6. Create additional graphs if desired to gate on cells of interest.
Recommended: Scatterplot of
Area
versus
Aspect Ratio
of brightfield to gate on cells and eliminate debris. Scatter
plots or histograms of
Raw Max Pixel
for the channels used in the experiment for setting laser power. Scatterplots or
histograms of Intensity for the channels used in the experiment.
To identify objects for inclusion in or exclusion from the acquiring data file the following features in any chan-
nel are available:
l
Area:The number of pixels in an image reported in square microns.
l
Max Gradient Intensity: The value of the largest slope spanning three pixels in an image. This feature
measures image contrast or focus quality.
l
Intensity: The integrated intensity of the entire object image; the sum of all pixel intensities in an image,
background subtracted.
l
Mean Pixel: The average pixel intensity in an image, background subtracted.
l
Raw Max Pixel: The intensity value of the brightest pixel in an image.
l
Raw Min Pixel: The intensity value of the dimmest pixel in an image.
l
Saturation Count: The number of pixels in an image that have an intensity value of 4096.
l
Saturation Percent: The percentage of pixels in an image that have an intensity value of 4096.
l
Area:The number of pixels in an image reported in square microns.
l
Max Gradient Intensity: The value of the largest slope spanning three pixels in an image. This feature
measures image contrast or focus quality.
l
Intensity: The integrated intensity of the entire object image; the sum of all pixel intensities in an image,
background subtracted.
l
Mean Pixel: The average pixel intensity in an image, background subtracted.
l
Raw Max Pixel: The intensity value of the brightest pixel in an image.
l
Raw Min Pixel: The intensity value of the dimmest pixel in an image.
l
Saturation Count: The number of pixels in an image that have an intensity value of 4096.
l
Saturation Percent: The percentage of pixels in an image that have an intensity value of 4096.
Collecting and Saving the Data Files
Once the sample is running and the FlowSight
®
Instrument is properly set up, you are ready to acquire the data as a raw
image file (.rif). This file contains uncompensated pixel data along with instrument settings and ASSIST information in a
modified TIFF format. The file includes only those objects defined by the population selected in the acquisition section. As
an option, a compensation matrix may be applied to the data as it is collected by either going through the compensation
wizard to create the matrix or choosing a matrix from the File menu.
For Research Use Only. Not for use in diagnostic procedures.
21
Amnis
®
FlowSight
®
Imaging Flow Cytometer User Manual
