Bidirectional Triple Light Sheet Microscopy
UltraMicroscope II
Six variable light sheets for perfect 3D fluorescence
microscopy from macro view to cellular resolution in organic solvents and aqueous buffers
The bidirectional triple light sheet technology of theUltraMicroscope II generates 6 focused light sheets toexcite samples from the side while the fluorescence lightis detected by a sCMOS camera perpendicular to theillumination plane. Moving the sample through the lightsheet generates a 3D image stack. Selective excitationof the focal plane reduces bleaching and photo toxicitysignificantly. The open setup allows the analysis of clearedsamples in any clearing solution or in vivo data acquisitionin aqueous media. The dynamic horizontal light sheetfocus guarantees excellent Z-resolution covering the entirefield of view.
• Imaging in all kinds of clearing solutions:CLARITY, CUBIC, DBE, iDISCO, uDISCO,Scale, SeeDB ...
• Imaging of living animals or cleared samples
• Whole brain, organ and embryo imaging at cellular resolution
• Sample size over 1 cm to few µm
• Easily accessible sample chamber
• Improved Z sectioning by dynamic horizontal light sheet focus
• Bidirectional triple light sheet microscopy for homogenous illumination
• High magnification objective lens setup
• 3D fast image acquisition of large samples
Mouse hippocampus, Thy-1 GFP; courtesy of Bianca Schmid, Max Planck Institute for Psychiatry, Munich, Germany.
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 a pin sharp 3D image of the sample.
A) Bidirectional fluorescence excitation with up to six light sheets perpendicular to the detection. B) Dynamic horizontal focus fluorescence excitation of the sample positioned by up to six focused light sheets (travel range 1 cm x 1 cm x 1 cm). C) Variable light sheet NA and dynamic horizontal focus.
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.
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.
in vivo imaging
The in vivo setup for the UltraMicroscope II ensures constant environmental conditions within the sample cuvette considering the temperature and the CO 2 /O 2 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
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.
The UltraMicroscope II Setup
The UltraMicroscope II configuration delivers superior imaging capabilities and user friendliness. Just choose the camera, objective lens and laser light source to adapt the UltraMicroscope II to your application:
• sCMOS camera technology for high quality images
• Double triple sheet excitation allows a consistent illumination
• High resolution microscopy with custom 20x objective lens
• Low magnification lenses for macro imaging
• Laser module with up to five laser diodes or supercontinuum white light laser for maximum flexibility
Infinity Corrected Optics Setup
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 with standard dipping objectives.
LVMI-Fluor 4x objective lens:
Objective lens
LVMI-Fluor
Magnification
4x
NA
0.3
Working Distance (WD)
6 mm
Immersion
organic solvents & aque-ous buffers
Refractive Index (RI)
1.33 - 1.56
Sheet optics
Illumination
Uni- & bidirectional
Number of light sheets
3 – 6
Thickness
4 µm – 24 µm
Width
1 mm – 20 mm
Numerical aperture
0.0135 – 0.135
Focus positioning
Dynamic
Refractive index matching
1.33 – 1.56
Zoom microscope for 2x
objective lens
Zoom
Mono zoom
Zoom ratio
0.63x – 6.3x (1:10)
Detection optics
Objective lenses
2x
4x
Numerical aperture
0.5
0.3
FOV diagonal (mm)
1.7 - 17.6
5.4
Total magnification (objective lens +
zoom ratio)
1.26x – 12.6x
(w/o Zoom)
Working distance
4 mm, 6 mm, 10 mm
6 mm
Refractive index matching
1.33 – 1.56
Chromatic detection
Seven filters
Chromatic correction
Dynamic 400 nm – 850 nm
Detector
Type
sCMOS
Pixel
2560 x 2160
Pixel size
6.5 µm x 6.5 µm
Maximum frame rate
100 fps @ full frame
Read noise
1 e-
Imaging chamber
Imaging solution
Aqueous buffers and organic solvents
Sample travel range (X, Y, Z)
1 cm, 1 cm, 1 cm
Sample size
µm range to cm range
Light source
Laser module
Max. 5 laser lines, 50 mW - 100 mW per diode
Supercontinuum laser
Emission 460 nm – 800 nm, 1 mW/nm – 3 mW/nm
Dimensions
54 cm x 70 cm x 65 cm (W x H x D)
Weight
47 kg (w/o controller and laser)