The light sheet microscope for fluorescence imaging from macro view to cellular resolution

In 2009 the first commercial light sheet microscope was launched by LaVision BioTec. Today we present the second generation which has been inspired by our user’s feedback. 

The bidirectional triple light sheet technology of the UltraMicroscope II generates 6 focused light sheets to excite samples from the side while the fluorescence light is detected by a sCMOS camera perpendicular to the illumination plane. Moving the sample through the light sheet generates a 3D image stack. Selective excitation of the focal plane reduces bleaching and photo toxicity significantly. The open setup allows the analysis of cleared samples in any clearing solution or in aqueous media. The dynamic horizontal light sheet focus guarantees excellent Z-resolution covering the entire field of view.

Technology: Six light sheets with an adjustable numerical aperture and horizontal focusing for a better homogenous illumination.

Clearness: The large and open sample  chamber is directly accessible and can be loaded with all current clearing solutions.

Usability: Samples are inserted from the top into the chamber. The sample exchange is done without removing the imaging solution.

Versatility: Available add-ons for in vivo imaging and for refractive index corrected objective lenses for higher magnification.

About the UltraMicroscope

Features and modules

  • Imaging in aqueous buffers and organic solvent
  • Optical zoom from 1.26x to 12.6x
  • Large sample uptake up to 30 mm x 30 mm x 15 mm
  • Dynamic horizontal focusing for better Z resolution
  • 6 light sheets for homogenous illumination
  • Easily accessible sample chamber
  • Large field of view with a diagonal of 17.6 mm
  • Additional modules for in vivo imaging or for infinity corrected optics

Imaging chamber for in vivo applications with connectors for environmental control.

LVMI-Fluor objective lenses

The UltraMicroscope II infinity corrected optics setup delivers superior imaging capabilities and user friendliness. It can be mounted directly to the focusing unit instead of the zoom body. It allows the implementation of infinity corrected objective lenses like the LaVision BioTec multi immersion objective lenses. These objectives were designed for different refractive indices ranging from water with a refractive index of 1.33 to 1.56 for benzyl ether. The LVMI-Fluor objectives can operate in organic solvents and in aqueous buffers. Today, diverse clearing protocols have been established. The UltraMicroscope II can be used for all current applications reaching from in vivo imaging to cleared tissue imaging. The modular UltraMicroscope with its flexible design offers the possibility to operate the microscope also with standard dipping objectives.

The UltraMicroscope module for in vivo imaging

The in vivo setup for the UltraMicroscope II ensures constant environmental conditions within the sample cuvette considering the temperature and the CO2/O2 atmosphere. All settings are controlled by a touch-screen. 
The sample feedback mode measures the temperature at the sample with an additional temperature sensor. Thus, the temperature of the thermostat is automatically adjusted to the feedback of this sensor. The heating element and the sample holder can be easily unmounted for autoclaving. This in vivo imaging setup includes:

  • in vivo sample cuvette
  • Temperature control unit
  • Active CO2 controller
  • Active humidity module
  • Touch-screen control panel
  • Feedback temperature sensor 

Ultramicroscope downloads

Literature on Light Sheet Microscopy

Multi refractive index compensation

Several different clearing procedures for fixed tissue have been developed and utilized. Most clearing reagents differ from protocol to protocol and so does the refractive index. Running a system with different clearing or imaging solutions induces the necessity to correct for different refractive indices. The refractive index compensation of the UltraMicroscope II is utilized via the software interface. The user chooses between current clearing techniques such as CLARITY* or Benzyl Ether. Water can also be selected for in vivo imaging. This technology guarantees the perfect setting for every imaging solution. The UltraMicroscope II is the only light sheet microscope handling organic clearing solutions as well as aqueous buffers. 

UltraMicroscope imaging chamber with horizontal double-sided excitation and perpendicular detection.

Bidirectional triple light sheet microscopy 

Exciting the fluorescent sample perpendicular to the detection by a focused light sheet offers two major advantages: First, it allows 3D microscopy utilizing a wide field microscope. A camera-based wide field microscope delivers much higher frame rates than any laser scanning microscope. Second, the combination of wide field microscopy and sheet excitation reduces bleaching and photo toxicity significantly. LaVision BioTec’s UltraMicroscope II comes with up to six light sheets that excite the sample from different angles. This means the fluorescence excitation is most homogenous and artifacts like dark areas and stripes are minimized.

 

Double-sided excitation for more homogenous sample illumination.

Variable light sheet parameter

The UltraMicroscope II is the flexible solution for a variety of applications and diverse samples. The UltraMicroscope II has an adjustable light sheet that allows the user to set width, NA and Rayleigh length of the focused light sheets. This unique feature helps to meet the demand for the highest flexibility. The user can choose the perfect matching settings for any kind of sample via the software. In fully automated mode, the software selects the settings. Together with the multi refractive index compensation and the chromatic correction from 400 nm to 800 nm, the UltraMicroscope II can be adapted to different samples and clearing protocols.

Dynamic horizontal focus

Custom-made optics form two triple light sheets that are focused into the sample. Z-resolution and contrast is best within the light sheet focus. To optimize this, the focus diameter and the focus length [Rayleigh length] can be adapted to the imaging conditions and will be optimized by software. Then, the UltraMicroscope II provides the dynamic horizontal focus that shifts the focus through the sample while imaging. In combination with advanced software algorithms, the UltraMicroscope II delivers pin sharp 3D image of the sample.

Horizontal dynamic focusing ensures a homogenous PSF over the entire field of view

Selected articles:

Nature 2019 Apr
Compartmentalized gut lymph node drainage dictates adaptive immune responses
Esterházy D, Canesso MCC, Mesin L, Muller PA,  de Castro TBR, Lockhart A,  ElJalby M, Faria AMC, Mucida D

J Biophotonics. 2018 May;11(5):e201700248. doi: 10.1002/jbio.201700248. Epub 2018 Jan 30.
Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.
Matryba P, Bozycki L, Pawłowska M, Kaczmarek L, Stefaniuk M

Nat Commun. 2018 Apr 12;9(1):1422. doi: 10.1038/s41467-018-03802-y.
GABAergic inhibition in dual-transmission cholinergic and GABAergic striatal interneurons is abolished in Parkinson disease.
Lozovaya N, Eftekhari S, Cloarec R, Gouty-Colomer LA, Dufour A, Riffault B, Billon-Grand M, Pons-Bennaceur A, Oumar N, Burnashev N, Ben-Ari Y, Hammond C.

J Neuroendocrinol. 2017 Dec;29(12). doi: 10.1111/jne.12551. PMID: 29044774
Three-dimensional distribution of tyrosine hydroxylase, vasopressin and oxytocin neurones in the transparent postnatal mouse brain.
Godefroy D, Dominici C, Hardin-Pouzet H, Anouar Y, Melik-Parsadaniantz S, Rostène W, Reaux-Le Goazigo A.