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SUPER-RESOLUTION MICROSCOPY

CF® Dyes with Superior Properties for Super-Resolution
Validated in dozens of publications for STORM, STED, SIM, 2-photon and other specialized applications
Validated for FLIM imaging by Stellaris 8 STED FALCON
Fluorescence lifetime data for CF® Dyes available for FLIM imaging
Specialized Labeling Kits and Antibody Conjugates for STORM
Unique kits and pre-labeled secondary antibody conjugates with a low degree of labeling optimal for STORM
Labeling Kits, Bioconjugates, and Reactive CF® Dyes
Featuring rapid and efficient Mix-n-Stain™ labeling kits, cytoskeleton probes, and probes for other cellular structures
Validated Membrane and Organelle Stains
Featuring MemBrite® Fix cell surface stains developed for STORM imaging
List of Cellular Stains for Super-Resolution
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). 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

Two-color (d)STORM super-resolution image of nuclear pores in a HeLa cell. Nuclear pore complex protein Nup133 (green) was detected with CF®583R-conjugated secondary antibodies (DOL = 4.18), and Nup62 (magenta). (d)STORM was performed in an oxygen scavenging imaging buffer containing 50 mM MEA. Courtesy of Dr. Leonid Andronov, Moerner Laboratory, Stanford University.
Nuclear pore complex protein Nup133 was detected with anti-Nup133 primary antibody (Abcam, ab155990) and Donkey Anti-Rabbit IgG (H+L), Highly Cross-Adsorbed, CF®583R Single Label for STORM (Cat. No. 20795). The images are reconstructed as 2D histograms of single-molecule localizations with a bin size of 12 nm x 12 nm. The color scale represents the number of single-molecule detections per bin. Courtesy of Dr. Leonid Andronov, Moerner Laboratory, Stanford University.

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

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.

 

 

 

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

Dye Abs/Em (nm) STORM STED SIM 2-Photon TIRF Other Applications Features
CF®405S 411/431 ✓ ✓ • Brighter than Alexa Fluor® 405
CF®405M 416/452 ✓ ✓ ✓ • More photostable than Pacific Blue™
• Excellent choice for SIM imaging
CF®440 433/512 ✓ • More photostable than spectrally similar dyes
CF®488A 490/516 ✓ ✓ ✓ ✓ ✓ DNA-PAINT • Less non-specific binding than Alexa Fluor® 488
CF®505 505/519 ✓ • Identical to ATTO 488
CF®535ST 535/569 ✓ • Orange dye designed specifically for STORM imaging
CF®555 554/568 ✓1 ✓1 • Brighter and more photostable than Cy®3
• Less non-specific binding than Alexa Fluor® 555
CF®568 562/584 ✓1 ✓ ✓ ✓ • 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®583R 585/609 ✓1 • One of two top-performing dyes specifically designed for STORM
with green laser (also see CF®597R) – see publication
CF®594 593/615 ✓ ✓ • Significantly brighter than Alexa Fluor® 594 and Texas Red®
• Extremely photostable
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
CF®633 629/650 ✓ FIONA, gSHRImp, SMT • Significantly brighter than spectrally similar far-red dyes
• Far more photostable than Alexa Fluor® 647
CF®640R 642/663 ✓ ✓ ✓ ✓ FLImP • Offers improved brightness and photostability over ATTO 647N
and spectrally similar dyes
CF®647 652/668 ✓1 • 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®660R 662/682 SMLM, DNA-PAINT • Much brighter than Alexa Fluor® 660
• The most photostable 660 nm dye
• Validated for use with DNA-PAINT SMLM – see publication
CF®660C 667/685 ✓1 MINFLUX • 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®680 681/698 ✓1 Dual-color 3D SMLM, MINFLUX • 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®680R 680/701 ✓1 ✓ ✓ Single-molecule spectroscopy, SMT • The most photostable 680 nm dye
• Suitable for labeling nucleic acids and small biomolecules
CF®750 755/779 ✓ • 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. Please contact techsupport@biotium.com for more information.

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 number Dye Specialized
applications
30092 MemBrite® Fix 405/430 SIM, TIRF
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
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

Extracellular vesicles (EVs), including exosomes, are lipid-bound vesicles that are released from cells. EVs display specific surface proteins and can carry nucleic acids and other cargo, allowing them to transfer biological information between cells in different parts of the body. Therefore, EVs are increasingly studied for their potential use in drug delivery and medical diagnostic applications.

ExoBrite™ STORM EV Membrane Stains were developed for STORM imaging of EVs and are available in four CF® Dyes validated for STORM.  ExoBrite™ 560/585 and ExoBrite™ STORM CF®647 have been validated for dSTORM on 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™ STORM EV Membrane Stain advantages:

  • Dive deeper into EV morphology and structure
  • Available in 4 CF® Dyes validated for STORM imaging
  • Allows antibody co-staining and localization studies with EV biomarkers

Super-resolution dSTORM images of human colorectal cancer cell line derived EVs. EVs were stained with anti-tetraspanin antibodies (an anti-CD9/CD63/CD81 cocktail, all conjugated to CF®647, shown in magenta) together with 1X ExoBrite™ 560/585 (in cyan). Samples were prepared using ONI’s EV Profiler Kit and acquired using the Nanoimager S Mark II from ONI (Oxford Nanoimaging, UK). Data was processed using a beta-release version of CODI, ONI’s cloud-based data analysis platform. Scale bars are 500 nm (zoomed out, left panel) and 50 nm for single-EV panels (right). Image courtesy of ONI.

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List of Cellular Stains Validated for Super-Resolution Microscopy

Localization Catalog Number Product Abs/Em (nm) STORM STED SIM 2-Photon TIRF Other Applications
Nucleus 40046, 40047 Hoechst 33342 352/458 (with DNA) ✓ ✓ ✓
40011, 40009, 40043 DAPI 358/461 (with DNA) ✓ ✓ ✓
40090 Oxazole Yellow Homodimer (YOYO®-1) 491/509 (with DNA) ✓
40081 NucSpot® Live 488 503/518 ✓ Multiview confocal microscopy
40082 NucSpot® Live 650 655/681 ✓ ✓ ✓
Membrane /Cell Surface 30092 MemBrite® Fix 405/430 405/430 ✓
30093 MemBrite® Fix 488/515 488/515 ✓ ✓ ✓
30095 MemBrite® Fix 568/580 568/580 ✓ ✓ ✓
30096 MemBrite® Fix 594/615 594/615 ✓
30097 MemBrite® Fix 640/660 640/660 ✓ ✓ FLImP
30099 MemBrite® Fix 680/700 680/700 ✓ ✓ ✓ Single-molecule imaging
30101 MemBrite® Fix-ST 650/665 650/665 ✓
30102 MemBrite® Fix-ST 667/685 667/685 ✓
30103 MemBrite® Fix-ST 681/698 681/698 ✓ Single-molecule imaging
30104 MemBrite® Fix-ST 755/777 755/777 ✓
30090 CellBrite® Fix 488 480/513 ✓
30088 CellBrite® Fix 555 542/570 ✓
30107 CellBrite® Steady 550 562/579 ✓
30108 CellBrite® Steady 650 656/676 ✓
30109 CellBrite® Steady 685 686/708 ✓
30023 CellBrite® Red 644/665 ✓
70020, 70022 SynaptoGreen™ C4 (FM®1-43) 480/598 (in membranes) ✓
Cytoplasm 30050 ViaFluor® CFSE 495/515 ✓
Microtubules 70063 ViaFluor® 647 650/675 ✓ ✓
Lysosomes 70067 LysoView ™ 488 506/532 ✓
70059 LysoView™ 650 650/675 ✓ ✓
Lipid Droplets 70065 LipidSpot™ 488 427/585 ✓
EVs & Exosomes 30115 ExoBrite™ STORM CF®505 505/519 ✓
30116 ExoBrite™ STORM CF®583R 583/609 ✓
30117 ExoBrite™ STORM CF®647 652/668 ✓
30118 ExoBrite™ STORM CF®680 681/698 ✓
Ion Indicators 50033, 50034,
50033-1		 Fura-2, AM Ester 363/512 (no Ca2+)
335/505 (high Ca2+)		 ✓
50029 Fura-2, AM Ester, 1 mM Solution ✓
50030 Fura-2, Pentaammonium Salt ✓
50031 Fura-2, Pentapotassium Salt ✓
50032 Fura-2, Pentasodium Salt ✓
Oxidative Stress 10061 10-Acetyl-3,7-dihydroxyphenoxazine
(Amplex® Red)		 571/585 (end product) ✓
Cell Tracing 80015 Lucifer Yellow CH, lithium salt 428/536 ✓
80026 Lucifer Yellow CH, lithium salt, 100 mM in water ✓
80016 Lucifer 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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