Follow molecular interactions with FLIM-FRET

Modern research investigates how molecules interact to accomplish vital tasks. FLIM-FRET is the gold standard to explore these interactions.

STELLARIS 8 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.

STELLARIS 8 FALCON provides access to information contained in fluorescence lifetime, even for ultrafast events such as membrane potential dynamics. This information complements the spectral fluorescent intensity imaging and TauSense modes delivered by the STELLARIS 8 system.

More reliable and sensitive metabolic imaging

Autofluorescence can be an issue in conventional imaging. STELLARIS 8 FALCON turns it into valuable information. You can now transform autofluorescence into a reporter for metabolic status, cell differentiation, and cancer development.

Moreover, STELLARIS 8 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.

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 STELLARIS 8 FALCON, you can exploit the full potential of fluorescence lifetime imaging to separate multiple fluorescent probes using exponential fitting, pattern fitting and the new FLIM phasor separation analysis.

Interactive 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.

STELLARIS 8 FALCON for fast lifetime imaging

The STELLARIS 8 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 STELLARIS 8 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.

Application note in Nature: SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM

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 STELLARIS 8 FALCON. Until now, researchers had to cope with complex wiring and cumbersome file transfer tasks. With STELLARIS 8 FALCON, you can integrate lifetime information into your standard confocal workflow.

STELLARIS 8 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. STELLARIS 8 FALCON gives you access to lifetime contrast in 3D-stacks, time-lapse sequences, and even large mosaic-tiling formats.

With 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 STELLARIS 8 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.

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: STELLARIS 8 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.

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