Fluorescence Lifetime-based Imaging Gallery

TauSense and FLIM with STELLARIS confocal platform

 tauSep_mneonMTgreen-NucRedSiRTub_3to2.jpg

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.

Calcium waves in mammalian cells

Calcium oscillation after mechanical stimulation in mammalian cells loaded with OregonGreen 488. The response in individual cells is recorded as a change in TauContrast. Time series acquired at 4.5 fps, TauContrast traces in different cells (ROI selected in different color). Image size: 256 x 256 pixles, Rainbow LUT (TauContrast): 1.0-4.0ns

Live NE-115 cells expressing mNeonGreen-LifeAct - STELLARIS

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

2 intensity channels.

4 lifetime-based channels

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 distingushed with TauSeparation tool. It results in 4 color image: Actin: LifeAct-mNeonGreen (left: yellow, right: red); mitochondria: MitoTracker Green (left: yellow, right: green); nuclei: NUC Red (left: gray, right: blue); and tubulin: SiR-tubulin (left: gray, right: magenta). Sample courtesy: Max Heydasch, University of Bern, and Spirochrome.

Fast 3D in vivo imaging of Nematostella vectensis

Fast 3D in vivo imaging of Nematostella vectensis (Cnidaria) showing endogenous signal for cilia, nematosomes and clusters of freely circulating cnidocytes (green) and dextran red fluorescence (magenta). Endogenous signals and fluorescence separated by TauGating on HyD S, 340 volumes acquired in 12 min 45 s. Scale bar, 50 μm. Sample courtesy of Anniek Stokkerman and Aissam Ikmi, EMBL Heidelberg.

Lifetime-based species separation in live cells 

Intensity

TauSeparation

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

Kita mycelia expressing mCherry at the membrane and in the interior​

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.

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