Time-correlated single photon counting is a technique used for measuring single-wavelength fluorescence decays with picosecond resolution.123 Our setup is home-built124 and its optical scheme is shown in Figure 2.1.
Figure 2.1 | Optical scheme of our TCSPC setup. Two different options for fluorescence detection are shown.
Three picosecond laser-diodes (PicoQuant LDH Series) emitting at 375, 395 and 470 nm are used as excitation light sources (pulse duration:
~50 ps, repetition rate: 40 MHz). The beam profile of all of these lasers is horizontally elongated, and therefore we send it through a Keplerian telescope for recollimation and reduce its size by a factor of ~3. A thin
microscope glass plate is used to reflect a small fraction of the beam (~5 %) which is attenuated by neutral density filters and then tightly focused on a fast photodiode PD (Becker&Hickl GmbH, active area: 0.25 mm2). The signal from the photodiode is amplified and directed to the SYNC channel of the electronic timing system to provide an external reference for the timing when a laser shot is fired. The use of an external trigger is crucial to obtain a high time resolution with a microchannel plate photomultiplier (MCP-PMT). The major fraction of the beam simply propagates through the glass plate. A multi-order half-wave plate (Thorlabs) is used in conjunction with a Glan-Taylor polarizer (Newport) in order to ensure that the excitation beam is vertically polarized in the laboratory frame and allow for adjustment of the excitation intensity at the sample. The sample is typically placed in a 10 mm quartz cuvette and fluorescence is collected at 90°.
There are two different options for emission detection. The ‘routine’
option is based on a PMT (Hamamatsu). Fluorescence from the sample is recollimated and propagates through a thin-film polarizer set at magic angle with respect to the vertical excitation. A bandpass interference filter lets through a narrow (typically 5-12 nm) part of the emission spectrum, which is focused tightly on the active area of the PMT. For such settings, the use of external SYNC channel is optional and internal electronic triggering can equally be used. The time resolution of the setup in this mode is <200 ps (as determined by full width at half maximum (FWHM) of the instrument response function (IRF)).
Another option, one more experimentally demanding but providing better time resolution, is to use an MCP-PMT detector (Hamamatsu). In this case, the time for the setup preparation is increased by at least three hours as the detection system must be purged with nitrogen gas and cooled using a Peltier element. The cuvette with the sample is laterally tilted and displaced from the beam in such a way that the latter is shifted with respect to the cuvette center. Such a geometry allows for optimization of the time resolution which could be obtained with the MCP detector. A narrow paper slit made out of black paper is inserted in the cuvette holder to limit the area from which fluorescence is collected. This trick minimizes the
spatial distribution of spots along the excitation pathway contributing to the observed fluorescence and improves the fluorescence imaging on the detector. The fluorescence is recollimated by an achromatic lens doublet and passed through a foil polarizer and an interference filter. Importantly, the fluorescence is not focused onto the MCP detector but evenly distributed over the entire 11 mm diameter photoactive area. This condition is crucial to achieve the best time resolution and contrasts with the use of the single-channel PMT detector. The power of the laser diode should be also adjusted to minimize after-pulsing and the temporal width of the IRF. All these adjustments and precautions allow one to reliably attain ~55-60 ps time resolution. In principle, such a fight for a better time resolution using photon counting may be considered questionable, but larger temporal overlap between femtosecond time-resolved fluorescence and photon counting allows one to check and extract more reliable time constants obtained from the same sample by comparison of the different methods.
A few things have to be said about the important details affecting SPC measurements. For the extraction of time constants that are shorter than
~10× the IRF width, an iterative reconvolution technique employing non-linear least squares fitting must be applied. Therefore, the measurement of a high-quality IRF is required and is a critical factor in high-resolution single photon counting experiments. A very dilute scattering solution or better a neat solvent in the same cuvette should be used to reduce artifacts in the IRF. The use of a piece of foil as a scattering material is highly discouraged as it adds sharp artificial reflection peaks to the genuine IRF.
For the best-quality fits a correction for the differential nonlinearity of the time-to-amplitude converter (TAC) must be performed. It stems from the size differences of the time bins of the TAC. When the signal originating from random events is recorded, such as illumination by a continuous light source, it should result in a flat line as there are no inherent correlations between photons arrival times to the detector. However, due to the TAC nonlinearity, the deviations from the flat line were observed as high as 15-20 % in the worst case, with typical values around 5-10 %. Therefore, such a curve should be recorded and used to correct both the IRF and
sample decay traces. Additionally, among all our group’s instruments, the single-photon counting experiment is probably based most heavily on electronics. Therefore, proper adjustment of the crucial settings of the electronic boards as well as timing of the electronic signals should be done before actual experiments. A comprehensive review of all the experimental and technical details can be found in Ref. 123.