Choose Your Excitation Wavelength

Time Correlated Single Photon Counting

April 13, 2011

Fluorescence Lifetime Imaging Microscopy (FLIM) is widely used to quantify protein-protein interaction by measuring the FRET (Förster Resonance Energy Transfer) occurring between two fluorophores spaced by a few nanometers. FLIM is also used in a large array of applications ranging from tissue imaging to fluorophore environment probing. Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including i) a pulsed laser source, ii) a photon counting card, and iii) a fast detector. Such technical requirements render TCSPC acquisitions difficult to perform and/or narrow down the choices of usable fluorophores.

The Leica TCS SP5 X overcomes these limitations since it combines a pulsed white light laser (WLL) with an ultrafast photo-multiplier (PMT), allowing tunable spectral detection. This system is highly versatile and user-friendly for FLIM experiments. In this paper, we illustrate its potential in two biological applications: interaction studies and autofluorescence multispectral imaging.

Technical description

Supercontinuum generation has attracted much attention since the first report of photonic crystal fibers in the late 1990s. It allows coherent white light production by pumping a highly non linear photonic crystal fiber with ultrashort laser pulses (picosecond or femtosecond). Due to its broad light spectrum, this source was commonly used for spectroscopic purposes and it was also recently implemented on a commercial microscope system: the Leica TCS SP5 X.

Equipped with an Acousto-Optical Beam Splitter (AOBS), this system provides a pulsed laser source easily tunable from 470 to 670 nm, allowing for the excitation of a large panel of fluorophores at their absorption maximum. In order to determine whether the WLL is a suitable source for FLIM experiments, we ran a calibration protocol based on the one validated for two-photon FLIM measurements [1]. Notably, we measured the system’s instrumental response function and obtained a full width at half maximum around 300 ps, perfectly compatible with fluorescence lifetime measurements in cells and tissues. We then tested its capability on the two major fields of FLIM application: i) interaction studies by FRET and ii) tissue autofluorescence imaging. To address both situations, we applied the following experimental conditions:

  • Lifetime acquisitions were performed using the Leica TCS SP5 X confocal and the internal PMT. Photon counting was performed using the Becker and Hickl SPC830 card and SPCImage was used for lifetime analysis.
  • Two excitation sources were used for FRET measurements: the Cameleon ultra2 Ti:Sa laser at 920 nm (8 mW : objective back pupil) and the WLL at 488 nm. Detection bandpass was set between 500 and 540 nm and acquisition time to 300 s.
  • For convalaria cross section imaging we also used two distinct excitation sources: the 405 pulsed laser diode and the WLL. The excitation and detection bandpass are reported in the third figure. The acquisition time was set to 60 s per image.
Fig. 1a: Non linear photonic crystal fiber cross section

Versatility for FLIM-FRET experiments

The Leica TCS SP5 X presents both a pulsed WLL and an ultrafast detector with spectral selection. The system we developed is also equipped with a photon counting card (SPC830, Becker and Hickl) and a Ti:Sa laser (chameleon ultra II), a state-of-the-art source for TCSPC experiments. FLIM is particularly adapted for visualizing and measuring FRET occurring between interacting proteins at the nanometer scale in tissues or cells. Thus, in order to validate the WLL as a source for FLIM, we performed TCSPC experiments on positive and negative FRET references alternately using Ti:Sa and WLL as excitation laser source on the same cells. As shown in Figure 2, both excitation sources give consistent results and allow quantitative FRET measurements. The WLL can then be used as a versatile source for FLIM-FRET experiments. Furthermore, in TCSPC mode, the lifetime accuracy is directly dependent on the number of acquired photons. Using this system, we can optimize the number of collected photons by i) optimizing the excitation wavelength to the fluorophore absorption maximum and ii) choosing the ideal detection range with maximum donor emission without acceptor fluorescence bleedthrough.

Fig. 2: Fluorescence lifetime imaging of both a negative (mb-eGFP) and positive (mb-eGFP-mCherry) FRET reference using either the WLL or the Ti:Sa laser. Acquisitions were performed using a 40x, NA 1.25 oil immersion objective. Scale bar: 40 µm.

Multi-Spectral Lifetime Imaging Microscopy (MSLIM)

Combining AOBS and spectral selection using the internal PMT, the system allows high versatility regarding both excitation and emission wavelength selection. As previously shown, correlated spectral and lifetime measurement (SLiM) is a promising and powerful technique for discriminating multi-labeled samples and for detecting molecular interactions inside heterogeneous and auto-fluorescent media such as tissues [2, 3]. The use of a WLL further extends the possibilities for studying complex fluorescent signals. Indeed, one can now perform Multi-Spectral Lifetime Imaging Microscopy (MSLIM) by tuning both excitation wavelength (WLL through AOBS) and observation range (moving the slit in front of the internal PMT). Here, we present an example of autofluorescence MSLIM using a fixed convalaria slide.

Fig. 3: Sequential fluorescence lifetime imaging of convalaria cross-section using various excitations (grey bar) and observation (dashed box) wavelength windows. Excitation is achieved using the WLL, except for 405 nm excitation, which is achieved by using a pulsed laser diode. Acquisitions were performed using a 10x, NA 0.3 dry objective. Scale bar: 500 µm.

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