Nuance

Nuance imaging is a self-serve instrument and you will need to be trained before you start your actual imaging. The Nuance system is located in WIMR Room 2214A. Contact Karla Esbona at kesbona@wisc.edu to book a training appointment.

Spectral imaging is an imaging format that captures a high-resolution optical spectrum at every pixel of an image, providing extensive analysis opportunities, especially with judicious selection of staining techniques.

Multispectral Imaging of a tissue that has undergone multiplex-IHC staining results in image cubes, or files, which in combination with specialized software algorithms allow the individual spectra of each marker signal to be separated from each other. This information, when “unmixed”, with additional software manipulation (Nuance or InForm) provides an enormous amount of information about the tissue and biomarkers.

Spectral library slides are necessary for unmixing the spectral information found in image cube files created by the Vectra or Nuance imaging systems.  Images from the slides are used to build a library of the individual stain components. When the final composite stain slide is imaged and analyzed the mixed signals can be unmixed and individual components analyzed.

Brightfield Imaging:

You need an individual slide demonstrating each individual chromogen or dye component used in the final composite staining protocol. This means that if you are using two chromogens plus a counterstain, you need one slide demonstrating the first chromogen and no counterstain, the same thing for the second chromogen, and a third slide for the counterstain only.

There is no need to use the markers you are using for your project, especially if they are weak or sparse (or expensive!). Choose a marker that is robust and plentiful, for example, cytokeratin or Ki67.

Fluorescent Imaging:

You need an individual slide demonstrating each individual fluorophore component used in the final composite staining protocol. This means that if you are using two fluorophores plus DAPI, you need one slide demonstrating a marker with the first fluorophore and no DAPI, the same thing for the second fluorophore, and a third slide for DAPI only.

There is no need to use the markers you are using for your project, especially if they are weak or sparse (or expensive!). Choose a marker that is robust and plentiful, for example, cytokeratin or Ki67.

Imaging files acquired by Vectra and Nuance are saved in multiple formats, one with an .im3 extension, designating it as an image “cube.” This 3D cube composed of stacked 2D images includes spatial (x, y coordinates) and depth (wave-length associated) information.

• Slides and coverslips must be clean.
• Recommended glass coverslip thickness is 1.5
• Be sure to use the appropriate mounting media for your application.
• Remove any excess “ooze” from the tops and edges of the coverslip.
• In the iLab form provide components of staining:
  • Type of chromogens/fluorophores
  • Number of spectral library slides (single color for each chromogen/fluorophore and an unstained slide of tissue of interest if IF)
  • Tissue Type
  • Antibodies used, link to datasheet, catalog number, species
• Projects require a minimum 15 minute project design discussion.
• Training is available for this equipment at the rental rate per hour plus labor cost per hour.
• You must be trained by TRIP staff in order to use this microscope. After training, you will be able to schedule this instrument on your own.

• External hard drive for imaging files storage-500GB capacity. TRIP can also provide external hard drives at an additional charge.
• Information on markers and detection systems used, or histology stain components
• Cellular location of each marker if IHC/ISH
• Level of abundance of each marker if IHC/ISH

Currently, only 25 x 75 mm microscope slides can be imaged.