TCSPC FLIM

Principles

More about FLIM applications

Principle of TCSPC FLIM

FLIM stands for Fluorescence Lifetime Imaging (Microscopy). The ingenious thing about this is that in addition to the mere fluorescence intensity, the arrival times of all photons in relation to the exciting laser pulse are measured and recorded. This provides you a fluorescence lifetime map of the sample.
The method originates in bh’s multi-dimensional TCSPC technique. The sample is scanned by a high-repetition rate pulsed laser beam, and single photons of the fluorescence signal returned from the sample are detected. Each photon is characterized by its time in the laser pulse period and the coordinates of the laser spot in the scanning area in the moment of its detection. The recording process builds up a photon distribution over these parameters. The result is equivalent to an array of pixels, each containing a full fluorescence decay curve in a large number of time channels.

TCSPC FLIM delivers a near-ideal photon efficiency, excellent time resolution, and is independent of the speed of the scanner. The general features are the same as for classic TCSPC. The signal-to-noise ratio depends only on the number of recorded photons, i.e. on the total acquisition time and the photon rate available from the sample. The time resolution depends on the laser pulse width and the transit-time-spreead of the detector. With fast hybrid detectors an IRF width of less than 20 ps (fwhm) is reached. Another important feature is the large number of time channels into which the decay data are resolved. In combination with the high time resolution, this allows the decay functions to be resolved into several decay components, with characteristic lifetimes and amplitudes. Even decay components that are normally hidden within the IRF can be determined. In life-science applications these parameters are often more important than the apparent ‘lifetime’ of the net decay function.

The technique can be extended by including additional parameters in the photon distribution. These can be the depth of the focus in the sample, the wavelength of the photons, the wavelength of the excitation, the time after a stimulation of the sample, or the time within the period of an additional modulation of the laser. These techniques are used to record Z stacks or mosaics of FLIM images, multi-wavelength FLIM images, images of physiological effects occurring in the sample, or to record simultaneously fluorescence and phosphorescence lifetime images. Please see ‘The bh TCSPC Handbook’, overview brochures ‘The bh FLIM Technique – More than Lifetime Imaging’ and ‘FLIM Systems for Laser Scanning Microscopes’, or overview brochures and handbooks of the individual systems.

bh FLIM systems work both with multiphoton excitation by femtosecond lasers and with and with one-photon excitation by ps diode lasers. Complete TCSPC FLIM systems with detectors, recording electronics, microscope, scanner, excitation source, and integrated data acquisition and data analysis software are available from bh, see versions of the DCS-120 confocal and multiphoton FLIM systems. Systems are also available for laser scanning microscopes of other manufacturers, scanning systems for clinical applications, and customer-specific systems for non-standard applications. Please see FLIM Systems for High-Resolution Fluorescence Lifetime Imaging on this website.

Pile Up

As an argument against TCSPC FLIM it is often stated that the technique is plagued by the ‘Pile-Up-Effect’, which means the detection of a second photon in the same signal period with a previous one, causing a distortion of the recorded optical waveform. Please don’t get confused by such statements. 

The commonly stated pile-up limit of 0.1% of the excitation rate is wrong. The correct value is 0.1 x Excitation Rate, or 10% of it. This is 100 times more than commonly believed! The limit of 0.1% stems from a typo in the TCSPC literature of the late 1970s. Since then, the mistake cas been copied uncritically again and again. With the correct value of the pile-up limit, the maximum count rate for 80 MHz repetition rate is 8 MHz (8×10⁶ photons per second). This is at least 10 times more than typical FLIM samples can deliver without photo-induced changes in their molecular structure.

A deriviation of the correct size of the pile-up error and the conclusion of its irrelevance for TCSPC and TCSPC FLIM can be found in [1] and [2]. How deeply the misconception of pile-up is fixed in the minds of the users becomes evident from the fact that [1] is constantly cited as a proof of the alledgedly devastating effect of pile-up on TCSPC FLIM. In fact, it proofs the opposite. Therefore, make sure that you cite the reference in the correct context. If you carelessly copy the mistake you show that you either have stolen the reference from another paper, or that you were unable to grasp the point of it – which even sub-average intelligence should have done.

Practical Implementation

For practical implementation please see ‘FLIM Systems for High-Resolution Fluorescence Lifetime Imaging‘ on this website, and ‘The bh TCSPC Handbook’, chapters ‘Implementation of the bh TCSPC Technique’ and ‘Time-Resolved Laser Scanning Microscopy’.

[1] W. Becker, Advanced Time-correlated Single Photon Counting Techniques, Springer, 2005
[2] W. Becker, The bh TCSPC Handbook, 8th edition, 2021

Products

Application Notes

• Two-Photon FLIM of Mushroom Spores Reveals Ultra-Fast Decay Component
• High-Resolution Measurement of NADH and FAD Fluorescence Decay with the DCS-120 MP
• Two-Photon FLIM of Pollen Grains Reveals Ultra-Fast Decay Component
• FLIM at a Time-Channel Width of 300 Femtoseconds
• Suppression of Lens Fluorescence in FLIO Images of Cataract Patients
• High-Resolution Multiphoton FLIM Reveals Ultra-Fast Fluorescence Decay in Human Hair