The Best Dyes for Super-Resolution
Studies show CF® dyes offer unrivaled performance for super-resolution applications.
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, photostability, 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.
As Lehmann and colleagues report in their publication in the Journal of Biophotonics:
CF® Dye Advantages for Super-Resolution
- Superior brightness, photostability, 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 30 fluorophores across visible spectrum with several near-IR options
- Unique dye options designed specifically for STORM
CF® dyes have been validated for fluorescence lifetime imaging microscopy (FLIM) by the Stellaris 8 STED FALCON microscope
|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®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|
|CF®633||630/650||✓||FIONA, gSHRImp, ExM, SMT|
|CF®680||681/698||✓1||Dual-color 3D SMLM|
|CF®680R||680/701||✓1||✓||✓||Single-molecule spectroscopy, SMT|
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™ Antibody Labeling Kits dramatically simplify the process of preparing fluorescently-labeled antibodies, offering bright and stable conjugates in only 30 minutes. The resulting conjugates offer comparable performance to antibodies labeled with other commercially available labeling kits.
- Label with your choice of 29 CF® dyes in only 30 minutes
- Stable covalent labeling with no purification required
- Compatible with common antibody stabilizers
- Labeling kits designed for Nanobodies® (ie. camelid single variable or VHH domains) also available
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.
- Succinimidyl esters for labeling amine-reactive NHS esters
- Maleimide and MTS CF® dyes for labeling thiols
- Aminooxy and hydrazide reactive CF® dyes for labeling carbonyl groups or carbohydrates
- Azides, alkynes, & BCN CF® dyes for highly specific bioorthogonal labeling
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:
Stains for Membranes, Lysosomes, and Lipid Droplets 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
|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,
|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.
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 email@example.com
List of Cellular Stains Validated for Super-Resolution Microscopy
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.