Quenching Autofluorescence
Last modified by Jussi Kenkkilä on 2024/03/18 10:49
Treatments to Quench Autofluoresence
- MaxBlockTM Autofluorescence Reducing Reagent Kit
- TrueBlackTM Lipofuscin Autofluorescence Quencher
- Sudan Black B (effective after 15 min incubation, maximum effect after 120 min)
- Ammonia/Ethanol
- TrueVIEWTM Autofluorescence Quenching Kit
- Sodium Borohydrate
- Copper Sulfate
- Eriochrome black T
- Chicago (Pontamine) blue
- Trypan Blue
- Tris glycine
- Ammonium chloride
- Pre-bleaching with wide-spectrum light (UV to visible light)
Sources of Autoflouresence
Lipofuscins
- Brown color
- Excitation: UV-violet (345-360 nm)
- Emission: blue-red (450-650 nm)
- Neurons, glial cells, cardiac muscle cells, etc.
- Predominant in post-mitotic cells
- Breakdown product of old red blood cells (“aging” pigment)
- Appears as small punctate intracellular structures
Elastin & Collagen
- Excitation: UV-violet (330-400 nm)
- Emission: blue-green (470-520 nm)
- Blood vessel walls
Flavins
- Excitation: UV-green (360-520 nm)
- Emission: green (500-560 nm)
NAD(P)H
- Excitation: UV-blue (340-460 nm)
- Emission: violet-blue (440-470 nm)
Fixative-Induced Fluorescence
- Excitation: UV-violet (355-435 nm)
- Emission: violet-blue (420-470 nm)
- Aldehyde fixatives react with amines and proteins
- Block with glycine/lysine
- Avoid aldehydes
- Pre-bleach for 12-48 h prior to labeling
(Autofluoresence: Causes and Cures from Wright Cell Imaging Facility)
Comparison of Efficacy
Test on Mouse Adrenal Cortex Tissue
Reduction in autofluorescence. NB! Trypan Blue did not reduce autofluorescence at 488 nm excitation but shifted it to longer wavelengths.
| Method | Excitation at 405 nm | Excitation at 488 nm |
|---|---|---|
| MaxBlockTM Autofluorescence Reducing Reagent Kit | 95% | 90% |
TrueBlackTM Lipofuscin Autofluorescence Quencher | 93% | 89% |
| Sudan Black B | 88% | 82% |
| Ammonia/Ethanol | 70% | 65% |
| TrueVIEWTM Autofluorescence Quenching Kit | 70% | 62% |
| Copper (II) Sulfate | 68% | 52% |
| Trypan Blue | 12% | 0% |
(Characterizing and Quenching Autofluorescence in Fixed Mouse Adrenal Cortex Tissue)
Test on Formalin-fixed Paraffin-embedded Human Respiratory Tissue
Relative scores for the tested methods, 3 is best, 0 is worst.
| Method | Reduction in Autoflorescence | Low Interference to Other Channels | Ease of Use | Total |
|---|---|---|---|---|
| Trypan blue | 0 | 3 | 3 | 6 |
| Eriochrome black T | 3 | 1 | 3 | 7 |
| Sudan black B | 3 | 0 | 0 | 3 |
| Chicago (Pontamine) blue | 2 | 2 | 3 | 7 |
| Sodium borohydrate | 3 | 3 | 2 | 8 |
| Tris glycine | 1 | 3 | 2 | 6 |
| Ammonium chloride | 0 | 3 | 2 | 5 |
| Ammonia ethanol | 2 | 3 | 3 | 8 |
| UVB transillumination | 1 | 3 | 2 | 6 |
Notes on channel interference
Eriochrome black T
- DAPI visibility reduced
- Rhodamine red autofluorescence increased
- Bright field shows black residue
Sudan black B
- DAPI visibility reduced
- Bright field has black residue and clarity is poor
- Boils under multi-photon excitation
Chicago (Pontamine) blue
- DAPI visibility reduced