Contact & Support

Follow molecular interactions with FLIM-FRET

Modern research investigates how molecules interact to accomplish vital tasks. Successful biomedical studies use FLIM-FRET to explore these interactions.

SP8 FALCON sets a new speed standard for FLIM instruments.

It enables FRET in highly dynamic cellular events. You can acquire and interpret FRET data in your daily experiments.

Caged cAMP in HeLa cells expressing EPAC mT2-dVenus FRET sensor. EPAC response to UV-mediated cAMP uncaging (central area). Movie recorded at 4 fps. Image size: 256 x256 pixels. Color bar scale (lifetime): ns. Courtesy Kees Jalink, Bram van den Broek, NKI Amsterdam.

Calcium oscillations after stimulation with thrombin activating peptide. The response in individual cells is recorded as a change in lifetime. Movie acquired at 4 pfs. Image size: 256 x 256 pixels. Color bar scale (lifetime): ns. Courtesy Kees Jalink, Bram van den Broek, NKI Amsterdam.

Monitor subtle and fast changes with biosensors

Biosensors are powerful reporters for metabolic activity, signaling mechanisms, pH and microenvironment changes.

SP8 FALCON provides access to this information contained in fluorescence lifetime, even for ultrafast events such as membrane potential dynamics. This information complements the already powerful spectral imaging delivered by the SP8 platform.

More reliable and sensitive metabolic imaging

Autofluorescence is an issue in conventional imaging.

SP8 FALCON turns it into valuable information.

You can now transform autofluorescence into a reporter for metabolic status, cell differentiation, and cancer development.

Moreover, SP8 FALCON enables imaging contrast in living tissue, where fluorescence labeling is often unspecific or destroys physiological conditions.

Autofluorescence in mammalian cells at non-physiological conditions (pH 8.5). The signal correlates with changes in the NAD/NADH endogenous pool. The development of oxidative stress reads out as decrease of fluorescence lifetime over time. Original image size: 512 x 512 pixels. Color bar scale (lifetime): ns. Two-photon image acquisition performed with SP8 DIVE and SP8 FALCON.

Fluorophore separation beyond the spectral options

Fluorescence labeling is the standard way to differentiate intracellular structures. Spectral separation is very powerful, but sometimes limited when the emission spectra are too close.

With SP8 FALCON, you can overcome this limit by using the additional fluorescence lifetime dimension. This unlocks the potential of separating multiple fluorescent probes.

You can enjoy even more freedom by combining options of the SP8 platform. Options include the white light laser excitation source, acousto-optical beam splitter and multichannel spectral detection.

Image: Cytoskeleton structures distinguished by lifetime contrast. Vimentin immunolabeled with Alexa Fluor 555 (green), and tubulin immunolabeled with Alexa Fluor 546 (blue). The fluorophores are spectrally very similar, but they are separated using the fluorescence lifetime information. Image size: 512 x 512 pixels.

SP8 FALCON Imaging is FAst Lifetime CONtrast

The SP8 FALCON microscope overcomes the speed limitation of FLIM and opens the door to fast lifetime data.

Up to now, functional information extracted from fluorescence lifetime data of fast processes was hard to get due to the technical constraints of FLIM. FLIM acquisition speed was at least 10 times slower than recording the confocal intensity.

Using SP8 FALCON fast lifetime contrast imaging, you can follow dynamic processes in cells at the appropriate pace. These tasks are possible thanks to a new way to measure time using TCSPC (Time-Correlated Single Photon Counting) together with intelligent algorithms for data handling and analysis.

Single channel image of fluorescent beads (magenta) in a solution containing Alexa Fluor 555 (green). Fluorophore separation based on fluorescence lifetime is possible at various speeds, for example 16 fps (top), 27 fps (video-rate, middle), and 83 fps (ultra-fast, bottom). Using lifetime information for dye separation is significantly more advantageous than intensity (grayscale). The movies show pixel-by-pixel fitting of the lifetime components. Frame size: 512 x 64 pixels. Scale bar: 10 µm.
Straightforward acquisition of complex samples. High resolution mouse embryo mosaic image of 722 tiles containing 190 Megapixels. FLIM data fitted with four characteristic fluorescence lifetimes, color coded. Acquisition: 1:23 h. Analysis: 1:00 h

All-in-one multimodal imaging

Combining FLIM with other modalities was never as easy as with the SP8 FALCON. Until now, researchers had to cope with complex wiring and cumbersome file transfer tasks. With SP8 FALCON, you can integrate lifetime information into your standard confocal workflow.

SP8 FALCON is fully integrated in the LAS X acquisition and analysis software. It can record FLIM in four spectral channels simultaneously and up to 10 channels sequentially. SP8 FALCON gives you access to lifetime contrast in 3D-stacks, time-lapse sequences, and even large mosaic-tiling formats.

With the new LAS X NAVIGATOR, you can expand your viewing area up to 10,000 times, saving precious time in identifying your regions of interest and exploring your samples in a whole new way.

Straightforward lifetime definition with phasors

Analysis with SP8 FALCON using FLIM phasors provides a 2D visualization of lifetime components. With FLIM phasors you can follow microenvironmental changes, select components to multiplex signal and determine FRET efficiency.

Phasor FLIM allows resolution improvement in STED images. It is possible to achieve the same resolution with less STED depletion power (Lanzanò et al., Nature 2015).

Cells labeled with Alexa555-Phalloidin and H2B mCherry. Separation performed using FLIM phasors. The phasor plot clearly shows two distributions. Courtesy: Dr. Martin Stöckl, Department of Biology, University Konstanz, Germany.
1-click philosophy to focus on your science: SP8 FALCON controlled by the LAS X software

The results you need – with one click

The LAS X software enables FLIM with a click and the same philosophy as the routine spectral imaging.

Even if you use microscopy as a complementary technique, you can find the essentials and start imaging right away.

More specialized functions are accessible as workflows and allow automation for your convenience.

Investigate molecular dynamics with SP8 FCS

Fluorescence Correlation Spectroscopy (FCS) is a quantitative technique in microscopy. It is applied to study particles concentration, diffusion coefficient, viscosity, molecular mass, binding constant and photo-physical properties based on intensity fluctuations.

Combining FCS with FLIM enables Fluorescence Lifetime Correlation Spectroscopy (FLCS) experiments. FLCS is used to study the interaction of molecules that are spectrally not separable and to eliminate background contribution.

STED decreases the focal volume. For this reason, STED FCS provides molecular information even at high concentration ranges (>100 nM) where confocal FCS encounters its limit.

Image top: Intensity traces and autocorrelation curves for different concentration of Atto488, the amplitude decreases by increasing the concentration. Image bottom: excitation volume in the case of confocal (left) and 3D STED (right). The smaller volume in the case of STED enables measurement of fluctuations at higher molecular concentration.
Prof Christian Eggeling, Prof. Enrico Gratton, Prof. Scott E. Fraser, Prof. Kees Jalink

The new Leica FLIM/FCS solution will boost their applicability - fantastically fast, flexible and straightforward to use." - Prof Christian Eggeling, University of Oxford

Most FLIM instruments are attachments, this is a total change of perspective: having a truly integrated system – very powerful!" - Prof. Enrico Gratton, UCI Samueli, University of California, Irvine

The Leica SP8 FALCON is the first commercial system that offers integrated confocal and lifetime imaging that I can imagine using in a core facility environment." - Prof. Scott E. Fraser, Ph. D., University of Southern California, Los Angeles

We have scrutinized the Leica SP8 FALCON. It is every bit as accurate as dedicated TCSPC solutions, but with an intuitive interface and at turbo-speed.” „In my mind, this is a game changer for functional imaging. Prof. Kees Jalink, Ph. D., Netherlands Cancer Institute, Amsterdam

Member of the TCS SP8 family

The SP8 FALCON is the latest addition to the established SP8 platform from Leica Microsystems. The platform can be configured to accommodate different research methods, ranging from confocal microscopy with super-resolution to STED nanoscopy.

For users, this delivers both versatility and investment protection.

All instruments of the SP8 platform are open and adaptable to the user's research, both now and in the future.

Top: Versatile platform SP8 (left), Nanoscopy (middle), Super-Resolution (right)
Bottom: CARS (left), Light Sheet (middle), Multiphoton (right)