UltraMicroscope II Introduction - LaVision BioTec GmbH

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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 light sheet microscope for fluorescence imaging from macro view to cellular resolution
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
Clearness
Usability
Versatility
6 light sheets with an adjustable numerical aperture and horizontal focusing for a better homogenous illumination.
The large and open sample chamber is directly accessible and can be loaded with all current clearing solutions. 
Samples are inserted from the top into the chamber. The sample exchange is done without removing the imaging solution. 
Available add-ons for in vivo imaging and for refractive index corrected objective lenses for higher magnification.

  
About the UltraMicroscope
The standard UltraMicroscope includes following features:
  • 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
 
 
Following add-ons for upgrade the UltraMicroscope are available:
  • The in vivo imaging add-on including environmental control units for live imaging.
  • The fixed microscope tube add-on for using refractive index corrected objective lenses for higher magnification imaging.



FAQ:
1) Can I image iDISCO samples with the UltraMicroscope?
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 Organs
Jonathan R. Epp, Yosuke Niibori, Hwa-Lin (Liz) Hsiang, Valentina Mercaldo, Karl Deisseroth, Sheena A. Josselyn, Paul W. Frankland
DOI: 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 3DISCO
Ali Ertürk, Klaus Becker, Nina Jährling, Christoph P Mauch, Caroline D Hojer, Jackson G Egen, Farida Hellal, Frank Bradke, Morgan Sheng & Hans-Ulrich Dodt
Nature protocols. 2012 Oct

2) Can I image BABB cleared samples with the UltraMicroscope?
Yes, there are several users doing BABB clearing. Here you may find a paper describing a BABB project:
Neoplasia. 2014 Jan
Multispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment response
Dobosz M, Ntziachristos V, Scheuer W, Strobel S

3) Will the usage of organic solvents destroy the UltraMicroscope?
No. The system was originally designed for using organic solvents.

4) Can I image an entire mouse brain?
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.

5) Do I have to cut a mouse brain to size so that it fits into the imaging chamber?
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.

6) How deep can I image into the tissue?
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.
 
7) How long does it take to image a sample?
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.
 
8) Can I do in vivo imaging with the UltraMicroscope?
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.
 
9) Can I use other objective lenses with the UltraMicroscope? For instance refractive index corrected objective lenses?
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.
 
10) Do I have to remove the imaging solution from the imaging chamber if I want to change the objective lens or the sample?
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.
 
11) Do I have to rotate the sample?
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.

12) What is the resolution of the UltraMircosope?
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.)
 
13) Can I do tiling with the UltraMicroscope?
Yes. Tiling is an integrated feature.
 
14) How large are the data stacks?
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.

15) What file format is used for the stacks?
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.




 
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