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Custom MERFISH read-out probes with cleavable fluorescent chemistries

Dye-labeled read-out probes, cleavable linkers, bDNA amplifier support, HPLC/UPLC purification, optional LC-MS QC, and per-round kitting for spatial transcriptomics workflows.

MERFISH Cleavable Dyes Disulfide Linkers Photocleavable Options bDNA Amplifier Support HPLC / LC-MS QC Per-Round Kitting
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Overview

Custom MERFISH read-out probes for cyclic spatial transcriptomics workflows

Bio-Synthesis offers custom MERFISH read-out probes with cleavable fluorescent dye chemistries, orthogonal barcode-compatible designs, optional bDNA amplifier support, and flexible purification workflows for spatial transcriptomics applications. Our team supports dye selection, linker chemistry, cyclic imaging workflows, pooled probe formats, and QC documentation for MERFISH, cyclic FISH, and multiplex RNA imaging assays.

What we make

  • Dye-labeled MERFISH read-out probes
  • Disulfide, photocleavable, or enzyme-cleavable designs
  • Single-dye or dual-dye configurations
  • Per-round pooled probe mixes

Why it matters

  • Cleaner signal reset between imaging rounds
  • Better round-to-round consistency
  • Reduced handling through kitted mixes
  • QC documentation for assay development
Best for: MERFISH, cyclic FISH, spatial transcriptomics, single-cell RNA imaging, multiplex RNA detection, and bDNA-amplified imaging workflows.
Image Reset
18–25 nt

Typical read-out length

SS / PC

Cleavable linker options

QC

HPLC, UPLC, LC-MS options

Kits

Per-round pools available

MERFISH Readout Probes

Read-out probe design, dyes, and QC

Read-out probes are short oligos, typically about 20 nt, that are complementary to encoding-probe docking tags. Dyes are commonly attached through a cleavable linker at the 5′ terminus or, when needed, the 3′ terminus for alternate geometry or dual-dye designs.

Design

Use orthogonal 20–25-mers with Tm about 5–8 °C above imaging temperature. Screen docking tags for cross-homology across the full codebook.

Dye Selection

Common choices include Alexa Fluor 568/594/647/700/750, Atto 550/647N, Cy5, and other microscope-compatible dyes.

QC

HPLC or UPLC purification is recommended. LC-MS, dye loading review, and cleavage verification can be added when appropriate.

Design tip: Add a short spacer, such as TEG, between dye and oligo to improve accessibility, wash behavior, and imaging consistency.
Probe Formats

Core MERFISH read-out probe options

Keep the product offering simple: dye-labeled read-outs, cleavable linker variants, and optional kitting aligned to the customer’s codebook.

Standard Read-Out Probes

Short dye-labeled oligos complementary to encoding-probe docking tags for cyclic imaging rounds.

Cleavable Read-Outs

Disulfide, photocleavable, enzyme-cleavable, or custom linker options for signal removal between rounds.

Kitted Probe Pools

Per-round pooled mixes matched to the customer’s MERFISH codebook, imaging channels, and workflow.

bDNA Amplifier Support

Branched DNA (bDNA) Amplifiers — Layered Signal Amplification

bDNA amplifier probe sets increase MERFISH signal by building a layered hybridization structure on the target complex. A pre-amplifier binds the capture/encoding scaffold, multiple amplifier branches bind to the pre-amplifier, and each amplifier recruits labeled read-out probes to generate a brighter signal without PCR amplification of the target.

  • No target PCR — signal amplification only
  • Layered hybridization: pre-amplifier → amplifier → labeled probes
  • Higher signal-to-noise for weak or low-abundance targets
  • Multiplex-friendly orthogonal amplifier sets
  • Adjustable branch factor and label density
  • Compatible with cleavable MERFISH read-out probes
Simple flow: Target → Encoding Probe → Pre-Amplifier → Amplifier → Labeled Read-Out Probes.

Signal Gain

Recruit multiple fluorescent read-outs per target complex.

Multiplex Ready

Use orthogonal amplifier/read-out designs for multi-target assays.

Low Targets

Improve detection when transcripts are weak, sparse, or difficult to image.

Layered bDNA Signal Amplification
Products & Ordering

MERFISH read-out probe products and ordering options

Choose the dye/linker format that best fits your cyclic imaging workflow, reset chemistry, tissue compatibility, and microscope channels.

Product / Modification Description Typical Use Notes Code
5′-Dye Disulfide-Cleavable Dye via disulfide at the 5′ terminus. Cyclic imaging / MERFISH resets Fast, widely used, dye-agnostic option. [Dye-SS-5′]
3′-Dye Disulfide-Cleavable Disulfide-tethered dye at the 3′ terminus. Alternate geometry / dual-dye designs Useful when sterics or probe orientation matter. [Dye-SS-3′]
Photocleavable Dye oNB, DMNB, coumarin, or related photo-labile linker. Rapid on-scope reset Requires light-dose and sample-sensitivity review. [Dye-PC-5′/3′]
Enzyme-Cleavable Dye Ester, peptide, or enzyme-labile linker design. Tissue-compatible workflows Useful when chemical cleavage is less desirable. [Dye-EC-5′/3′]
Dual-Dye Cleavable Read-Out Two dyes configured with a cleavable strategy. Higher SNR or two-color rounds Verify crosstalk and channel balance. [2×Dye-SS/PC]
bDNA Amplifier Probe Set Nested branched oligos that multiply fluor binding sites. Low-abundance targets / high-background samples Two- or three-stage designs using pre-amp and amp layers. [bDNA-Amp-Set]
Cleavable Chemistries

Choose the trigger that best fits your sample and microscope

Disulfide is the field-standard reset option for MERFISH and cyclic FISH. Photocleavable and enzyme-cleavable triggers are useful alternatives when fast on-scope reset or sample-specific compatibility is needed.

Linker Trigger Speed Pros Considerations Best-fit Uses Code
Disulfide 10–50 mM TCEP/DTT 2–10 min Gentle, dye-agnostic, widely adopted 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 Phototoxicity management Time-critical cycles [PC-5′/3′]
Photocleavable (Coumarin) Violet / near-UV <1–2 min Lower UV dose Optimize fluence Delicate tissues [cPC-5′/3′]
Enzyme-Cleavable Esterase / protease 5–30 min Biocompatible reset Requires enzyme sourcing Live-cell adjacent or sensitive samples [EC-5′/3′]
Acid / Base Labile pH shift 5–15 min Simple buffer trigger Check oligo and dye stability Robust fixed samples [pH-Labile]
Technology Workflow

MERFISH technology workflow

MERFISH uses sequential hybridization and imaging rounds to identify RNA molecules by barcode. Each cycle adds fluorescent read-out probes, captures the image, and removes the signal before the next round.

1

Encode

Hybridize encoding probes containing docking tags and target-binding regions.

2

Read-out

Add fluorescent read-out probes that bind selected docking tags for the current imaging round.

3

Reset

Trigger cleavage by reductive, photo, or enzyme chemistry to remove fluorescence and prepare the next cycle.

Deliverables: custom sequences, dye/linker configurations, recommended cleavage conditions, storage guidance, and lot-linked QC.
Workflow

Workflow: cyclic hybridize → image → strip

This practical workflow summarizes the major steps customers typically plan around when ordering MERFISH read-out probes and cleavable dye formats.

Step Description Notes
1. Barcode & Encoding Probe Design Assign robust bit barcodes per gene and synthesize encoding probes carrying readout sites. Use GC-balanced bit sites and error-robust layouts.
2. Primary Hybridization Hybridize encoding probes to fixed cells or tissue and wash to reduce background. Keep temperature and salt conditions gentle to preserve morphology.
3. Readout Hybridization Hybridize fluorescent readouts that match selected bit sites across channels. 5′ cleavable linkers such as disulfide are common for fast resets.
4. Imaging Acquire multichannel images and detect puncta for each color. Maintain exposure uniformity; include fiducials if needed.
5. Fluorophore Removal Cleave dyes with TCEP/DTT or use photo, enzyme, or alternate strip chemistry. Leave encoding probes intact and confirm low residual signal.
6. Subsequent Rounds Repeat hybridize, image, and strip until all bits are read. Log per-round QC, drift, and background.
7. Barcode Decoding Call presence or absence across rounds and channels to decode each barcode. Error-robust decoding helps mitigate dropouts.
8. Spatial Analysis Assign transcripts to cells and compute cell types, neighborhoods, and pathways. Export single-cell tables, expression maps, and region statistics.
How to Order

What to include for a faster quote

Probe Design

  • Read-out sequence or docking tag
  • Desired length and Tm range
  • 5′ or 3′ dye placement

Chemistry

  • Dye/channel selection
  • Cleavage mode
  • Spacer or linker requirements

Delivery

  • Scale and purification
  • Tube, plate, or pooled format
  • QC and documentation needs

Frequently asked questions

FAQ

What is a MERFISH read-out probe?
A MERFISH read-out probe is a short dye-labeled oligonucleotide that binds a docking sequence on encoding probes during an imaging round.
What is the common read-out probe length?
 Many MERFISH read-out probes are around 18–25 nucleotides, but length and Tm should be matched to the imaging buffer and workflow.
Which cleavage chemistry should I use?
 Disulfide cleavage is common for cyclic imaging. Photocleavable and enzyme-cleavable approaches can be considered for specialized workflows.
Can Bio-Synthesis help with bDNA amplifier probe sets?
 Yes. Bio-Synthesis can review pre-amplifier, amplifier, branch factor, orthogonal amplifier set, and labeled read-out probe requirements for layered bDNA signal amplification workflows.
Can probes be delivered as pooled round mixes?
 Yes. Read-out probes can be delivered individually or as per-round pooled mixes aligned to a MERFISH codebook.
What QC options are available?
 Common QC options include HPLC or UPLC purification, OD quantitation, dye loading review, and LC-MS when applicable.
More FAQ'sAll FAQ's
Contact & Quote Request

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MERFISH Read-Outs
Cleavable Dyes
QC + Kitting
MERFISH Read-Out Probes Cleavable Fluorescent Imaging Support

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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
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