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Image Gallery: Tunable Multicolor Deep In Vivo Imaging

With the SP8 DIVE Multiphoton Microscope

Today’s life science research focusses on complex biological processes, such as the causes of cancer and other human diseases. A deep look into tissues and living specimens is vital to understanding the conditions and mechanisms in cells and finding answers to crucial questions challenging the life sciences.

The SP8 DIVE Deep In Vivo Explorer is a multiphoton microscope with spectrally tunable non-descanned detection that provides unlimited flexibility, so that users can optimally distinguish components and structures deep in tissues and living organisms. The SP8 DIVE comes with the unique 4Tune detection that offers full spectral freedom and allows the detection of multiple transgenic markers with perfect color separation in a single experiment.

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See the image gallery with examples from a range of applications that the SP8 DIVE can be used for.

Deep in vivo multicolor imaging

Small intestines of a confetti mouse, fluorescently labeled and lineage traced from a multi-color tracer. The gray color codes for the SHG signal from collagen. Lineage traced stem cells are shown in cyan (CFP), green (GFP), yellow (YFP), and red (RFP). Image size is approximately 700x700x150 μm³. Recorded with two-photon excitation using the SP8 DIVE. Sample courtesy of J. van Rheenen, Netherlands Cancer Institute, Amsterdam (the Netherlands).
Transgenic and transiently labeled mouse brain. Different types of nerve cells are shown in green (microglia, GFP), yellow (neurons, YFP), and blue (astrocytes, sulforhodamine), while the blood system is shown in red (Alexa680-dextran). Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE Bonn (Germany).

Label-free imaging: Second (SHG) and third harmonic generation (THG)

The small intestine of Lgr5CreERT2-GFP confetti mice. Gray: SHG signal (collagen 1) which is used as an orientation landmark. Green: Lgr5+ stem cells. Red, cyan, and yellow, traced cells that are progeny of the Lgr5 stem cells. Sample courtesy of Prof. Jacco van Rheenen, Netherlands Cancer Institute, Amsterdam (the Netherlands).
Mouse brain cortex. Thy1-eYFP cortical neurons, THG blood cells. Excitation: 1300 nm. Objective: IRAPO 25x1.0 W motCorr. 70 fps, 256x256 px. Sample courtesy of Kevin Keppler, Light Microscopy Center, DZNE, Bonn, Germany.

Spectral unmixing

Effortless separation of GFP (green) and YFP (red) expressed in the cortex of a live mouse by capturing either two channels simultaneously or conducting a spectral emission scan and applying channel unmixing in the LAS X software. Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE, Bonn (Germany).

Three photon excitation

The brain cortex of a live mouse. Thy1-eYFP is expressed in the cortical neurons. Imaged at 450 μm depth in the cortex with 1300 nm excitation wavelength. Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE, Bonn (Germany).

Deep in vivo colocalization

Striated muscle from a mouse tibia. Morphological analysis of neuromuscular junction development. Green: GFP – Rab 5 (GTPase, endosomes). Red: mCherry – Bif 1. Gray: AF647- Alpha bungarotoxin (NMJ). Sample courtesy of Prof. Rudolf, University of Applied Science, Mannheim (Germany).
Mouse intestine. Blue: Alexa Fluor 350 - wheat germ agglutinin mucus of goblet cells. Green: SYTOX Green - Nuclei. Red: Alexa Fluor 568-Phalloidin - F-Actin, prevalent in the brush border. White: SHG. Sample courtesy of Molecular Probes.

FLIM

Autofluorescence in mammalian cells under non-physiological conditions (pH 8.5). The signal correlates with changes in the NAD/NADH endogenous pool. The development of oxidative stress over time reads out as decrease of fluorescence lifetime. Images size: 512 x 512 pixels. Color scale bar has units of ns. Images are extracts from a movie taken with the SP8 FALCON and SP8 DIVE.

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