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Image Gallery: FLIM for high-speed investigation of molecular interactions

Fluorescence Lifetime Imaging with the SP8 FALCON platform.

SP8 FALCON (FAst Lifetime CONtrast) is the fast and fully integrated platform for fluorescence lifetime imaging (FLIM) and analysis. It enables the observation of fast dynamic cellular events with lifetime contrasts between fluorescent molecules down to sub-second time scales. With the SP8 FALCON platform, it is now possible to measure molecular interactions with Förster Resonance Energy Transfer (FRET) by exploiting FLIM at confocal speed.

The lifetime of autofluorescence and of fluorescent dyes changes depending on the local environmental conditions, such as pH, ion concentration, binding to other molecules, etc. The SP8 FALCON platform can measure these fast changes and provide metabolic/functional imaging. The additional lifetime contrast SP8 FALCON allows fluorophore separation.

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Find FLIM images and data acquired with the SP8 FALCON, assorted by the main application.

Dye separation: Rabbit tongue

Differentiation of different structures in histological stainings.

The combination of LASX Navigator and SP8 FALCON allows the acquisition of 50 Mpixels images (2326 µm x 1739 µm) in 14 x 18 tiles with additional lifetime information. Transverse histological slice of a rabbit tongue (Filiform Papillae). Lifetime gives an additional contrast that allows to differentiate different structures in histological stainings.

Dye Separation: Sepal of Silverberry

Lifetime Movie of Sepal of Silverberry

The lifetime contrast of sepal of silverberry allows to separate different structure within the sample. The intensity image (yellow image) doesn't show any specific structure while the FastFLIM (rainbow scale image) and the lifetime fitted images (RGB, three component image) give insigth into the different part of the sample. The end of the video shows the three structures determined by the lifetime fit split into three channels (RGB).

Dye separation: Kidney slice

Observation of different microenvironments in Kidney slice with Lifetime Imaging.

Kidney slice (FluoCells® prepared slide #3): The cell membranes are stained with WGA-Alexa Fluor488. The lifetime contrast displayed in rainbow scale indicates that the membranes are located in very different micro-environmental conditions, e.g. different pH or ion-concentrations. Image size: 2048 x 2048 pixels. Color bar scale (lifetime): ns.

Dye separation: Murine pancreatic Organoids

Differentiation of different structures in multiphoton images.

Multiphoton image of murine pancreatic organoids
Murine pancreatic Organoids imaged with the combination of SP8 DIVE and FALCON. The Lifetime information gives the multiphoton images an additional contrast. The cells of the Organoids (short lifetime = green) can be easily separated from the background fluorescence of the surrounding matrigel (longer lifetime = red).Courtesy of Till Moreth, Lotta Hof, Francesco Pampaloni - Prof. Ernst Stelzer, Buchmann Institute for Molecular Life Sciences.

Dye separation: Speed

Observing physiological processes at increased speed, see here the movement of chloroplasts in life Egeria plant.

The increased speed of SP8 FALCON enables the observation of fast physiological processes, such as the movement of chloroplasts in life Egeria plant. The autofluorescence of the plant was imaged life at 1 frame per second in one spectral channel 500 ‒ 600 nm. The lifetime information allows to separate between chloroplasts (green) and other background autofluorescence (red) at video rate. 256x256 pixels, 1fps.

FLIM-FRET: Metabolic changes observed over time

The Lifetime measurement of FRET-sensors allows fast, easy and robust observation of the binding and unbinding of metabolic molecules, in this example: cAMP fluctuations.

Fluctuation of cAMP in HeLa cells expressing EPAC mT2-dVenus FRET sensor. EPAC response to cAMP-raising agent IBMX and Forskolin. Movie recorded at 2 fps. Image size: 512 x 512 pixels. Color bar scale (lifetime): ns. Courtesy Kees Jalink, Bram van den Broek, NKI Amsterdam.

FLIM-FRET: Single Molecule FRET 

Measuring interaction at the single molecule level.

Measuring interaction at the single molecule level. FLIM-FRET measurement of fluorescently tagged receptor-bound G proteins in detergent micelles, immobilised on a glass coverslip for fluorescence lifetime imaging. A. Intensity of Donor (green) and Acceptor (red), B. FastFLIM image, mean arrival time, C. FRET efficiency image calculated using fluorescence lifetime imaging. Courtesy of Sebastian Furness and Cameron Nowell, Monash Institute of Pharmaceutical Sciences, Monash University https://research.monash.edu/en/persons/sebastian-furness/publications/ https://research.monash.edu/en/persons/cameron-nowell

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