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Cleavable Fluorescent Probes

Cleavable Probes - Full Chemistry Menu for MERFISH Readout Probes & Cyclic Spatial RNA Imaging

Build multiplexed, error‑robust FISH panels with dye‑labeled readout probes that cleanly detach after imaging. Choose reductive S–S linkers, photocleavable spacers, or enzyme‑labile chemistries—delivered with custom design support, RUO‑to‑GMP‑like production, and full QC.

Speak to Scientist How cleavable readouts work
Overview MERFISH Readout Cleavable Chemistries Workflow Products Technical Workflow FAQ References Speak

Overview

Cleavable fluorescent probe design

Cleavable probes are dye‑labeled oligonucleotides that reversibly attach to a target sequence or to an unlabeled “encoding” probe. After imaging, the dye is removed by a triggered cleavage—commonly S–S reduction, light exposure (photolysis), or enzyme treatment—so the same specimen can be reused across many hybridization rounds. This strategy underlies MERFISH (Multiplexed Error‑Robust FISH) and related high‑plex spatial transcriptomics.

MERFISH readout probe
Disulfide‑cleavable dye
Photocleavable (oNB / coumarin)
Enzyme‑cleavable linkers
Dual‑dye barcodes
QC: HPLC • LC‑MS
10–10,000+ barcodes
Supported per panel (design‑dependent) for high‑plex MERFISH/cyclic FISH.
<10% CV fluor load
Typical batch‑to‑batch dye loading uniformity for consistent rounds.
RUO → GMP‑like scale
Documentation & scale‑up paths to fit R&D through regulated builds.
Why cleavable?
  • Reset fluorescence between rounds—enable large barcode spaces.
  • Lower background by removing residual signals.
  • Compatible with fixed cells, tissues, and many microscopes.
Typical build
  • Short readout oligo (e.g., 20–25 nt) complementary to encoding probe tag.
  • Terminal dye (or dual dyes) via cleavable linker.
  • Synthesis options: DNA, LNA‑mix, 2′‑OMe RNA, or mixed backbones.
QC & docs
  • HPLC/UPLC purification; LC‑MS confirmation.
  • Fluor:absorbance ratio and dye loading report.
  • RUO→GMP‑like: CoA, method summary, and lot record on request.

Selection Guide

Linker type Trigger Speed Pros Considerations Best‑fit uses Code
Disulfide 10–50 mM DTT/TCEP 2–10 min Gentle, dye‑agnostic, widely adopted Requires reducing step; avoid free thiols pre‑imaging MERFISH/cyclic FISH resets [SS‑5′], [SS‑3′]
Photocleavable (oNB/DMNB) UV/violet light <1–2 min Fast on‑scope reset Light control; potential photodamage Time‑critical cycles, automation [PC‑5′], [PC‑3′]
Photocleavable (Coumarin) Violet/near‑UV <1–2 min Lower UV dose vs oNB (setup‑dependent) Optimize fluence Delicate samples [cPC‑5′], [cPC‑3′]
Enzyme‑cleavable Esterase/protease 5–30 min Biocompatible triggers Enzyme sourcing; longer cycles Tissue‑friendly workflows [EC‑5′], [EC‑3′]
Acid‑labile pH < 6 5–15 min Simple buffers Sample tolerance to pH Robust cells & materials [AL‑5′], [AL‑3′]
Base‑labile mild base 5–15 min Orthogonal to acid Backbone compatibility Specialty releases [BL‑5′], [BL‑3′]
Click‑to‑cleave* Bioorthogonal trigger 1–10 min Spatiotemporal control Design‑specific availability Advanced/custom workflows [CtC‑5′], [CtC‑3′]

*Click‑to‑cleave denotes designs where a secondary click reaction initiates cleavage; available as a custom engagement.

MERFISH Readout Probes

In MERFISH, barcoded “encoding” probes bind target RNAs. Across sequential rounds, readout probes carrying cleavable dyes hybridize to the probe barcodes to reveal a subset of bits. After imaging, the dye is cleaved off, returning the sample to a low‑background state for the next readout cycle. Error‑robust codes improve detection accuracy over many rounds.

Barcodes & cycles

Design supports Hamming‑style codes and multi‑color rounds; 8–16+ cycles are common depending on plex and error tolerance.

Signal control

Cleavable linkers keep intensities consistent, reduce carryover, and protect tissue morphology across rounds.

Dye choices

Select from FITC/Alexa 488, Cy3, Atto 550, Alexa 568, Texas Red, Alexa 594, Cy5/Atto 647N, Alexa 647, Alexa 700, etc., balanced to your filters and laser lines.

Tip: Pair readout Tm about 5–8 °C above imaging temperature to minimize off‑rates while keeping wash times short.

Cleavable Chemistries

Below is the full menu in one place. Use this with the Selection Guide table above for quick trigger/speed comparisons.

Reductive (Disulfide)

Dye tethered via –S–S–; cleave with 10–50 mM DTT/TCEP in PBS/TBS (pH 7–8). Gentle and widely adopted for cyclic imaging.

Fast (minutes) Dye‑agnostic Low equipment burden
Photocleavable (oNB/DMNB)

o‑Nitrobenzyl family; sub‑minute resets possible with UV/violet illumination. Protect from stray light between rounds.

Very fast Automation‑friendly Verify phototoxicity
Photocleavable (Coumarin)

Absorbs at slightly longer wavelengths than classic oNB—useful for delicate tissues in some systems.

Gentler spectra LED‑friendly Tune fluence
Enzyme‑Cleavable

Ester or peptide linkers removed by mild enzymes (e.g., esterases/proteases). Prioritize buffer/sample compatibility.

Biocompatible Orthogonal to redox/light Longer cycles
Acid/Base‑Labile

Trityl‑like, acetal/ketal, carbonate, etc., tuned for selective cleavage at low or high pH (check backbone stability).

Simple buffers Orthogonal options Materials / robust cells
Click‑to‑Cleave (Custom)

Bioorthogonal reaction initiates bond scission. Great for tight spatiotemporal control in advanced assays (availability/IP applies).

Highly programmable Custom feasibility
Tip: Start with disulfide if you want a gentle, dye‑agnostic baseline; move to photo or enzyme triggers to fit instrument and tissue constraints.

Technology Workflow

1
2
3
1
Encode

Hybridize barcoded encoding probes to target RNAs (per panel design; include readout binding sites).

2
Readout

Add dye‑labeled readout probes (cleavable); image per round/channel for your codebook.

3
Reset

Trigger cleavage (reductive, photo, or enzyme) to remove fluorescence and prep the next cycle.

✔
  Deliverables. Custom sequences, dye/load confirmation, recommended cleavage conditions, storage guidance, and optional barcode map spreadsheet.

Products & Ordering

Product / Modification Description Typical Use Notes Code
5′‑Dye (Disulfide‑Cleavable) Dye via disulfide at 5′ terminus Cyclic imaging / MERFISH resets Fast, dye‑agnostic [Dye‑SS‑5′]
3′‑Dye (Disulfide‑Cleavable) Disulfide‑tethered dye at 3′ Alternative geometry / dual‑dye Balances sterics [Dye‑SS‑3′]
Photocleavable Dye (oNB/DMNB) o‑Nitrobenzyl family linker Rapid on‑scope reset Protect from stray light [Dye‑PC‑5′/3′]
Photocleavable Dye (Coumarin) Violet‑sensitive coumarin linker Lower UV dose (setup‑dependent) Tune illumination [Dye‑cPC‑5′/3′]
Enzyme‑Cleavable Dye Ester/peptide‑based cleavage Tissue‑compatible workflows Longer cycle time [Dye‑EC‑5′/3′]
Acid‑Labile Dye Cleavage under mild acidity Materials & robust cells Check backbone stability [Dye‑AL‑5′/3′]
Base‑Labile Dye Cleavage under mild base Orthogonal release Backbone compatibility [Dye‑BL‑5′/3′]
Dual‑Dye Cleavable Readout Two dyes per oligo with cleavable strategy High SNR / two‑color rounds Verify crosstalk [2×Dye‑SS/PC]
Biotin/Reporter (Cleavable) Cleavable biotin or hapten (e.g., DIG) Capture‑and‑release assays Orthogonal triggers available [Bio‑SS/PC‑5′/3′]
Customizations: internal dye sites, PEG/hexa‑EG spacers, mixed backbones (LNA or 2′‑OMe), desalting‑to‑HPLC purifications, aliquots, and kit packaging.

Ready to design your Cleavable MERFISH readout Probe?

Ordering checklist
  • Sequences & length, desired Tm
  • Cleavage mode (SS / PC / enzyme / pH / custom)
  • Dyes & channels (e.g., 488, 568, 647)
  • Purification (Desalt, HPLC/UPLC) & scale
  • Buffer constraints (live/fixed, tissue type)
Speak to a Scientist

FAQ

What is a read‑out probe?

A read‑out probe is a short, dye‑labeled oligonucleotide that hybridizes to a docking or readout sequence on your encoding/primary probes. It is imaged during each round and then removed (often via a cleavable fluorescent probe linker such as disulfide, photocleavable, or enzyme‑cleavable) so the next round can proceed. Typical length is 18–25 nt with a Tm slightly above the imaging temperature.

What is a MERFISH probe?

In MERFISH (Multiplexed Error‑Robust FISH), there are two probe types: (1) encoding (gene‑targeting) probes that carry error‑robust barcodes, and (2) read‑out probes that bind the barcode docking sequences and carry fluorophores for sequential imaging. The combination of codebook, rounds, and read‑out probes enables highly multiplexed detection with error correction.

Do you provide branched cleavable read‑out probes?

Yes. We can manufacture branched/multi‑arm read‑out probes (e.g., 2–4 arms) for signal boosting, with each arm optionally carrying a cleavable linker (disulfide, photocleavable, or enzyme‑cleavable). These are delivered with HPLC/UPLC purification and optional LC‑MS, and are available from screening to RUO→GMP‑like scales.

How do I design a MERFISH read‑out probe?

1) Start with your codebook: determine the number of rounds/channels and the docking sequences per barcode. 2) Choose read‑out length & Tm: 18–25 nt is common; aim for a Tm 5–8 °C above imaging temperature in your buffer. 3) Minimize cross‑talk: check specificity across all docking tags; avoid strong secondary structures. 4) Pick dye & linker: select bright, stable dyes (e.g., Alexa/Atto/647N family) and a cleavable linker (disulfide/photo/enzyme) to reset fluorescence between rounds. 5) Optimize attachment: 5′ labeling with a spacer (e.g., TEG) can improve accessibility; confirm wash/cleave conditions. 6) Purification & QC: HPLC/UPLC is recommended; LC‑MS available. We can review your codebook and return a manufacturable bill‑of‑materials for custom MERFISH read‑out probe design.

What length and Tm are recommended for readout probes?

Common designs are 18–25 nt with Tm 5–8 °C above imaging temperature (accounting for ionic strength). LNA or 2′‑OMe motifs can raise Tm for shorter, faster‑washing probes.

How fast is disulfide cleavage vs photocleavage?

Disulfide cleavage with 10–50 mM TCEP/DTT typically completes in a few minutes; photocleavage can be sub‑minute with adequate illumination. Choose based on sample tolerance and instrument setup.

Can I reuse the same readout probe across many rounds?

Yes—readouts can be cycled across rounds as long as carryover is controlled. Cleavable linkers reduce bleed‑through compared with non‑cleavable dyes.

Which dyes are most robust for multi‑round imaging?

Atto 550/647N, Alexa 568/594/647 are popular for brightness and stability. Match to your excitation/emission hardware and verify crosstalk empirically.

Do you provide sequences for MERFISH readout tags?

We can synthesize customer‑provided designs or assist with custom tag sets compatible with your encoding probe architecture and codebook.

Speak to a Scientist

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Key References

  1. Moffitt, J.R., et al. (2016). High-performance multiplexed fluorescence in situ hybridization with error-robust barcoding and clearing. Proceedings of the National Academy of Sciences (PNAS) 113(39):11046–11051. https://doi.org/10.1073/pnas.1617699113
    Note: “Readout probes were purchased from Biosynthesis.” Disulfide-linked readouts for cyclic imaging.
  2. Wang, G., et al. (2018). Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Scientific Reports 8:4847. https://doi.org/10.1038/s41598-018-22297-7
    Note: “Readout probes conjugated to the desired dye via a disulfide linkage were synthesized and purified by Bio-synthesis, Inc.”
  3. Zhang, M., et al. (2023). Molecularly defined and spatially resolved cell atlas of the whole mouse brain. Nature 615:117–124. https://www.nature.com/articles/s41586-023-06808-9
    Note: “These readout probes were synthesized and purified by Bio-synthesis,” dye-conjugated via disulfide linkers.
  4. Bhattacherjee, A., et al. (2023). Spatial transcriptomics reveals the distinct organization of inhibitory and excitatory neurons in human cortex. Nature Neuroscience 26: (pagination varies online). https://www.nature.com/articles/s41593-023-01455-9
    Note: “Readout probes were synthesized by Biosynthesis and contained Cy5 or Alexa750 via a disulfide bond,” enabling reductive cleavage after imaging.
  5. Wang, C., et al. (2019). Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization. PNAS 116(39): 19490–19499. https://doi.org/10.1073/pnas.1903808116
    Note: “All dye-labeled readout probes based on disulfide linkage were obtained from Bio-Synthesis.”
  6. Luce, J.J., et al. (2025). Protocol optimization improves the performance of MERFISH readout probes in tissue. Scientific Reports 15: (early online). https://www.nature.com/articles/s41598-025-17477-1
    Note: “Readout probes … were synthesized by Biosynthesis, Inc.” Discusses non-specific binding and mitigation.
  7. Chaves-Perez, A., et al. (2025). Metabolic adaptations direct cell fate during tissue regeneration. Nature (early online). https://www.nature.com/articles/s41586-025-09097-6
    Note: “Fluorescent conjugated probes were purchased from Bio-Synthesis,” fluorophores attached via disulfide bond.
  8. Moffitt, J.R. (2018). MERFISH – Imaging (protocol v1 PDF). protocols.io / Zhuang Lab. PDF link
    Note: “Prepare readout probes — designed and ordered from Bio-synthesis, Inc. …” Also details cleavage/stripping between rounds.

We can crosswalk your design to literature‑style readout sequences on request.

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Greetings Researchers,

Please be advised that any information, guidelines, writeups, and oral/video/graphic content on our website are intended solely as general guidelines.
It is the researcher's sole responsibility to conduct thorough research on the designs of the products intended for their experiments before submitting
their designs to Biosynthesis for review of production feasibility.
Thank you for your understanding.

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