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Confetti Mouse Small Intestine. CFP, GFP, YFP and RFP
Sample courtesy of Jacco van Rheenen, University of Utrecht, the Netherlands

Enjoy spectral freedom with 4Tune

The number of transgenic markers evolves constantly and rapidly.  The SP8 DIVE keeps apace with these developments and can adapt to existing and new transgenic markers with just a few mouse clicks.

Spectral freedom results from the new groundbreaking 4Tune technology for non-descanned detection. The 4Tune detector allows you to adapt to the full emission and to separate strongly overlapping spectra – without mathematical restoration.

The SP8 DIVE enables you to capture twice as much of the fluorescence signal, which translates into improved penetration depth, imaging speed, or less 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 the wavelength for each fluorophore you want to detect."

Prof. Dr. Jacco van Rheenen. University of Utrecht, the Netherlands

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

“Its 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 its very interesting.”

Kees Jalink

Netherlands Cancer Institute

"I just got a demo and I am flabbergasted. I think its 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. 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 its 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."

DIVE with ease – the 4Tune detector

The 4Tune non-descanned detector can be equipped with 2 to 4 detection units 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. The tuning ranges from 380 up to 800 nm wavelength.

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 graph: Rapid increase of markers
Bottom: 4Tune user interface
Mouse leg. Muscle fiber labeled with Rab5-GFP. IRAPO 40x 1.1. Left: Best resolution. Right: Deep penetration. Using the deep penetration setting approx. doubles the brightness. As a consequence, penetration depth is increased. Sample courtesy of Prof. Rudolf, University of Applied Science, Mannheim.

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 (top image)

You can choose the best individual balance between resolution and energy input by tuning the diameter of the beam. When you widen the beam, you obtain homogeneous illumination of the back focal plane. If used in combination with a high numerical aperture, maximal resolution is reached.

Narrowing the beam will allow more energy input into the focal volume, resulting in a better penetration of the sample and deeper imaging. With the VBE, you can adjust the beams independently, for up to four different IR (infrared) wavelengths, to find the best balance between resolution and depth.

Tunable beam divergence for full color correction (bottom image)

Introducing an input-divergence corrects for the axial color aberration of different IR wavelengths. This allows you to excite at the same focal point in the specimen with up to four IR wavelengths.

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

Multicolor excitation: 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 tunable excitation lines simultaneously, balanced by acousto optical modulation. Since the lasers are tunable up to 1300 nm, you can even use red and far red dyes for multiphoton experiments. Less scattering enables deeper penetration and results in bright images full of detail from deep layers.

TuneIR - Consistantly perfect instrument conditions allow decisive insights: For constantly exact results at any time, reliable and stable conditions for the instrument and the IR beams are paramount. To that, we combine a novel beam catcher unit with our Vario Beam Expander. We make sure you get reproducible and perfect imaging conditions for up to three tunable excitation lines and perfect colocalization of multiple markers.

DIVE – a member of the SP8 platform

The SP8 DIVE is the latest addition to the established SP8 platform from Leica. The platform can be configured to accommodate different research methods ranging from confocal microscopy with Super-Resolution to STED Nanoscopy.

For users, this delivers both versatility and investment protection.

All instruments of the SP8 platform are open and adaptable to the user’s research, both now and in the future.

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