AF610-X
Alexa Fluor 610-X fluorophore on FISHscope
modeling performance of alternative filter sets using Semrock Searchlight https://searchlight.semrock.com/
20211214U: GM started modeling performace of our current and potential hardware for Alexa Fluor 610-X fluorophore. Had found in Maillard 2020 Chem Sci
FISHscope web page http://confocal.jhu.edu/current-equipment/fishscope
Multiplex page http://confocal.jhu.edu/mctips/multiplex
Maillard 2020 info
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Maillard J et al 2021 Universal quenching of common fluorescent probes by water and alcohols. Chem. Sci. 12: 1352. https://pubs.rsc.org/en/content/articlelanding/2021/sc/d0sc05431c Alexa Fluor 610-X highest Brightness B=121 in H2O (highest Brightness of the 42 dyes), over 2x that of Alexa Fluor 594, B=54 in H2O. https://www.linkedin.com/pulse/maillard2020-chemsci-42-fluorophores-paper-values-george-mcnamara/ H2O vs D2O ... ~10% improvement for AF610-X in D2O ... H2O is a weak quencher (probably the -OH) but at 55 Molar, a lot of H2O (~200 molecules in the "sphere of influence) -- vibrational energy state(s) of red and NIR excited fluorophores overlap with H2O, resulting in some quenching in solution ... 20241217Tue: occured to me that the theme song for experiments using D2O should be the Beach Boys Good Vibrations https://youtu.be/oMfjgQ8ptrE
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Brightness of "far red" and "near near infrared" dyes from Maillard 2020 Chem Sci B Dye Ex Em 39 Cy3B 558 572 |
Inputs
Basics (center column of "quantitatively evaluate ..."):
Fluorophore: Alexa Fluor 610
FL Abs: Alexa Fluor 610 absorption
FL Em: Alexa Fluor 610 emission
Light source: Spectra III 360 575-25 normalized
Detector: CMOS ORCA-FlASH 4.0 V2 (peak QE 82%, our 4.0LT peak QY 70%)
| Dye | Alexa Fluor 610-X | Alexa Fluor -610-X | Alexa Fluor 610-X | ATTO594 |
ATTO594 (Bin Wu likes) |
Cy3.5 | Cy5 (crosstalk) | Cy5 (optimal filter set; Lumencor 635 ex) |
| Filter set | LED-DAPI/FI/TR/Cy5/Cy7-A | LED-mCherry-A (semrock) |
Olympus FMCHE (modelled) |
Olympus FMCHE (modelled) | Olympus FMCHE (modelled) | Olympus FMCHE (modelled) | Olympus FMCHE (modelled) | LED-Cy5-A (Semrock |
| ex | (554) FF01-378/474/554/635/735 | FF01-578/21 | 575/20x | 575/20x | 575/20x | 575/20x | 575/20x | FF01-635/18 |
| em | (595) FF01-432/515/595/681/809 | FF02-641/75 | 595DM | 595DM | 595DM | 595DM | 595DM | FF01-680/42 |
| dm | (573) FF409/493/573/652/759-Di01 | FF596-Di01 | 645/90m | 645/90m | 645/90m | 645/90m | 645/90m | FF652-Di01 |
| Outputs | ||||||||
| Fluorescence Signal (mW): | 1.10x10-6 | 1.35x10-5 | 1.29x10-5 | 7.12x10-6 | 1.10x10-5 | 1.07x10-5 | 1.94x10-6 | 7.54x10-6 |
| Excitation Light Noise (mW): | 3.88x10-9 | 1.51x10-10 | 8.53x10-8 | 8.53x10-8 | 8.53x10-8 | 8.53x10-8 | 8.53x10-8 | 3.32x10-9 |
| Autofluorescence Noise (mW) | 3.25x10-6 | 5.17x10-6 | 5.23x10-6 | 3.03x10-6 | 3.03x10-6 | 3.03x10-6 | 3.03x10-6 | 1.78x10-6 |
| Signal-to-Noise Ratio: | 0.338 | 2.60 | 2.42 | 2.28 | 3.53 | 3.44 | 0.622 | 4.24 |
| Maillard 2020 Brightness (in H2O) of dye | 121 | 121 | 121 | 97 | n/a | 15 | 73 | 73 |
| FS * B (all "e-4") | 1.33 | 1.63 | 15.6 | 6.91 | n/a | 1.61 | 1.42 | 5.5 |
GM notes:
* Semrock Searchlight does not have AF-610-X so using AF610.
* Bin Wu likes AF590 (AF594?), Maillard lacks data on this fluorophore.
* GM calculated Maillard's Brightness values from data in their supplemental Excel file. GM happy to email current version of the modified Excel file.
* It is unclear to GM whether Semrock Searchlight has Extinction coefficient (abs) and quantum yield (fl emission) of every fluorophore, so using Maillard's values for Brightness (last row).
