There are two main paths that customers follow to have TCSPC capability:
First, you can upgrade existing equipment with the appropriate capabilities. This approach is mist common, and our solutions include a wide range of electronics, detectors, lasers, confocal scanners, software, installation support, etc. We provide the tools to build a TCSPC capability for your system.
Second, you can purchase a complete turnkey solution; this approach is common for Fluorescence Lifetime Imaging Microscopy (FLIM):
You can buy a FLIM-ready microscope from Nikon, Zeiss or others.
Or, you can buy a confocal scanner from us and attach it to a camera port of a light microscope, add our excitation, detection and TCSPC signal processing, and let us set it up for you to make it a confocal scanning microscope.
Or you can do it yourself (especially, convert an existing research grade microscope) by buying the key modules from us – we will tell you how to put it together painlessly.
Fluorescence Lifetime Imaging Microscopy (FLIM)
The largest single application of TCSPC at this time is FLIM, using a laser scanning confocal microscope and a high rep rate pico- or femtosecond laser such as a pulsed diode for one-photon or a Ti: Sapphire laser for two-photon excitation. Another hot topic is Fluorescence Correlation Spectroscopy (FCS). The latest version of the Becker & Hickl software (bundled at no cost with the electronics) includes a new FCS module.
For a comprehensive introduction to TCSPC the latest version of the B&H Manual (TCSPC Handbook) is available by clicking here. This is a very comprehensive tutorial handbook for the serious user.
No matter how much you download, print out and read, nothing is as useful in making the decision to buy or not as talking to us. Please phone, fax or email us. Let us know what you have, and what you want to accomplish, and we will tell you what you need and where to get it.
What Is Time Correlated Single Photon Counting (TCSPC)?
Time-Correlated Single Photon Counting (TCSPC) is a technique to record low level light signals with picosecond time resolution. Typical applications are
Ultra-Fast Recording of Optical Waveforms
Fluorescence Lifetime Measurements
Detection and Identification of Single Molecules
DNA Sequencing
Optical Tomography
Fluorescence Lifetime Imaging
The method has some striking benefits:
Ultra-High Time Resolution – 25 ps FWHM with the best detectors
Ultra-High Sensitivity – down to the Single Photon Level
Short Measurement Times
High Dynamic Range – Limited by Photon Statistics only
High Linearity
Excellent Signal-to-Noise Ratio
High Gain Stability
Suppression of Detector Leakage Currents
TCSPC works best for
High Repetition Rate Signals (MHz Range)
Wavelengths from 160 nm to 1500 nm
Measurement Principle
Time-Correlated Single Photon Counting is based on the detection of single photons of a periodical light signal, the measurement of the detection times of the individual photons and the reconstruction of the waveform from the individual time measurements. The method makes use of the fact that for low level, high repetition rate signals the light intensity is usually so low that the probability of detecting one photon in one signal period is much less than one. Therefore, the detection of several photons can be neglected and the principle shown in the figure below can be used: The detector signal consists of a train of randomly distributed pulses due to the detection of the individual photons. There are many signal periods without photons, other signal periods contain one photon pulse. Periods with more than one photons are very rare. When a photon is detected, the time of the corresponding detector pulse is measured. The events are collected in memory by adding a ‘1’ in a memory location with an address proportional to the detection time. After many photons the histogram of the detection times, i.e. the waveform of the optical pulse, builds up in the memory. Although this principle looks complicated at first, it is very efficient and accurate since the accuracy of the time measurement is not limited by the width of the detector pulse. Thus, the time resolution is much better than with the same detector used in front of an oscilloscope or another linear signal acquisition device. Furthermore, all detected photons contribute to the result of the measurement. There is no loss due to ‘gating’ as in ‘Boxcar’ devices or gated image intensified CCDs.
Sensitivity
The sensitivity of the SPC method is limited mainly by the dark count rate of the detector. Defining the sensitivity as the intensity at which the signal is equal to the noise of the dark signal the following equation applies:
S = (Rd*N/T)1/2/Q
where Rd = dark count rate, N = number of time channels, T = overall measurement time and Q = quantum efficiency of the detector. Typical values (uncooled PMT with multialkali cathode) are Rd=300s-1, N=256, Q=0.1 and T=100s. This yields a sensitivity of S=280 photons/second. This value is by a factor of 1015 smaller than the intensity of a typical laser (1018photons/second). Thus, when a sample is excited by the laser and the emitted light is measured, the emission is still detectable for a conversion efficiency of 10-15.
Time resolution
The SPC method differs from methods with analog signal processing in that the time resolution is not limited by the width of the detector impulse response. For the SPC method only the timing accuracy in the detection channel is essential. This accuracy is determined by the transit time spread of the single photon pulses in the detector and the trigger accuracy in the electronic system. The timing accuracy can be up to 10 times better than the half width of the detector impulse response. Some typical values for different detector types are given below.
The accuracy of the measurement is given by the standard deviation of the number of collected photons in a particular time channel. For a given number of photons N the signal-to-noise ratio is expressed as SNR = N-1/2. If the light intensity is not too high, nearly all detected photons contribute to the result. Therefore, the SPC yields a very good signal-to-noise ratio at a given intensity and measurement time. Furthermore, in the SPC method, noise due leakage currents, gain instabilities, and the stochastic gain mechanism of the detector does not appear in the result. This yields an additional SNR improvement compared to analog signal processing methods.
Recording Speed
The TCSPC method was once thought to suffer from slow recording speed and long measurement times. This bad reputation comes from traditional TCSPC devices built up from nuclear instrumentation (“NIM”) modules which had a maximum count rate of some 104 photons per second. State-of-the-art TCSPC devices from Becker & Hickl achieve count rates of some >>106 photons per seconds. Thus, 1000 photons can be collected in less than 0.1 ms, and the devices can be used for such high speed applications as the detection of single molecules flowing through a capillary, for fast image scanning, for the investigation of unstable samples or simply as optical oscilloscopes.
Multichannel and Multidetector Capability
Becker & Hickl has introduced multichannel and multidetector capabilities in their TCSPC modules. In the device memory space is provided for several waveforms, and the destination of each individual photon is controlled by an external signal. In conjunction with a fast scanning device, time resolved images are obtained with up to 128 x 128 pixels containing a complete waveform each. Furthermore, several detectors can be used with one TCSPC module. This technique makes use of the fact that the simultaneous detection of several photons in different detectors is very unlikely. Thus, the output pulses of several detectors are combined and an external ‘Routing’ device determines in which detector a particular photon was detected. This information is used to route the photons into different memory blocks containing the waveforms for the individual detectors.
