FLIM startup, from optomicroquant course

Gather materials

Rhodamine110 solution, slower lifetime: used for comparing the signals between parallel and perpendicular detectors, and for calibrating the system at the start (comparing to historical values)

Erythrosin solution + KI (saturated), has an extremely short lifetime, used for measuring our IRF

40x water objective, dedicated to spectroscopy (remove DIC filters from it!)

Number 1.5 coverslips (system is calibrated for that)

Laser power meter, to determine laser dosage at the objective lens: should not exceed 1 µW!

Black-paper cover for over the sample area, blocking out environmental light

ZEN first, set up a simple FCS measurement

Prepare a coverslip with a droplet of rhodamine, and find focus using the edge of the drop (that is a sharp line, then go into the drop a bit and start measuring)

Perform a continuous FCS measurement existing pre-set settings: 488 nm, point-scan, cw laser (argon)

this happens at 500 KHz repetition (of detector), but is with CW laser so you can't draw lifetime conclusions

Get diffusion time. During the course, we saw 32 µs for rhodamine.

Reveal count rate: how many events per molecule do we detect in the setup?

Correction collar: Maximize this number by adjusting the objective correction collar. That is the optimal correction.

SymPhoTime first, start observing photons

Make sure the relevant PC is also switched on...

Create a new workspace ("file" menu), make sure it is saved in the right location

Select "point" for a point-measurement. We are running a parked laser using ZEN already.

The software now collects data all the time. You can open the individual detectors (there are 4) count rates window, to make them bigger (double click)

Inspect detector performance: how do the count rates compare? For the GFP detectors (parallel, perpendicular) we find (240000, 230000) counts.

ZEN and SymPhoTime, set up for acquiring FLIM data, checking channels

Use pre-set settings to pick the 485 pulsed laser (diode), at 60% intensity, point measurement. Then click start!

At this point, you can measure laser power. The attenuator knobs (course, fine) on the laser combiner box, can be used to correct the laser power to 1 µW or less.

Regardless of power metering, using the laser power attenuators should lead to a visible change in counts in the live displays on symphotime. Confirm with the students.

Set up a first 10-second measurement in SymPhoTime, have a cursory look at the correlation curves of GFP and RFP-channel signals. By now, make sure to have familiarized yourself with which channel is which (1, 2, 3, 4 correspond to GFPpar GFPperp, RFPpar RFPperp)

Compare intensities between channels. Are they close? Yes, any small differences that appear can be corrected in analysis steps. Trying to adjust the beam splitter usually makes it worse anyway.

Get a measured IRF using Erythrosin

Prepare a droplet of Erythrosin+KI solution on top of a coverslip, mount sample onto microscope.

Start experiment in ZEN, start detection in SymPhoTime, for a 10-sec experiment

Look at count rates! Compare parallel to perpendicular signals. We found for