UltraMicroscope II - Introduction
In 2009 the first commercial light sheet microscope was launched by LaVision BioTec. Today we present the second generation of our light sheet microscope which has been inspired by our user’s feedback. This new Ultramicroscope utilizes 6 thin light sheets to excite samples while the fluorescence light is detected with a sCMOS equipped microscope that is mounted perpendicular to the plane illumination.
The UltraMicroscope II combines latest imaging components and proprietary light sheet technologies. Recent features including the Dynamic Horizontal Focus (DHF) and the Multi Refractive Index Compensation (RIC) round out one of the most versatile and successful light sheet microscopes.
The UltraMicroscope II serves diverse applications. They share the fact that imaging only a minor part of the sample is not sufficient and distorting artifacts introduced by sectioning have to be excluded. Researchers who need artifact free data from overview to a specific region of interest with cellular resolution implement this technology into their projects. Some samples from neuroscience, developmental biology, immunolgy and oncology can be found here.
Different protocols for clearing samples are currently avialable. Here we give an brief overview of some methods as they are implemented in several diverse fields of applications. The clearing protocol has always to be adapted to the individual sample.
The planar illumination of the focal plane enables 3D image acquisition from macro to micro. View our gallery of samples from different fields of application. If you miss your application please contact us to inquire for more details.
Here you will find links to some of the recent publications of our UltraMicroscope users. If you miss an article which should be added to the list, please contact us.
The UltraMicroscope II configuration delivers superior imaging capabilities and user friendliness. By choosing different modules the light sheet microscope can be adapted to the individual application. The UltraMicrosope II series includes the UM II ST, the UM II HR and the UM II live. This new family of light sheet microscopes covers divers applications.
Yes. The UltraMicroscope is capable to image samples cleared with organic solvents but also aqueous buffer cleared samples. The following UltraMicroscope papers give an overview of the clearing procedures:Optimization of CLARITY for Clearing Whole-Brain and Other Intact OrgansJonathan R. Epp, Yosuke Niibori, Hwa-Lin (Liz) Hsiang, Valentina Mercaldo, Karl Deisseroth, Sheena A. Josselyn, Paul W. FranklandDOI: 10.1523/ENEURO.0022-15.2015
Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR.Nat Protoc. 2015 Nov;10(11):1709-27. doi: 10.1038/nprot.2015.085. Epub 2015 Oct 8.iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M. Cell. 2014 Nov 6;159(4):896-910
Three-dimensional imaging of solvent-cleared organs using 3DISCOAli Ertürk, Klaus Becker, Nina Jährling, Christoph P Mauch, Caroline D Hojer, Jackson G Egen, Farida Hellal, Frank Bradke, Morgan Sheng & Hans-Ulrich DodtNature protocols. 2012 Oct
Yes, there are several users doing BABB clearing. Here you may find a paper describing a BABB project:Neoplasia. 2014 JanMultispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment responseDobosz M, Ntziachristos V, Scheuer W, Strobel S
No. The system was originally designed for using organic solvents.
Yes, you can image an entire adult mouse brain if it was cleared with organic solvents. At lower zoom factor you can image the brain without tiling. The diagonal of the field of view is 17 mm. If the brain was cleared with an aqueous buffer you may have to do tiling.
In case that the brain is not thicker than 15 mm it will fit into the chamber. Every sample which is in size not larger than 30 mm x 30 mm x 15 mm fits directly into the imaging chamber.
This depends on the working distance of the implemented objective lens. With the standard setup of the UltraMicroscope you can image down to 4, 6 or 10 mm depending on the dipping cap mounted to the objective lens. Please consider that even within a perfectly cleared sample the image quality is affected if the signal has to pass 9 mm of tissue.
It is a tradeoff between speed and quality. If you want to screen a rodent brain, a lung or an embryo for your lab book image acquisition may take about 5 minutes in case you are recording only one color. Every additional color will take about additional 5 minutes. If you want to acquire a data stack for a report you will increase the amount of Z planes and you will illuminate the sample from both sites. In that case it will take about 20 to 30 minutes. For publication quality the data acquisition of the same stack will take about 60 minutes. In some cases if users do tiling with multiple colors image acquisition may also take several hours. Due to the large field of view of the UltraMicroscope this is a pretty rare case but it may occur.
Yes. The in vivo add-on for the UltraMicroscope enables the user to do live imaging. This add-on includes an additional sample chamber with environmental control. Every existing UltraMicroscope can be upgraded with this module.
Yes. The fixed microscope tube module was designed to combine refractive index and infinity corrected objective lenses from different suppliers like Leica, Nikon, Olympus or Zeiss.
No. If you have acquired the data of your first sample you can easily remove the sample and insert the next sample. Also changing objective lenses does not require draining of the imaging chamber.
No. With the UltraMicroscope there is no need to rotate the sample. The system is designed for larger samples. To guarantee a homogenous illumination we have implemented a dynamic horizontal focus. This feature enables the user to acquire data on large field of view with a high NA very homogenously.
The standard setup achieves a lateral resolution of 1 µm. The fixed microscope tube add-on enables higher resolutions depending on the objective lenses. The Z step size can be decreased down to 1 or 2 µm:A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System.Belle M, Godefroy D, Dominici C, Heitz-Marchaland C, Zelina P, Hellal F, Bradke F, Chédotal A(Cell Rep. 2014 Nov 20;9(4):1191-201.)
Yes. Tiling is an integrated feature.
The size of a data stack correlates with the amount of Z planes. The majority of the image files are between 2 and 20 GB in size. Large stacks which are not that frequently acquired may reach about 50 to 80 GB.
Stacks can be exported as tiff and the tiff.ome file formats or directly converted and saved as .ims (Imaris proprietary file format based on hdf5 technology). For subsequent 3D visualization and rendering of stacks exported as .ims it is recommended to use Imaris software. The tiff and ome-tiff formats can be transferred to most of the existing 3D rendering software solutions of third parties like Imaris, Volocity, Amira or Arivis. Also open source software solutions like ImageJ or Fiji can handle these file formats.