667/685 | ✓1 | • Much brighter and more photostable than Alexa Fluor® 660 • Ideal for long high-intensity 3D (d)STORM image acquisitions with minimal photobleaching – see publication |
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| CF®680 | 681/698 | ✓1 | Dual-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 |
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| CF®680R | 680/701 | ✓1 | ✓ | ✓ | Single-molecule spectroscopy, SMT | • The most photostable 680 nm dye • Suitable for labeling nucleic acids and small biomolecules |
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| CF®750 | 755/779 | ✓ | • Exceptionally bright and photostable near-IR dye • Patented pegylated dye for superior performance |
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:
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.
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 number | Dye | Specialized applications |
|---|---|---|
| 30092 | MemBrite® Fix 405/430 | SIM |
| 30093 | MemBrite® Fix 488/515 | STED, TIRF, 2-photon microscopy |
| 30094 | MemBrite® Fix 543/560 | N/A |
| 30095 | MemBrite® Fix 568/580 | SIM, STORM, TIRF |
| 30096 | MemBrite® Fix 594/615 | 2-photon microscopy |
| 30097 | MemBrite® Fix 640/660 | FLImP, SIM, TIRF |
| 30098 | MemBrite® Fix 660/680 | N/A |
| 30099 | MemBrite® Fix 680/700 | STORM1, Single-molecule imaging, STED, 2-photon microscopy |
| 30101 | MemBrite® Fix-ST 650/665 | STORM |
| 30102 | MemBrite® Fix-ST 667/685 | STORM |
| 30103 | MemBrite® Fix-ST 681/698 | Single-molecule imaging, STORM |
| 30104 | MemBrite® Fix-ST 755/777 | STORM |
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.
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
List of Cellular Stains Validated for Super-Resolution Microscopy
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.comFLImP: 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.