Measuring Picosecond Fluorescence Lifetimes

The measurement of fluorescence lifetimes provides a wealth of information on the sample under study, from the local environment of biomolecules in living cells to charge-carrier lifetimes in semiconductors. Short fluorescence lifetimes on a picosecond timescale must be measured in a number of these studies.  

The measurement of sub 20 picosecond lifetimes using the FLS1000 Photoluminescence Spectrometer equipped with a hybrid photodetector is outlined in this article. The effect that the configuration of the FLS1000 has on the minimum lifetime which can be measured is also discussed.

FLS1000 Photoluminescence Spectrometer.

Figure 1. FLS1000 Photoluminescence Spectrometer. 


The hemicyanine dye trans-4-[4-(dimethylamino)-styryl]-1-methyl-pyridiniumiodide (4-DASPI) was selected to show the measurement of short lifetimes by utilizing the FLS1000.

Usually, DASPI dyes are utilized as a laser gain media and as short lifetime fluorescence probes. 4‑DASPI was acquired from Sigma Aldrich and dissolved in either distilled water or anhydrous ethanol.

Molecular structure of 4-DASPI .1

Figure 2. Molecular structure of 4-DASPI .1

Experimental Configuration

The time-correlated single-photon counting (TCPSC) method is utilized to measure fluorescent lifetimes in the FLS1000. The temporal response of the detection system and the temporal width of the excitation pulse would both be infinitely sharp (delta functions) in an ideal TCSPC system.

Yet, as lasers have finite pulse widths and detectors have transit response times this is never the case. This stray from the ideal is characterized by the instrument response function (IRF) of the TCSPC system.

The IRF can be considered a temporal response that the TCSPC system records for an infinitely short fluorescence lifetime. The measured fluorescence decay is convoluted with the IRF, and the temporal width of IRF (FWHMIRF) establishes the minimum lifetime which can be measured successfully.

A helpful rule of thumb is that the minimum fluorescence lifetime that can be established via reconvolution analysis is approximately 1/10th of the FWHMIRF. So, when measuring short fluorescence lifetimes, it is crucial to minimize the FWHMIRF.

The IRF has four main components which combine to give the complete width of the IRF:

The FLS1000 can be configured to suit different research requirements and is a completely modular spectrometer. The width of the IRF can be adjusted to meet the minimum measurable fluorescence lifetime needed in different research applications by modifying the configuration of the FLS1000 or the light source.

Due to the transit time spread (TTS) of the detector, FWHMdetector is broadening and has the biggest influence on the overall width of the IRF. The TTS is because of the photon-to-photon variation in the delay between the absorption of a photon at the photocathode of the detector and the electrical output pulse.

The PMT-900 is the standard detector of the FLS1000, which is a conventional side window photomultiplier tube (PMT). It has a TTS of ~600 ps and limits the minimum lifetime to > ~60 ps. The FLS1000 can be equipped with high-speed PMTs (HS-PMT) which have TTS of ~180 ps in order to measure shorter lifetimes.

For a number of years, the only choice for measuring shorter fluorescence lifetimes was utilizing a microchannel plate PMT (MCP-PMT). They have excellent TTS of <25 ps but are fragile and expensive.

The hybrid photodetector (HPD) is a new type of fast detector that has recently been developed. It combines an avalanche photodiode with components of a traditional PMT to make a robust detector with a TTS as low as an MCP‑PMT.

The FLS1000 was equipped with an HPD detector for the measurement of 4-DASPI. The combination of low groove density grating, HPD detector, and femtosecond laser in the FLS1000 resulted in a FWHMIRF of only 34 ps.

FWHMlaser is the pulse width of the laser excitation source. For TCSPC measurements, picosecond pulsed diode lasers are the most common light source. Edinburgh Instruments has developed an EPL series of diode lasers which have pulse widths down to 55 ps and are ideal for most scenarios.

In addition, the FLS1000 has laser ports to enable the user to couple third party lasers into the spectrometer. A femtosecond fiber laser, the Chromacity 520,3 which has a pulse width of 150 fs was utilized for the measurement of 4-DASPI to narrow the IRF and show this flexibility.

A fraction of the laser pulse was picked off and directed into the OT900 Optical Trigger Module To trigger the TCSPC electronics.

The broadening of the IRF due to the electronic jitter of the TCSPC timing electronics is FWHMelectronics. This contribution to the IRF is small, as every FLS1000 utilizes the advanced Edinburgh Instruments TCC2 counting electronics module which has a jitter as small as 20 ps.

FWHMdispersion is the temporal dispersion of the light after passing through the emission monochromator. The extent of the dispersion is dependent on the width of the monochromator slits and the groove density of the monochromator grating. The higher the groove density the bigger the temporal and spectral dispersion.

Therefore, for high-resolution spectral measurements, high groove density gratings are ideal but they broaden the IRF. Each monochromator in the FLS1000 can house up to three gratings at the same time, permitting an optional low groove density grating to be installed for measuring short lifetimes.

Using the Fluoracle® operating software, the user can switch quickly between the spectral (typically 1200 gr/mm) and lifetime (300 gr/mm) gratings. The 300 gr/mm lifetime grating was employed for the measurement of 4‑DASPI in the following section.

A subtractive double monochromator could be used to lower the temporal dispersion even more, as found in the Lifespec II Spectrometer, which has zero temporal dispersion.

Results and Discussion

Figure 3a shows the fluorescence decay of 4-DASPI in ethanol. The IRF was measured at the laser wavelength (520 nm) using a scattering dispersion of colloidal silica (LUDOX) and the decay was measured at the emission maximum (600 nm).

Fluorescence decays of 4-DASPI in (a) ethanol and (b) water measured using TCSPC

Figure 3. Fluorescence decays of 4-DASPI in (a) ethanol and (b) water measured using TCSPC.

By using revonvolution fitting in the Edinburgh Instruments FAST advanced lifetime analysis software, the decay was fit with a single exponential. A single exponential decay models the fluorescence well and the lifetime of 57 ps is in good agreement to previous reports.1

It is known that when 4-DASPI is dissolved in water its fluorescence lifetime is decreased fivefold,1 so a solution of 4‑DASPI in water was also measured to give a more challenging sample to test the short lifetime capabilities of the FLS1000.

Figure 3b shows the fluorescence decay and fit of 4-DASPI in water, revealing a lifetime of 11 ps which is in perfect agreement to the 11 ps lifetime reported in the literature previously.2


By measuring the fluorescence lifetime of 4‑DASPI in ethanol (57 ps) and water (11 ps), the capability of the FLS1000 Photoluminescence Spectrometer to measure short picosecond lifetimes was demonstrated.

The minimum measurable lifetime can be modified to meet the requirements of a wide range of different research areas through customization of the light sources, gratings, and detectors of the modular FLS1000.

References and Further Reading

  1. Kim, M. Lee, J -H. Yang, & J -H. Choy, Photophysical Properties of Hemicyanine Dyes Intercalated in Na-Fluorine Mica, J. Phys. Chem. A 104 1388-1392 (2000)
  2. Kim & M. Lee, Excited-State Photophysics and Dynamics of a Hemicyanine Dye in AOT Reverse Micelles, J. Phys. Chem. A 103 3378-3382 (1999)
  3. Chromacity 520 Data Sheet,

This information has been sourced, reviewed and adapted from materials provided by Edinburgh Instruments.

For more information on this source, please visit Edinburgh Instruments.


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