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Fluorescence Lifetime-based Imaging Gallery

TauSense and FLIM with STELLARIS confocal platform

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Confocal microscopy relies on the effective excitation of fluorescence probes and the efficient collection of photons emitted from the fluorescence process. One aspect of fluorescence is the emission wavelength (spectral characteristics of a fluorophore). Another one, very powerful albeit less explored, is the fluorescence lifetime (the time a fluorophore stays in the excited state). Information based on fluorescence lifetime adds an additional dimension to confocal experiments, revealing information about the fluorophore microenvironment and enabling multiplex of spectrally overlapping species.

FLIM imaging with STELLARIS confocal platform

This image gallery shows applications of TauSense and fluorescence lifetime imaging (FLIM) on the STELLARIS confocal platform.

3D confocal image of C. elegans neurons labelled with four red fluorescent proteins.

Image acquired with STELLARIS 8 FALCON with a single excitation line and a single detector. The 4 distinct signals were obtained with phasor separation. The use of a single laser line and single detector improves live imaging speed and lowers the laser dose. Grey: Intensity only. Colors: Merged phasor-separated signals. Scale bar: 20 μm. Tracking 4 neuronal types with 4 red fluorescent proteins on a single detector with FLIM.

3D confocal image of C. elegans neurons 3D confocal image of C. elegans neurons

3D confocal image of C. elegans neurons labelled with four red fluorescent proteins

Transcription activity in mice gastric stem cell derived organoid

Hoechst channel labelling the nucleus. The differences in fluorescence lifetime corresponds to difference in DNA compaction. The longer lifetimes (red) correspond to cells with more open reading frames and an active DNA transcription such as stem cells. Video acquired with multiphoton (DIVE) and lifetime (FALCON).

Endocytic pathway and mitochondria dynamics

Mitochondria labelled with MitoView Green. Membrane vesicles labelled with CellBright NIR750. The distinct endocytic steps are revealed with TauSeparation with lifetime component separation based on pH maturation steps correlated to endocytic maturation of vesicles

Live NE-115 cells expressing mNeonGreen-LifeAct - STELLARIS

Live NE-115 cells expressing mNeonGreen-LifeAct are stained with MitoTracker Green, NucRed and SiR Tubulin.

Live NE-115 cells expressing mNeonGreen-LifeAct are stained with MitoTracker Green, NucRed and SiR Tubulin. Signals are acquired with 2 detectors for two fluorophores each. Intensity image shows in yellow and gray. Signal in each detector will be distingu

Exploring Chloroplast activity with multiphoton and fluorescence lifetime imaging

Image acquired with a STELLARIS 8 DIVE FALCON.

The multiphoton transmission image shows the leaf structure, the endogenous chloroplast fluorescence is shown in green. Then the Fluorescence lifetime imaging fingerprint from a phasor analysis converts this endogenous to colors depending on the oxidative potential for each chloroplast.

Mammalian cells expressing LifeAct-GFP (manufactured by ibidi GmbH) and labeled with a MitoTracker Green. Acquisition with one detector, separation performed by TauSeparation.

Root-hypocotyl junction of Arabidopsis thaliana

Nature is multi-dimensional. To truly comprehend its complexity, we need to look in multiple dimensions. This detailed multicolor image of Arabadopsis thaliana root junction was obtained with just one click and one detector. With conventional confocal microscopy, it would be black and white. With STELLARIS what we see is transformed by an extra dimension of lifetime information. Not only is the remarkable architectural diversity of the actin-containing root system revealed with more clarity, but also its relationship to the cell wall and chloroplasts—structures that would be difficult or impossible to distinguish using the intensity data alone. The result is not just a snapshot of nature’s beauty, but an information-rich image that progresses our understanding of plant biology.

Root-hypocotyl junction of Arabidopsis thaliana
Root-hypocotyl junction of Arabidopsis thaliana: actin (Lifeact-Venus, Era et al. Plant Cell Physiol., 2009), chloroplasts (endogenous fluorescence), and cell wall (Propidium iodide). The intricate details of the actin network, cell wall and chloroplasts come to light using fluorescence lifetime-based information. Image acquired with TauContrast. Sample courtesy: Dr. Melanie Krebs, COS, University of Heidelberg.

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