Leica TCS SP8 SMD, User Manual

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Summarized manual for FLIM experiments
17.12.1.3 With Fluorescence Mode
This is the preferred mode for MP and pulsed VIS or UV excitation, because the conditions
of the IRF measurement match the measurement conditions of the real experiment.
1. Use a dye with a very short lifetime (a few picoseconds) in a measurement chamber. A
dye can be used for IRF measurements if the fluorescence decay time is much shorter
than the IRF width. An example for such a dye is picric acid.
2. For external FLIM detection select an appropriate SMD filter cube which will be used for
your final measurement. For SP FLIM detection, make sure the fluorifier disc is in the
correct position (
Chapter 16.2.3 , Table 19 ).
3. An additional attenuation filter in the filter holder in front of the external detector may not
be required.
Dyes with a very short lifetime can be made with the following recipe: A saturated aqueous
dye solution is created from a spectrally suitable dye with good water solubility. Potassium
iodide is added to this solution until the dye solution is saturated with it. The potassium
iodide usually quenches the fluorescence so strongly that the fluorescence lifetime of the
solution sinks to below 100 ps FWHM and thus can also be used for IRF measurements. The
advantage of this method is that the IRF can be measured at the same emission wavelengths
as it is in the final measurement. This method can also be used for multi-photon excitation
(refer also to Szabelski M. et al., Collisional quenching of Erythrosine B as a potential
reference dye for impulse response function evaluation, Applied Spectroscopy, 63, 2009).
17.12.1.4 With SHG (Second Harmonic Generation – Possible for MP Lasers Only)
1. Use a SHG-capable specimen without lifetime or with very short lifetime (a few
picoseconds) in a measurement chamber. A typical example of a SHG specimen is
muscle tissue, collagen or urea powder mounted on a cover slip.
2. Emission happens at roughly half the excitation wavelength (for instance excitation at
900 nm, emission at 450 nm). For external FLIM detection, select an appropriate SMD
filter cube that allows SHG emission to pass through.
3. The fluorifier disc has to be in the in position SP700 or SP680 .
4. For SP FLIM detectors, move the spectral slider such that the emitted light hits the SP
FLIM detector.
5. For external detection, remove all additional filters from the filter holder in front of the
detector.
WARNING Risk of severe injuries from picric acid
Picric acid is poisonous and causes a danger of explosion. Observe your
country-specific regulations, which are indicated on the safety data
sheet.