SP8 DIVE — DEEP IN VIVO EXPLORER The world’s first tunable deep imaging solution

Confetti mouse small intestines, 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 ca. 700x700x150 µm³. Recorded with two-photon excitation, using the SP8 DIVE. Sample courtesy of J. van Rheenen, Netherlands Cancer Institute, Amsterdam (the Netherlands). Title page: Mouse brain, transgenic and transiently labeled. Different types of nerve cells are shown in green (microglia, GFP), yellow (neurons, YFP), blue (astrocytes,...

SP8 DIVE SPECTRAL FREEDOM FOR MULTICOLOR DEEP IN VIVO IMAGING Spectral Freedom with 4Tune Up to now, the possibilities to do multicolor deep in vivo experiments were limited by the choice of dichroic filters. The SP8 with the brand new and unique 4Tune detector is the world’s first fully spectrally tunable multiphoton microscope. Another degree of spectral freedom results from the newly implemented possibility to use up to three IR excitation lines. Today’s research focuses on complex biological processes such as human diseases. Complexity mainly results from the number of components involved:...

4Tune enables new dye combinations: Confetti mouse small intestine. Cell lineage tracer. Cyan: CFP, green: GFP, yellow: YFP, red: RFP. Colon cancer research. Sample courtesy of Prof. Jacco van Rheenen, Netherlands Cancer Institute, Amsterdam (the Netherlands). ENJOY SPECTRAL FREEDOM Tune your detection and collect more fluorescence with 4Tune Adapt to any transgenic marker The evolution of new fluorescent transgenic markers in combination with genetic tools like the CRISPR/Cas system speed up many research areas and challenges traditional multiphoton imaging technologies. CFP, YFP & RFP with...

♦ Cooled Standard * MP-EYFPi I The intuitive 4Tune user interface of the Leica LAS X software. Up to four channels can be defined and imaged simultaneously - unlimited in the sequential mode. DIVE WITH EASEExperimental set-up made easy with Leica LAS X Optimize your settings live and with minimal effort The intuitive 4Tune user interface lets you optimize the emission settings of multiple fluorophores live and on the fly. Due to its clear and user-friendly design, operation is easy and requires minimal training. If more than four markers are investigated or overlapping spectra require sequential...

LASER RADIATION LASER RADIATION LASER RADIATION LASER RADIATION VISIBLE VISIBLE AND INVISIBLE-3B AND INVISIBLE- CLASS CLASS 3B AVOID DIRECT EXPOSURE TO BEAM BEAM AVOID DIRECT EXPOSURE TO VISIBLE VISIBLE AND INVISIBLE-4CLASS 4 AND INVISIBLE- CLASS AVOID EYE OR SKINOR SKIN EXPOSURE TO AVOID EYE EXPOSURE TO DIRECT OR SCATTERED RADIATION DIRECT OR SCATTERED RADIATION

4 REASONS FOR 4TUNE Flexible Deployment of Fluorescent Markers 4Tune, the heart of the SP8 DIVE, is a unique spectral non-descanned detection unit that enables you to define your detection band freely between 380 and 800 nm with variable bandwidth. The detection can be adjusted live by simple drag and drop. Efficient Photon Collection 4Tune, located at the non-descanned site, enables multicolor deep in vivo imaging in 1 mm depth and beyond. In contrast, confocal detection strongly limits penetration depth in multiphoton mode. Equip your 4 Tune detection with HyD detection and tune the detection...

DIVE ENABLES NEW RESEARCH Lambda Square Scanning Fully explore the photonic landscape of your sample by carrying out an excitation-emission scan using 4Tune detection. Emission spectra are captured exploiting the tunable 4Tune bands, excitation spectra acquisition becomes easy using the newly introduced constant power mode. You can investigate the autofluorescence of your sample or hunt for new fluorescent markers. With only a few mouse-clicks you receive a two-dimensional plot of excitation versus emission for each pixel of your image – a spectral fingerprint. You can either use this to adjust...

Comparison of 2PE with 920 nm (left) and 3PE with 1300 nm (right), 800 pm deep in the live mouse brain cortex. Thyl-eYFP cortical neurons, IRAPO 25x1.0 W motCorr, both images autoscaled from darkest to brightest pixel. Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE, Bonn (Germany). Simplified principles of one-, two - and three-photon excitation. Three photon excitation The new SP8 DIVE can be equipped with laser sources that allow excitation up to 1300 nm. This is the ideal wavelength for three-photon excitation (3PE) of GFP, YFP or other markers within the same spectral range...

LABEL — FREE IMAGING WITH DIVE Second and third harmonics on the fly Second harmonic generation (SHG) has emerged as a powerful label-free imaging modality to visualize fibrillar collagen in diverse tissues. SHG signals occur from large non-centrosymmetric structures with a periodic alignment. These structures double the frequency of the infrared pulsed excitation light used for multiphoton imaging. In conclusion, no labels need to be incorporated and since the light is not absorbed but scattered, photodamage does not occur. The information from this channel can be used in various ways: Either...

Like SHG, third harmonic generation (THG) is a label-free imaging method, however it occurs at exactly one third of the incoming pulsed laser light wavelength. By equipping the SP8 DIVE with a laser source that provides wavelengths up to 1300 nm, THG can be detected in the visible range at 430 nm. Combine THG with Ultra-fast imaging Blood flow happens at very fast time scales. To enable live blood flow tracking, the optional 12 kHz resonant scanner with frame rates up to 428 fps, is the ideal tool. In combination with the ultrasensitive HyD detection even weak signals can be picked up. With 4Tune...

Mouse brain cortex., Thy1-eYFP, Tx Red. IRAPO 25x1.0 W motCorr. Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE Bonn (Germany).