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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 ca. 700x700x150 μm³. Recorded with two-photon excitation, using the SP8 DIVE. Sample courtesy of J. van Rheenen, Netherlands Cancer Institute, Amsterdam (the Netherlands).

Enjoy spectral freedom with 4Tune

The number of transgenic and synthetic markers evolves constantly and rapidly. With the SP8 DIVE you can keep up with these developments and adapt to existing and new fluorescent markers with just a few mouse clicks!

Spectral freedom results from the patent-pending, groundbreaking 4Tune technology for non-descanned detection. 4Tune detection allows you to adapt to the full emission and to separate strongly overlapping spectra.

The SP8 DIVE enables you to optimally adapt to the emission of your markers – you can capture twice as much of the fluorescence signal, improve penetration depth, imaging speed and reduce phototoxicity for in vivo imaging.

The conventional dichroics are never optimal to distinguish all fluorophores, but with the spectral detectors, this is now possible and much easier, since we can really optimize for each fluorophore the wavelengths you want to detect.

Prof. Dr. Jacco van Rheenen. Netherlands Cancer Institute, Amsterdam (the Netherlands).

DIVE with ease – the 4Tune detector

The 4Tune non-descanned detection system can be equipped with 2 to 4 detectors and is freely configurable with hybrid detectors (HyDs), photomultipliers (PMTs), or a mix of both. The emission light is separated by a combination of variable dichroics and bandpass filters. Freely tune your detection over the whole visible spectrum (380 – 800 nm)!

The 4Tune user interface allows you to optimize the emission setting for multiple transgenic markers by simple drag and drop. Due to its clear and intuitive design, operating it is easy and requires minimal training.

With the SP8 DIVE, you are equipped for every existing and newly developed transgenic marker and future proved for new developments!

Top: 4Tune non-descanned detection system: 1) Variable Dichroic (VD). 2) Variable Bandpass (VB). 3) HyD or PMT.
Bottom: The intuitive 4Tune user interface allows easy setting of the detection windows for all colors from 380 up to 800 nm.
Mouse brain cortex, Thy1-eYFP. 20% improved penetration depth using Best Depth setting. IRAPO 25x1.0 W motCorr. Sample courtesy of Kevin Keppler, Light Microscope Facility, DZNE Bonn (Germany).

Explore new dimensions in depth

With the SP8 DIVE, you can tune for the deepest insight and finest detail. All excitation beams can be optimally adjusted independently for any objective using the new Vario Beam Expander (VBE).

The VBE allows for optimized colocalization and the right balance of resolution and depth in line with your research question.

Optimize for depth and resolution with the Vario Beam Expander

The Leica Vario Beam Expander VBE combines tunable beam diameter and tunable divergence. This offers you maximum depth, best resolution and full color correction.

Tunable beam diameter for best balance of resolution and depth

The SP8 DIVE allows you to adapt to your sample requirements. Using the Vario Beam Expander, you can choose: Maximum resolution – which results from a fully illuminated back aperture of your objective lens – and maximized penetration depth – resulting from slightly underfilling the back aperture. Underfilling the back aperture results in a larger focal volume and a reduced pathlength which leads to more efficient excitation.

Tunable beam divergence for full color correction

Our IR APO objectives do not suffer from chromatic aberations over the IR range. However with SP8 DIVE you are ready to use any objective lens suitable for IR along with multiple IR laser lines: The Vario Beam Expander can be used to correct for chromatic shifts and enable meaningful multicolor experiments.

Tunable Vario Beam Expander (VBE)
Mouse tibia. Striated muscle. Morphological analysis of neuromuscular junction development. Green: GFP – Rab 5 (GTPase, endosomes). Red: mCherry – Bif 1. Grey: AF647- Alpha bungarotoxin (NMJ). Sample courtesy of Prof. Rudolf, University of Applied Science, Mannheim

Reproducible results for multicolor deep in vivo imaging

Excite multiple transgenic markers in a single experiment with perfect color separation. Or even do localized high precision photomanipulation and simultaneous imaging in a diffraction limited volume. The SP8 DIVE can be equipped with up to three excitation lines simultaneously. With lasers tunable up to 1300 nm, you can even use red and far red dyes for multiphoton experiments. With higher wavelengths, less scattering enables deeper penetration and results in bright images full of detail from deep layers.

Meaningful multicolor experiments require stable conditions. Great mechanical stability combined with a beamcatcher ensure reliable performance. The beamcatcher easily accounts for drifts that are not in your hands – laser tuning, temperature fluctuations or construction work next door – the beamcatcher restores the overlay of IR and visible laser lines by easy software operation. For the most precise alignment expert users can even fine tune more – deep in vivo co-localization becomes feasible!

Get more information from your sample with fluorescence lifetime

4Tune can completely take advantage of fluorescence lifetime imaging (FLIM) using SP8 FALCON (FAstLifetime CONtrast).

This additional flexibility adds a new dimension to multiphoton imaging, enabling signal multiplexing and metabolic imaging.

Image: Autofluorescence multiphoton imaging of wt zebrafish embryo. Lifetime contrast is given by different cofactors and vitamins. In this case NADH (free and protein bound), retinoids and FADH (free and protein bound). Excitation: 740 nm. Emission: 501 - 580 nm. Sample courtesy: Francesco Cutrale, University of Southern California, Los Angeles.

Fluorescence intensity image
FLIM image
The SP8 DIVE with the DM8 CS microscope stand, which is mounted on a cascaded table, offers extended workspace for behavioral research.

Spatial flexibility for behavioral research

For behavioral experiments, equip your SP8 DIVE with the DM8 CS microscope stand with extended workspace. The DM8 CS is mounted on a cascaded table which can be customized to your experimental requirements. The microscope stand provides you with the spatial flexibility for large and complex setups.

The SP8 DIVE with the DM8 CS microscope stand enables you to perform experiments such as the monitoring of brain activities on awake animals.

DIVE – a member of the SP8 platform

The SP8 DIVE is integrated in the established SP8 platform from Leica allowing combinations with other modalities – team it up with SP8 FALCON for additional contrast or add LIGHTNING image information extraction!

Top: Versatile platform SP8 (left), Nanoscopy (middle), Super-Resolution (right)
Bottom: CARS (left), Light Sheet (middle), SMD (right)

Alberto Diaspro

Istituto Italiano di Tecnologia (IIT), Genua

“Great for further disseminating multiphoton microscopy, today. It is a big jump related to the increased range  of applications and the solution of a lot of problems related to the variety of samples. It is worth noting the integration of SHG and two-photon images in an effective way.

I think that the possibility of coupling with other Leica solutions and products, including STED modules, will make this architecture tunable for the different experimental needs from spatial to temporal resolution to forthcoming fluorophores and methods.  

I love the possibility of controlling and visualizing the filling of the back focal plane enlarging or shrinking the two-photon excitation illumination beam. This is great.”

Sophie Allart

Ingénieur de Recherche
INSERM, Centre de physiopathologie de Toulouse Purpan

“It's amazing because of the modularity of the features and because the use of filter cubes in front of NDD detectors impact on the choice of fluorochromes, and sometimes we are obliged to adapt the sample preparation and change fluorochromes. Now it will be much easier.

No, I am convinced. I am doing two-photons for 10 years now, four colors and so it's very interesting.”

Kees Jalink

Netherlands Cancer Institute

"I just got a demo and I am flabbergasted. I think it's absolutely marvelous and you bring anything we had for visible light now to the infrared. You did a really great job."

Ron Hoebe

LCAM, van Leeuwenhoek Centre for Advanced Microscopy, University Amsterdam

“I like the new dual photon spectral detector. What I like most is how it is embedded in the software.

I am managing an imaging center, and the easier it is for the user, the better. We have 200+ users and training them on very complicated machines takes its time.

So the easier it is, the more intuitive it is, the better.”

Ammasi Periasami

W.M. Keck Center for Cellular Imaging, VA

“I like the wavelengths setting from 740 to 920 nm. That is a very good option of what can use. I mean how many wavelengths they want to use depending on the fluorophore. And yes, if they use one wavelength and then have two or three detectors to play around and tune, a lot of options.

The signal to noise ration is very good, flexibility on the emission side.

There is no doubt, it is very important to see the user friendliness of the system. So that's what I am looking for. Since I work with Leica, I follow the user friendliness of these operation of the two photon operation vs. one photon operations."

[During this demonstration at the Focus on Microscopy 2017, data of 4 labels were sequentially acquired using the SP8 DIVE equipped with one IR-laser and two spectral HyD-NDD-detectors. 740 nm excitation (Alexa Fluor 350 for mucus of goblet cells, Sytox Green for cell nuclei) 920 nm excitation (SHG for Collagen, Alexa Fluor 568 phalloidin for F-Aktin).] 

Marc van Zandvoort

Maastricht University, Genetics and Cell Biology

“Very nice extension of the current existing two photon systems. This extension makes the system much more flexible in use of the two photon direct detectors.

So far, that use was very limited, due to the need of separate filters. In that sense it's a very good extension.”

Urs Ziegler

Head of Facility, Center for Microscopy and Image Analysis, University of Zurich

"It's very flexible and very easy to use. You don't have to manually change filters and you can use all the dyes you can think of.
What I appreciate is that, basically, in the software you don't have to train someone who already knows confocal scanning microscopy. You don't have to manually change filters – DIVE is easy to use because it takes care of the different combinations of filters and dyes, which although possible, is not so easy to do. So this is already a major advantage.

And then obviously, in live tissue you have various dyes you can use, second and third harmonics. Using SP8 DIVE this can be done very, very easily. I think this is a major step forward."