Time Correlated Single Photon Counting (TCSPC) has been one of the best ways of measuring fluorescence decay times since the method was conceived in 1961 by Bollinger and Thomas, Rev. Sci. Instrum. 32, 1044 (1961). The most comprehensive text is that of D.V.O'Connor & D. Phillips, "Time-Correlated Single Photon Counting", Academic Press, London, 1984.
The Laser Apparatus |
Experimental Operating Procedures |
A Coherent Antares A76s Neodymium:YLF cw-modelocked laser acts as the pump for the lasers used in this technique. Its fundamental frequency of 1053 is frequency doubled in a KTP (potassium tytanil phosphate) crystal to give a 527nm 76MHz rep-rate pulse train which is used to synchronously pump the dye lasers. The pulse width is on the order of 100ps.
One of two Coherent 700 series dye lasers are employed. Dyes used in the lasers are Rhodamine 6G and Pyridine-1 which give wavelengths in the ranges 565-620nm and 680-720nm respectively. A Coherent 7220 cavity dumper is used to reduce the repetition rate and decrease the pulsewidth to about 6ps. Two BBO (beta barium borate) crystals cut at different angles are used to frequency double the dye laser outputs (290-310nm and 340-360nm respectively) and also to allow sum frequency generation of 413-428nm by adding the 680-720nm output to the 1053nm Antares fundamental.
The TCSPC technique is essentially a "stopwatch" technique. The excitation pulse is split such that a Photodiode is triggered at the same time that the sample is excited. It can be thought that a stopwatch is started at this point. When a fluorescence photon is detected by a Microchannel Plate Photomultiplier Tube (MCP or PMT) the stopwatch is stopped and the "time" measured is collected.
An emission photon is detected by the Micro-Channel Plate PhotoMultiplier Tube . The exact position in time of a pulse from the MCP-PMT, representing the time of the photon event is crucial. This is determined by the constant fraction discriminator (CFD) which sends a precisely timed signal to START the charging of a linear voltage ramp in the Time-to-Amplitude Convertor or TAC.
The charging linear voltage ramp of the time-to-amplitude coverter, or TAC, is STOPPED by the regular electronic output of the photodiode which represents the repetition rate of the optical excitation. A pulse is then output from the TAC, the amplitude of which is proportional to the charge on the ramp and hence the time between start and stop. The time difference between two separate pulses from the cavity dumper is known and so a 'decay time' is represented by the pulse amplitude from the TAC, figure 2.8. The TAC is run in inverted mode, like this, so that each photon that is detected is counted. It takes a finite time to reset the voltage ramp and if the TAC was started by each laser trigger many counts would be lost whilst the TAC was reset. This is why inverted mode is clearly the most efficient way of running the experiment. It is also why the decay that is measured is actually backwards on the screen. The time between each pulse is constant, accurate to one part in 10 million.
The TAC output may or may not be sent through a Biased Amplifier which effectively adds a small known voltage to the amplitude of the true TAC output. Manipulating the pulse in this way acts as a further, variable, analogue delay to the signal. A gain control on the amplifier acts as a multiplier so that the time resolution can also be controlled in a variable, analogue fashion by stretching the true TAC signal.
The pulse height is digitised by the analogue-to-digital converter and a count is stored in the multichannel analyser in an address corresponding to that number. These components are contained on a PC card. The multichannel analyser uses 1024 channels in our system. The experiment is repeated as described until the histogram of number of counts against address number represents to a required precision, given as a fixed number of counts at the maximum channel, the decay curve of the sample. A standard maximum count at the peak is 20000.
The TCSPC measurement relies on the concept that the probability distribution for emission of a single photon after an excitation yields the actual intensity against time distribution of all the photons emitted as a result of the excitation. By sampling the single photon emission after a large number of excitation flashes, the experiment constructs this probability distribution. Ideally one fluorescence photon is observed every few hundred excitation laser pulses. This can be achieved by reducing the power of the excitation light or by closing the monochromater slits such that a count rate of about 8000s-1 is achieved. This corresponds to one fluorescence photon being detected for 250 excitation pulses.
In principal one could greatly increase the count rate to the limit that one photon is detected for each excitation. However the chances that there are in fact 2 photons that reach the MCP-PMT are great. In this case it will always be the first photon that arrives that will be detected. The upshot of high count rates therefore is a bias towards early photons and shorter decay times which are inaccurate.