ADVERTISING FEATURE

APPLICATION NOTE

SP8 FALCON: a novel concept in fluorescence lifetime
imaging enabling video-rate confocal FLIM
The Leica SP8 FALCON (FAst Lifetime CONtrast) is a fast and completely integrated fluorescence lifetime
imaging microscopy (FLIM) confocal platform. SP8 FALCON delivers video-rate FLIM with pixel-by-pixel
quantification, thanks to a novel concept for measuring fluorescence lifetimes built on fast electronics
and sensitive spectral hybrid detectors. Photon arrival times are recorded at count rates typical forstandard confocal imaging. The system has ultra-short dead time and powerful built-in algorithms fordata acquisition and analysis. The deep integration of FLIM into the confocal platform provides easy
access to complex FLIM experiments.
Confocal imaging is the current standard for fluorescence imaging

arrival pulses from each detector are digitized at very high speed

in the life sciences. Functional imaging goes beyond the traditionalwith a temporal resolution of 97 ps. The data flow as a bitstream

recording of the location and concentration of molecular species,that is analyzed by an FPGA-controlled pattern recognitionand enables further investigation of molecular function: theiralgorithm. This step determines the photon arrival time frominteractions with other biomolecules as well as their activity,

the difference between the detection pulse and the laser pulse

conformation, molecular environment and post-translationalmodifications. Ideally, this must be accomplished at higha

spatiotemporal resolution. Fluorescence lifetimes are exquisitely
suited to report on biomolecular functional states, because thetime a molecule stays in the excited state is highly dependent on
its environment and interactions with other nearby species¹ ,². As
lifetime information is independent of fluorophore concentration,
it is a method of choice for functional imaging³. However, several
factors have limited the widespread application of FLIM. For
one, traditional time-correlated single photon counting (TCSPC)
solutions are intrinsically slow and difficult to implement,
particularly for complex imaging workflows. Therefore, FLIM
imaging has been limited so far to specialized laboratories and,beven with expert knowledge, traditional TCSPC has been unable todeliver the speed for biological processes occurring at time scalesbelow tens of seconds.
To overcome such limitations in speed and experimental
complexity, we developed an innovative concept for measuring
lifetime (Fig. 1a). The FLIM approach on SP8 FALCON is based ona confocal scan head with field-programmable gate array (FPGA)
electronics, pulsed laser excitation and fast, spectral single-photon
counting detectors. The signals of both laser pulses and photon

Luis A. J. Alvarez¹, Bernd Widzgowski¹, Giulia Ossato¹,Bram van den Broek², Kees Jalink², Lioba Kuschel¹, M. Julia
Roberti¹ and Frank Hecht¹
1Leica Microsystems CMS GmbH, Mannheim, Germany.
2Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
e-mail:luis.alvarez@leica-microsystems.com

Fig. 1 | Inside the Leica SP8 FALCON. a, Analogue signals from pulsed
laser excitation and emitted photons are recorded, digitized with separate
comparators, and fed into 10-GHz samplers. Pattern recognition analysis of theresulting bitstreams transforms it into photon arrival times (Fast FLIM). Results
are given as Fast FLIM, fluorescence intensity images, overall decay curves andfitted lifetime values. The example images are U2OS cells immunostained withα-tubulin (AF546) and vimentin (AF555), respectively. Color bar, 0–3 ns. Scalebar, 5 μm. b, The system can combine the detected photons from multiple
detectors into one for working at higher photon fluxes.

NATURE METHODS | OCTOBER 2019

ADVERTISING FEATURE

APPLICATION NOTE
arrival times. Jitter artifacts are avoided by measuring the timeof the individual detector bitstreams so they can be analyzed

difference from the laser pulse to the detector pulse. These steps are

together or separately at any time. This multidetector approach,performed directly at the system electronics, ensuring maximum

together with the overall system dead time below 1.5 ns, enables

speed and preserving signal quality. The data are then transferred toaccurate FLIM measurements at rates of 1 photon per pulse (Fig.

the computer to generate the image, build the decay curve from the2e, see below for details; each detector can reach up to 80 Mcps

total photon arrival times and calculate the fluorescence lifetime.

when used with 80 MHz pulsed lasers). The second approach is

These direct measurements from the differences in arrival times

the implementation of a high-speed FLIM photon filter to avoid

between detection and excitation pulses are rendered directly

pile-up effects. This filter ensures a faithful representation of theonline as ‘Fast FLIM’ images.

overall lifetime decay at high photon fluxes.

The speed limitations of traditional TCSPC arise from long

In addition to the high-speed FLIM filter, we implemented twoelectronics and/or detector dead times, making the system unableadditional modes to assess the quality of the data (Fig. 2): a ‘firstto keep up with the photon flux typical of modern confocal

photon’ filter and an ‘all photons’ mode. The ‘first photon’ filter

regimes. To avoid the underestimation of lifetimes due to the(Fig. 2a) builds the fluorescence decay exclusively from the firstso-called pile-up effect4,5, traditional TCSPC systems are operated

photon detected after each pulse. This filter mimics traditional

at photon fluxes between 0.01 and 0.1 photons per laser pulse5,6,

TCSPC and its inherent pile-up effect (Fig. 2b), with the resultingwith measurement errors around 2.5% 7. Efforts to correct pile-up

underestimation of the lifetime values. At higher counting rates,

effects have been previously described4,8–12.

the measured lifetimes artificially decrease2 (Fig. 2e). The ‘all

The SP8 FALCON implements two complementary approaches

photons’ mode (Fig. 2a) builds the fluorescence decay with allthat enable lifetime acquisitions at high photon fluxes. The firstthe photons detected. Although this option has the advantage

approach consists of combining multiple detectors and treatingof significantly increasing photon statistics, it also introducesthem as one (Fig. 1b). This is possible thanks to the full spectral

some bias and, if left uncorrected, it might lead to erroneous

flexibility of the SP8 platform and the SP8 FALCON electronics.

lifetimes (Fig. 2e). Nevertheless the ‘all photons’ approach yields

The spectral flexibility enables a free adjustment of the detectors’

a measured lifetime with a smaller error than traditional TCSPC.

range to cover a single emission spectrum, and the electronics

Using the high-speed FLIM filter (Fig. 2a), we achieve highhandle the information coming from the multiple detectors as

photon statistics and accurate lifetime values, with minimal error,

if it was coming from a single one. This addition of detectors

at the same time (Fig. 2e). With this filter, we ensure that only

increases the photon flux, which can be set before the acquisition

single-photon events between pulses are used for building andor at the analysis step. In fact, SP8 FALCON keeps the information

fitting the overall decay. Then, the SP8 FALCON electronics andadbceFig. 2 | Accuracy of lifetime measurements with SP8 FALCON. a, Schematics of the SP8 FALCON FLIM modalities. b, Pile-up and dead-time effects on the lifetime
decay built with the different photon filters. c, Flipper-TR® in U2OS cells. Color bar, 2-4 ns; scale bar, 10 µm. Overall decay versus pixel-based decay curves. d, cAMPsignaling in HeLa cells transfected with the EPAC FLIM-FRET biosensor. FLIM data quality at high photon flux with the three photon modalities. Color bar, 2.5–3.7 ns. Scalebar, 30 μm. e, Rhodamine B lifetime measurements (reference value of 1.47 ns) with increasing photon fluxes (in mega counts per second, Mcps). Red squares: resultswith high speed FLIM filter. Blue triangles: results from first photon filter (classical TCSPC behavior; open). Grey diamonds: unfiltered results. The grey box indicates theregion of 2.5% error. Open symbols, results with a single detector; filled symbols with 2 paired detectors.

NATURE METHODS | OCTOBER 2019

ADVERTISING FEATURE

APPLICATION NOTEnovel architecture allows us to directly use this overall model andMcps. When using the ‘all photons’ approach, the measured lifetimeequation (1) from Patting et al.12 for the pixel-by-pixel FLIM image

comes closer to the reference value but still yields lower values. Thefit. This leads to:standard high-speed FLIM filter on the SP8 FALCON (red squaresin Fig. 2e) allows a consistent rhodamine B lifetime determination
at photon regimes of up to 225 Mcps, with an error between 0.2%
and 1.6%. The data show that SP8 FALCON significantly increases
the photon flux regimes available for reliable lifetime measurements.

where the fluorescence decay, f(t), is corrected with the model

The high-speed FLIM recording of the SP8 FALCON is a crucial asset

function; with IRF, the instrument response function; A i, thefor demanding biological experiments. For example, it enabled time-amplitudes of the lifetime components; B, the background; P, thelapse recording of ERK activity in live-patient-derived colorectal cancer

number of laser pulses; τ, the lifetime; and td, the dead time.

organoids14 (Fig. 3). The ERK signaling, which is an important driver

After obtaining an accurate overall decay curve from the high-of malignant cell growth in colorectal and several other cancer types,

speed FLIM filter, we can apply the proper mathematical model for

was recorded using a modified version of the EKAREV biosensor15.

the pixel-by-pixel calculation. At each pixel, we use the unfiltered

A 3D-rendered movie is accessible at www.leica-microsystems.com/

data to build the decay and the overall mathematical model for fitting.

SP8-FALCON-3DLiveOrganoids. Another example is the recording ofcalcium oscillations with a commercially available calcium sensor16
in live cells with video-rate FLIM contrast (movie available at www.

-leica-microsystems.com/ SP8-FALCON-fastCalcium). In summary, thisnew-found speed performance in confocal and multiphoton functionalimaging, along with seamless integration of lifetime imaging into
all intensity-based confocal imaging and processing tools, enables
studying biological processes at time scales previously unattainable.
References1.

Lakowicz, J. R. Principles of Fluorescence Spectroscopy 3rd edn (Springer, 2006).

2.

Valeur, B. Molecular Fluorescence Principles and Applications (Wiley-VCH, 2001).

3.

Szmacinski, H. & Lakowicz, J. R. in Topics in Fluorescence Spectroscopy Vol. 4 (ed.
Lakowicz, J. R.) 295–334 (2006).

Fig. 3 | Live 3D FLIM on patient-derived organoid. Monitoring cancerderived increased of signal transduction activity in live organoids with an ERK
FRET-FLIM biosensor14 (right). Corresponding fluorescence intensity image withno functional information. Functional information showing FRET efficiency of
the ERK biosensor. Higher FRET efficiency indicates higher ERK activity. Colorbar, 2–12% FRET efficiency. Scale bar, 20 μm.

4.

Harris, C. & Selinger, B. Aus. J. Chem. 32, 2111–2129 (1979).

5.

Becker, W. The bh TCSPC Handbook, Pile-Up. 279 (Becker & Hickle, 2017).

6.

Birch, D. J. S. & Imhof, R. E. in Topics in Fluorescence Spectroscopy (ed. Lakowicz, J.
R.) 1–95 (Springer, 1999).

7.

Becker, W. The bh TCSPC Handbook. 280 (Becker & Hickle, 2017).

8.

Walker, J. G. Opt. Comm. 201, 271–277 (2002).

With this dual approach, we ensure that the best possible fitting model

9.

Coates, P. B. J. Phys. E Sci. Instrum. 5, 148–150 (1972).

is used, together with the appropriate photon statistics (Fig. 2c). This

10.

Driscoll, J. D. et al. J. Neurophysiol. 105, 3106–3113 (2011).

is particularly useful for samples that exhibit sub-regions with very

11.

Bajzer, Ž., Therneau, T. M., Sharp, J. C. & Prendergast, F. G. Eur. Biophys. J. 20, 247–different photon regimes. To illustrate this point, we imaged HeLa cellswith very variable expression of the EPAC cAMP FLIM sensor13 (Fig.

262 (1991).
12.

2d). All cells have equal cAMP levels, corresponding to a measured
lifetime of ~3.5 ns (yellow pixels in Fig. 2d). The ‘first photons’ and ‘allPatting, M., Wahl, M., Kapusta, P. & Erdmann, R. in International Congress on Optics
and Optoelectronics Vol. 6583 (ed. Dusek, M. et al.) 658307 (SPIE, 2007).

13.

photons’ filters show severe mistakes in the apparent lifetime at high-Klarenbeek, J., Goedhart, J., van Batenburg, A., Groenewald, D. & Jalink, K. PLoS
ONE 10, e0122513 (2015).

photon fluxes. The high-speed FLIM filter captures accurate lifetime

14.

van de Wetering, M. et al. Cell 161, 933–945 (2015).

values for all photon fluxes, up to 2.5 photons per pulse.

15.

Komatsu, N. et al.. Mol. Biol. Cell 22, 4647–4656 (2011).

16.

Raspe, M. et al. Nat. Meth. 13, 501 (2016).

To quantify the effects of each filter, we performed lifetime
measurements with a rhodamine B solution at increasing laser
powers, with a minimum of 1 million counts per decay (Fig. 2e). We
initially performed a measurement with 0.01 photons per pulse andset it as the reference lifetime value. These measurements allowed us
to directly see the effect of increasing count rates on the accuracyof lifetime determination. We note that with the ‘first photon’ filter,
the pile-up effect already becomes a significant issue at 20–40

This article was submitted to Nature Methods by a commercial organizationand has not been peer reviewed. Nature Methods takes no responsibility forthe accuracy or otherwise of the information provided.

NATURE METHODS | OCTOBER 2019