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Sep 16, 2026
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kb/literature/ gets its first entry: TRITC's lifetime, and G20 stops refusing #2

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19 changes: 19 additions & 0 deletions data/fluorophores.yaml
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Expand Up @@ -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"]
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4 changes: 3 additions & 1 deletion kb/INDEX.md
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Expand Up @@ -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

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260 changes: 260 additions & 0 deletions kb/literature/lifetime-ns-tritc.md
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---
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: <initials>
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.
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