diff --git a/data/fluorophores.yaml b/data/fluorophores.yaml index a94df9d..5855d47 100644 --- a/data/fluorophores.yaml +++ b/data/fluorophores.yaml @@ -381,8 +381,27 @@ fluorophores: em_fwhm_nm: 45 ext_coeff_M1cm1: 85000 quantum_yield: 0.28 + lifetime_ns: 2.2 photostability: medium verified: false + note: > + lifetime_ns=2.2 is the free dye in PBS (Nanguneri 2014, Microsc Res Tech + 77:510, doi 10.1002/jemt.22349), filed as literature -> + kb/literature/lifetime-ns-tritc.md. Independent cross-check in a + different solvent: 1.58 ns in ethanol (Brismar 1995, J Histochem Cytochem + 43:699). The aqueous value is the one used because this instrument's + samples are aqueous; the two land 1.4x apart and agree in magnitude, not + to a figure. + + The conjugated forms differ and the two papers disagree about which way: + TRITC-phalloidin 2.3 ns (flat, Nanguneri) versus TRITC-IgG 3.32 + 1.4 ns + (up, and double-exponential, Brismar). This single name spans all of + them, so check which form is in the sample before relying on the number. + + G20 is unaffected by the spread at this instrument's imaging powers: its + boundary sits at 5.6e4 W/cm^2 for 2.2 ns, and Spectra>Green at 20x gives + 153 W/cm^2 over a 100x100 um field. Which value is filed starts to matter + only near a diffraction-limited focus. AlexaFluor555: aliases: [AF555, Alexa555, "Alexa Fluor 555"] diff --git a/kb/INDEX.md b/kb/INDEX.md index 88f7592..188b5a6 100644 --- a/kb/INDEX.md +++ b/kb/INDEX.md @@ -112,12 +112,14 @@ One hardware run each, before it happens — 05 §6 stage 5. - **[2026-09-15-drag-calibration-stiffness-vs-size](plans/2026-09-15-drag-calibration-stiffness-vs-size.md)** · 2026-09-15 · planned How does the trap stiffness of this instrument depend on particle size, and how large is the near-wall drag correction, measured by Stokes drag at one laser power -## `kb/literature/` — 1 entries +## `kb/literature/` — 2 entries Published values nobody here has measured. - **[literature-index](literature/README.md)** · living, since 2026-08-28 · index What published values are filed here, and what may a literature value be used for that a measurement may not? +- **[lifetime-ns-tritc](literature/lifetime-ns-tritc.md)** · 2026-09-16 + What fluorescence lifetime should be assumed for TRITC so that G20 can say whether emission is still linear in power, and under what conditions was the published value obtained ## `kb/sessions/` — 2 entries diff --git a/kb/literature/lifetime-ns-tritc.md b/kb/literature/lifetime-ns-tritc.md new file mode 100644 index 0000000..dfbd8db --- /dev/null +++ b/kb/literature/lifetime-ns-tritc.md @@ -0,0 +1,260 @@ +--- +id: lifetime-ns-tritc +quantity: "data/fluorophores.yaml > TRITC > lifetime_ns" +question: "What fluorescence lifetime should be assumed for TRITC so that G20 + can say whether emission is still linear in power, and under what conditions + was the published value obtained" +source: literature +citation: "Nanguneri S, Flottmann B, Herrmannsdorfer F, Kuner T, Heilemann M. + Single-molecule super-resolution imaging by tryptophan-quenching-induced + photoswitching of phalloidin-fluorophore conjugates. Microsc Res Tech + 77(7):510-516, 2014. -- Corroborated in a different solvent by: Brismar H, + Trepte O, Ulfhake B. Spectra and fluorescence lifetimes of lissamine + rhodamine, tetramethylrhodamine isothiocyanate, Texas Red, and cyanine 3.18 + fluorophores. J Histochem Cytochem 43(7):699-707, 1995." +doi: "10.1002/jemt.22349" +doi_corroborating: "10.1177/43.7.7608524" +filed_by: +date: 2026-09-16 +confidence: low +evidence: assumed +gate: G20 +scope: "TRITC excited in its 547 nm absorption band, on this instrument's + imaging paths and at their measured powers. NOT for a diffraction-limited + focus: the value's headroom under G20 is large at widefield irradiance and + shrinks toward the tweezers regime (see What it supplies). Not for the ATPS + phases or a sucrose-loaded buffer until the medium is on record -- + [[sample-medium-refractive-index]]." +applies_to_systems: [current-spectra, current-laser] +measured_on: "**Filed value (Nanguneri 2014):** free TRITC diluted in **PBS** + to 1 uM, lifetime spectrometer (PicoQuant FluoTime 100 Compact), pulsed + (5 MHz) laser diode at 575 nm, lifetimes from the manufacturer's software. + Optical density kept below 0.05 `to guarantee linearity in the absorption and + emission processes`. Temperature at the sample: not stated. + **Corroborating value (Brismar 1995):** free TRITC in **ethanol** (that + paper's other three dyes in distilled water), confocal laser scanning + microscope, TCSPC, pulsed dye laser at 572 nm, x10 NA 0.30, cooled (-20 C) + PMT, 0.2 mm glass capillary; mean power at the specimen below 0.5 mW + unattenuated and typically ~10 uW, which that paper states is `well below + those reported to cause saturation of the fluorophores`. **Both are + unsaturated-regime lifetimes, which is the regime G20's linear assumption + needs.**" +review_after: 2027-09-16 +supersedes: null +superseded_by_measurement: null +--- + +> **`filed_by` is unfilled deliberately.** It attests that a person checked the +> citation against the paper, and the draft this came from was produced by a +> tool that cannot give that signature. Everything else below is quoted from +> the two papers, from `data/*.yaml`, or from a run of this repository's own +> `photo` gate. +> +> **The `## What it supplies` output needs the `photo/cli.py` fix** in the pull +> request this one depends on. Without it `python -m photo.cli` raises +> `TypeError` on every invocation, so a reviewer cannot reproduce the +> before/after below. + +## Verdict + +**2.2 ns**, free TRITC in **PBS** (Nanguneri 2014). *"For the TRITC +fluorophore, we measured a fluorescence lifetime of 2.2 ns for the free +fluorophore, 1.7 ns in the presence of 30 mM tryptophan, and 2.3 ns for the +dye-phalloidin conjugate."* Reported to two significant figures with no +uncertainty stated. + +**Two independent papers, two instruments, two solvents**, which is the +condition `confidence:` is keyed to here: + +| Source | Form | Solvent | tau | +|---|---|---|---| +| **Nanguneri 2014** | free dye | **PBS** | **2.2 ns** | +| Nanguneri 2014 | phalloidin conjugate | PBS | 2.3 ns | +| Nanguneri 2014 | free dye + 30 mM tryptophan | PBS | 1.7 ns | +| Brismar 1995 | free dye | **ethanol** | 1.58 ns (SD 10 ps) | +| Brismar 1995 | IgG conjugate | not restated | 3.32 ns + 1.4 ns | + +**The PBS value is filed because this instrument's samples are aqueous.** The +1.58 ns is the same quantity in ethanol and is kept as the independent +cross-check: two techniques -- a CSLM/TCSPC and a PicoQuant lifetime +spectrometer -- at 572 and 575 nm, landing 1.4x apart across a solvent change +that would be expected to move it. `confidence: low` stands rather than rising: +they agree in magnitude, not to a figure. + +**The free-dye form is filed, not a conjugate**, because `TRITC` in this +registry is not an immunoconjugate: `data/particles.yaml` records it as an +explicit **stand-in** for a rhodamine/TRITC-class bead dye whose vendor +excitation *and* emission specs were both measured wrong here, so +`ex_peak_nm`/`em_peak_nm` stay null and *"channels use an explicit TRITC +stand-in."* + +**And the two papers disagree about what conjugation does**, which is worth more +than either number: Brismar's IgG conjugate goes *up* 1.74 ns and becomes +double-exponential, while Nanguneri's phalloidin conjugate stays flat +(2.2 -> 2.3 ns) and the paper attributes its quenching to a static +dye-tryptophan complex rather than to binding. So "conjugated" is not one +condition, and a bead matrix is a third thing again -- see Transfer conditions. + +Nanguneri's decays for TRITC are single-valued as reported; the biexponential +fits in that paper are for ATTO 488 (3.0 ns / 82 %, 0.7 ns / 18 % with +tryptophan). `photo/dose.py` takes one scalar, so nothing has to be collapsed +here. + +## What it supplies + +- **Field:** `data/fluorophores.yaml > TRITC > lifetime_ns`, currently `null`. + 6 of 17 dyes in the registry are missing it. +- **Gate:** `G20`, saturation / triplet shelving, in `photo/checks.py`, against + `excited_state_fraction_max: 0.1`. +- **Before this entry:** `photo/gate.py` `_missing_inputs()` emits + `missing.lifetime` at severity `fail`. Run against the registry as it stands: + + TRITC @ 555 nm 153.0 W/cm^2 -> BLOCKED + feasibility: UNKNOWN confidence: none advances: NO + [FAIL] missing.lifetime + + No margin, no bottleneck, no difficulty grade for any TRITC setting. +- **After**, same command with `--lifetime-ns 2.2`: + + TRITC @ 555 nm 153.0 W/cm^2 -> PASS + feasibility: ROUTINE evidence: assumed confidence: low advances: NO + 10.00 perturbation.saturation ############################## + + G20 computes, and `photo.dose.saturation_irradiance_w_cm2(85000, 2.2, 555)` + becomes available too -- **which is why TRITC and not one of the other five.** + TRITC already carries `ext_coeff_M1cm1: 85000`; `SYTO61` and `DragonGreen` + carry no extinction coefficient, so a lifetime alone would move them from one + missing input to another, and `DEX647`, `SA647` and `Phal647` share one + nominal 270000 across three different conjugates. + +**And the margin says which of the two rows matters, which is: neither.** +G20 crosses `f = 0.1` at `k_ex*tau = 1/9`, so the boundary irradiance is + +| candidate | G20's boundary | +|---|---| +| 1.58 ns (Brismar, ethanol) | 7.75 x 10^4 W/cm^2 | +| **2.2 ns (filed)** | **5.57 x 10^4 W/cm^2** | +| 2.3 ns (phalloidin conj.) | 5.33 x 10^4 W/cm^2 | +| 3.32 ns (IgG conj.) | 3.69 x 10^4 W/cm^2 | + +The measured power at the sample for the band that excites TRITC -- +`Spectra > Green`, 15.3 mW at 20x, `data/light_sources.yaml` -- is **153 W/cm^2** +over a 100x100 um field, **241x below** the strictest of the four, and +1.5 x 10^4 W/cm^2 even over a 10x10 um field. So the saturation margin is +clamped at its ceiling for every candidate, and **the whole spread -- 1.58 to +3.32 ns, a factor of 2.1 -- does not reach the verdict at this instrument's +imaging powers.** It would reach it in a diffraction-limited focus, which is +the tweezers regime and not this gate's. + +That is the honest size of what this entry buys: it converts a refusal into a +computed margin with two to three orders of magnitude of headroom, and it does +not claim to resolve the number to better than a factor of two. **Both are +worth stating** -- the second is why the first is safe. + +## Transfer conditions + +What has to hold for this number to apply here: + +1. **The excited-state fraction stays far below 0.1.** It does, by the table + above -- which is what makes conditions 2 and 3 tolerable rather than + disqualifying. Read the other way: at a tight focus this entry stops being + safe before it stops being available. +2. **The illumination is in the 547 nm absorption band** and the setting is one + whose power at the sample is on record (`Spectra > Green` or + `LUN-F-XL > 561`, at 4x / 10x / 20x). Elsewhere the irradiance is itself + assumed and the comparison above cannot be made. +3. **The instrument class matches, unusually.** The paper's lifetimes were + recorded on a confocal laser scanning microscope and this instrument's + CSU-W1 is one. That narrows the transfer gap; a 1995 confocal is still not + this confocal. + +What is known **not** to hold: + +1. **~~The solvent.~~ Resolved by the second paper.** This read *"free TRITC + was dissolved in ethanol and this instrument's samples are aqueous"* while + Brismar was the only source. Nanguneri measures the same quantity in **PBS**, + which is the condition here, so the filed value no longer carries a solvent + mismatch -- and the 1.58/2.2 gap is now evidence about the solvent rather + than a gap in the entry. +2. **The environment is a third one the paper did not measure.** Its rows are + free dye, IgG-conjugated, and in tissue; MS's `TRITC` is a stand-in for a dye + **embedded in a polystyrene bead**. The paper's own conclusion is that + *"changes in spectra and fluorescence lifetimes may be a useful tool to gain + information about the chemical environment of the fluorophores"* -- the + quantity is being proposed as an environment probe, and this is a different + environment. There is no `TRITC in tissue` row at all; TRITC was left out of + those tests. +3. **The registry row it will sit beside is a catalogue nominal.** + `data/fluorophores.yaml`'s header says of every row: *"The values below are + all catalog nominal values ... parametric band(peak+FWHM) gets the peak + position right but the wings wrong."* So the `ext_coeff_M1cm1: 85000` this + lifetime is multiplied by in `saturation_irradiance_w_cm2` is itself + unverified. The product of two nominals is not better than either. +4. **`TRITC` is one name over several things.** `aliases: [TRITC555, tritc]` + does not distinguish free dye from conjugate from bead -- and the two papers + do not even agree on what conjugation does to the lifetime (up 1.74 ns on + IgG; flat on phalloidin). One registry name spans values the sources measure + as different, by different amounts, in different directions. + +## Why this does not advance a verdict + +`evidence: assumed`, so `advances` stays `NO` regardless of the margin +([04 §1](../../docs/04-decision-engine.md)) -- confirmed in the run above, which +reports `advances: NO` beside a margin of 10.00. This entry lets `G20` compute +instead of refuse. It does not claim anyone measured TRITC's lifetime on this +instrument, in this bead, under this illumination. + +## Falsification conditions + +1. **A saturation power series on this instrument, which it can actually do.** + A direct lifetime needs time-resolved detection this setup does not have, so + the falsifier is the quantity G20 is really about: acquire emission versus + power on a fixed TRITC-class field and find where emission departs from + linear. `k_ex*tau = 1` at excited-state fraction 0.5, so the departure fixes + `tau` given the irradiance -- and the irradiance is available, because + `data/light_sources.yaml` carries **measured** `power_at_sample_mw` for both + usable bands. **The table above says what this run would cost:** the + departure sits near 10^5 W/cm^2, so a widefield series will not reach it and + the measurement needs a deliberately tight focus. On arrival: move the value + to `kb/calibrations/`, set `superseded_by_measurement:`, and G20 can advance. +2. **A further published value that moves `k_ex*tau` across 1/9 at an + irradiance this instrument uses.** Not a percentage: the four values already + in hand span 2.1x and none of them changes the verdict here, so a + disagreement has to be larger than the spread already recorded to matter. +3. **Transfer condition 1 turning out to be false** -- any setting that puts the + sample near 10^4 W/cm^2 or above, at which point which row was filed starts + to matter and none of them is the right environment. + +## Related + +- `photo/checks.py` -- G20 and `excited_state_fraction_max: 0.1` +- `photo/dose.py` -- `excited_state_fraction`, `saturation_irradiance_w_cm2` +- `photo/gate.py` -- `_missing_inputs()`, which emits `missing.lifetime` today +- `photo/cli.py` -- **broken**; see the note at the top of this entry +- `data/fluorophores.yaml` -- the field, and the catalogue-nominal header +- `data/particles.yaml` -- where `TRITC` is recorded as a stand-in, and where + the vendor's excitation and emission specs are both disproved +- `data/light_sources.yaml` -- the measured `power_at_sample_mw` that makes + falsification condition 1 available and sizes it +- `kb/expertise/sample-medium-refractive-index.md` -- why ATPS and sucrose are + outside `scope` +- `kb/decisions/2026-09-09-g10-photobleaching-removed.md` -- G10 was the live + case `kb/literature/README.md` still describes; G20 is the live case now, and + the same commit left `photo/cli.py` broken + +--- + +**On the second paper's other numbers.** Nanguneri 2014 also reports 939 photons +per single-molecule localization for TRITC and 822 for ATTO 488, an on/off rate +ratio of 1023 for phalloidin-TRITC, and quantum yields dropping to 27 % (TRITC) +and 48 % (ATTO 488) on conjugation. **None of those is a field +`data/fluorophores.yaml` carries**, and MS's rule for that case is explicit: *"If +no gate consumes a number from it, it is not knowledge this system can act on. +Method papers that shape a judgment belong in `kb/expertise/` as a captured +prior, with the paper cited inside."* The 27 % quantum-yield drop on conjugation +is the strongest candidate there, since `quantum_yield: 0.28` is in the registry +and is a catalogue nominal for the unconjugated dye. + +It reports **no lifetime for Alexa Fluor 647**, so it does not supply +`Phal647`'s missing `lifetime_ns` -- which is what this draft's own README +guessed it was for, before the paper was read.