Re: What is the right way to record an IRF in FLIM system?

I have been using the system for a while but got much less fret
efficiencies than in the more conventional methods on same constructs.
After eliminating some "biological" reasons I gave a deeper view in the
IRF and understood that I have some optical problems. The system was
not in use before, so we did not come across these problems before.
From all immersion oils I had that I could use for tirf - all gave auto
fluorescence in 405nm (my excitation) and above all the most intruding
problem is that I actually have reflections on my optical path. So it
seems that we put our efforts now trying to solve these reflections so
I can return to biology...

Thanks for you good advices,

Adi

Christian Wilms wrote:
adi76 <adi.raveh@xxxxxxxxxxxxxx> wrote:

Thanks Chris,
I will check it with a scatterer from now on,
You're welcome - I hope that works as well for you as it has for us.

May be you can also direct me regarding another issue. Using a 406nm
laser line, we get a relatively strong scattering of our laser, cause
us a delayed secondary IRF-like pulse which is really annoying us.

I have seen reflections causing a "shadow" IRF. So far I've always found
it helpful to measure the time between the two peaks and calculate the
distance between the two pulses (thumbing 1ns to 30cm). This way you
already have an idea of the distance between the two reflecting surfaces
which are causing your second pulse.

I'm finding this a bit hard to explain, eventhough the idea is very
simple. In case I haven't made myself understandable yet, I'll give a
brief example: I had a second peak in my IRF with a delay of about
500ps. That corresponds to 15cm between the two pulses, meaning the two
reflecting surfaces causing the second pulse were about 7 to 8cm apart.
With this information I started looking for reflective surfaces meeting
that criterium and identified the beam-expander I was using as the
culprit.

In general any optic surface, that isn't slightly tilted will lead to
disturbing reflections. Especially when using one photon excitation this
can be a bit unnerving :-).

Are you just setting up, or have you been using the system for a while
already?

Regards, Chris

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