RRC-RHTIC: Vectra Training

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The CRI Vectra is a multi-spectral imaging system for use in both brightfield and fluorescence applications. The Vectra requires that samples be prepared on standard 1" x 3" microscope slides. Samples can be cultured cells, cell spreads, tissue sections or tissue microarrays. Unlike the Aperio which scans the entire slide, the Vectra intelligently samples the tissue using algorithms developed in a companion application called inForm. inForm classifiers can be created to tell the Vectra how you want to sample your tissue at 20x magnification. Additional inForm classifiers are used to segment each 20x image into tissue compartments (epithelium, stroma, blank slide etc...) where you can then do separate measurements of protein expression.
The specifications of the fluorescence filters currently installed on the Vectra are detailed in this file: Vectra Filters. Care needs to be taken to ensure that you select fluorophores that can be excited by the filters on the Vectra and that enough of the emission profile can also be captured. I recommend using the SpectraViewer application on the Invitrogen website to verify that you are choosing proper fluorophores. http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html. You need to keep in mind that the emission filters on the Vectra are long pass filters that go out beyond 700 nm. We recommend Dapi, Alexa 488, Alexa 546 and Alexa 647.
The data output for the Vectra depends on whether you are using brightfield or fluorescence imaging. For fluorescence, we recommend using the "normalized intensity counts" setting in the inForm analysis software. This ensures that your scores are normalized for exposure time, bit depth and any binning. The general formula is: normalized counts per second = counts /[full-scale* exposure time * gain * binning area]. "Full scale" means 256 for 8-bit images and 4096 for 12 bit images. "Binning area" means 1 for 1x1, 4 for 2x2 etc, while exposure time is in seconds. This ensures that the same sample will give the same output regardless of the exposure time, gain, binning or bit depth used to acquire the image.
Brightfield data follows the Beer-Lambert Law which is described on page 11 of the Nuance Manual. Each pixel in the image is scored as a negative base 10 log of the intensity of the stain on the slide being evaluated divided by the intensity of the brightfield reference image that is collected for each slide. The final component signal is the average across all wavelengths.
(-logā”10 ((Stain Intensity)/(Reference Intensity)) )

Training Documentation

Training is available for both brightfield and fluorescence tissue and TMA research studies on the Vectra at the rate of $60 an hour. The files below are step by step instructions that will aid you in completing your research using the Vectra. Information on the different high power field sampling strategies used in tissue imaging can be found here.
Brightfield Tissue Documentation
Brightfield TMA Documentation
Fluorescence Tissue Documentation
Fluorescence TMA Documentation

User Manuals

1. Vectra User Manual v 1.4
2. inForm Manual v 1.3
3. Nuance Quick Start Guide
4. Nuance User Manual v 2.6
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