LFI-3751 step-and-hold + FRET

1 · Connection

baud data parity stop addr

Emulation mode — simulated LFI-3751, no hardware

No instrument connected? Turn on Emulation to try the whole interface against a simulated controller — nothing needs to be plugged in.

FT232RL shows as a usbserial-… port. Settings are fixed at 19200 · 8 · N · 1; set addr to the unit's RS-232 address (front panel, default 1).

2 · Instrument state

setpoint—

actual T—

equilibration—

poll every s

Polling reads ACT T and feeds the live actual-T trace on the ramp plot. The unit refreshes ~every 600 ms — don't poll faster than ~1 Hz.

3 · Limits & output

high limit °C low limit °C

allow setpoints to °C (software guard — setpoints outside are refused)

Widen the high limit (≥ your top setpoint) before a 60 °C run. Enabling output drives current to the Peltier — set limits and verify a Read first.

4 · Step-and-hold ramp

start °C end °C step °C initial equil min per-step equil min

#	T (°C)	equil (M:SS)

Planned ramp Actual ramp Recent (zoom)

Planned setpoint staircase vs. cumulative equilibration time.

step —/— · —

Start run → wait for READY → collect spectrum + enter readings (panel 5) → Next. Stop run when finished to save the FRET data and clear for the next experiment. Actual T & equil show live in the sticky header.

5 · FRET readings & live plot

temperature °C tag

Nap direct
427/545

Nap→DiD
427/664

TFPC direct
499/551

TFPC→DiD
499/664

DiD direct
646/664

Flow: Start measurement (marks the time & reminds you to trigger FluorEssence) → run the scan → type the five values → Log. Tab across fields, Enter logs. Skipping Start measurement just timestamps at Log. Log again at the same T to re-measure.

FRET equations & channel definitions

Five single-point readings I(ex/em), in entry order:

I₁ = Nap direct (427/545) — donor, ND pair
I₂ = Nap→DiD (427/664) — sensitized acceptor, ND pair
I₃ = TFPC direct (499/551) — donor, TD pair
I₄ = TFPC→DiD (499/664) — sensitized acceptor, TD pair
I₅ = DiD direct (646/664) — acceptor (shared)

Normalized sensitized acceptor emission (Chaisson Eq. 1) — sensitized acceptor ÷ geometric mean of the donor-direct and acceptor-direct channels:

FRET_ND = I₂ / √(I₁ · I₅)
FRET_TD = I₄ / √(I₃ · I₅)
FRET_R = FRET_ND / FRET_TD

Relative/dimensionless. Readings used as entered — no background or crosstalk subtraction (do that upstream if your protocol requires it).

T	427/545	427/664	499/551	499/664	646/664	ND	TD	FRET_R	tag

Ratio ND & TD auto-flag > % from median

Points = individual acquisitions — filled = accepted, open = rejected; ND ● / TD ■. The line connects the reported per-T value (mean of accepted replicates; outliers auto-flagged when a T has ≥3 rows, and any point manually rejectable — excluded from the value but kept in the record).

6 · Diagnostics (read-only)

Builds and sends the frame for you; reply appears below. No hand-typing, no state changes.

7 · Documentation & links

LFI-3751 User Guide (PDF) — full manual incl. the RS-232 command reference
Fluorolog-3 manual (PDF) — spectrofluorometer operation & FluorEssence reference
Wavelength Electronics — LFI-3751 (5 A Digital) — manufacturer product page: manual, LabVIEW drivers, Benchlink
Web Serial API (MDN) — browser support & troubleshooting

Point the manual link at wherever you host the PDF on heberlelab.com. Add lab-specific links (SOPs, the FRET protocol) here as needed.