Time-resolved single-molecule spectroscopy setup

Time Correlated Single Photon Counting (TCSPC) technique is used to measure fluorescence kinetics of single molecules. For time-resolved measurements we use pulsed excitation of the sample by, for example, the second harmonic of 150 fs pulse from Ti-Sapphire laser (Tsunami, 80 MHz repetition rate) with spectral maximum at 916 nm. The light of the second harmonic (spectral maximum at 458 nm) is passing through the same set of filters (“filter cube”) as 458 nm Ar-ion laser line resulting in excitation spectrum with maximum at 458 nm and ≈10 nm spectral widths.

By flipping up the motorized mirror M1 (see figure 6) the image can be obtained at the pinhole plane (A₂^*). By adjusting the position the image in the pinhole plane a fluorescence light from a desired part of the image can be directed through the 100 μ pinhole (see the figure). In practice we find a pixel at the CCD camera (X_pinhole, Y_pinhole) which is conjugated to the pinhole. Then in order to get light from a particular molecule though the pinhole we just move the sample (using the sample stage) or the image itself (using motorized lens L2) to place the molecule of interest to the position (X_pinhole, Y_pinhole) at the CCD camera. After that we flip up the mirror and can detect fluorescent photons by a fast avalanche photodiode. We use photon-counting module from Micro Photon Devices, crystal size 100 μ, 250 cps dark counts.

Pulses from APD are then counted by PicoHarp 300 (PicoQuant GmbH). The whole system operating in time-correlated single photon counting regime had a response function of about 50 ps. Time-tagged time-resolved (TTTR) mode can be used to detect absolute arrival times of all photons together with synchronization pulses from the laser in order to obtain time resolved fluorescence decay kinetics and fluorescence intensity at any given time of a fluorescence transient.

Figure 6. Setup for time-resolved measurements