Leica MM MULTI WAVELENGTH CELL SCORING User Manual

Leica MM Multi Wavelength
Cell Scoring powered by MetaMorph

Analysis Software Drop-in for Leica MM AF

• Multi-Parameter analysis of up to seven wavelengths

• Identication of subpopulations of cells

• Adaptive background correction™ for improved segmentation

• Field and Cell-by-Cell measurements

The Leica MM Multi Wavelength Cell Scoring Application Module
for Leica MM AF software is designed for the scoring of cells with
up to seven uorescent stains.

This exible module is ideal for counting and logging measure­ments of cells in multiple wavelength experiments. Using a uo­rescent marker for the nucleus and additional markers for the cy­toplasm or the entire cell, each wavelength is analyzed and cells
are assigned scoring proles for the presence or absence of each
marker.

The module makes a number of additional cell-by-cell measure­ments including per-wavelength number and percentage of cells
scored as positive or negative, scoring proles, area and inten­sity.

The module utilizes Adaptive Background Correction adapting the
cell detection algorithm to the local intensity ranges between and
within cells to provide the most robust segmentation available.
This technique enables probe detection even with highly variable
background uorescence within a single image.

The simple interface minimizes setup efforts and at the same time
enables users to customize the settings and measurements to ob­tain the best possible results specic to the type of cells used in
the experiments.

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Multiple wavelengths

Leica MM AF provides a exible integrated
solution for multi wavelength image acquisition
and analysis. Up to seven wavelengths can be
analyzed simultaneously by the module. U2OS
rat β-arrestin 2-RrGFP cells treated with 1 μm
isoproterenol. Blue: Hoechst 33342, red: Anti­Phospho-Histone H3 (Ser28), green: Transu­or® vesicles.

Configuration for analysis

Set the number of wavelengths used and repeat
the following steps for each wavelength:

1. Select the image of interest

2. Choose the stained area (nucleus,
cytoplasm, or both)

3. Specify approximate minimum and maxi­mum widths of the objects to be detected

4. Adjust detection sensitivity by specifying the
intensity above local background

5. Set positive scoring criterion (minimum stai­ned area)

6. Optionally specify the reporting parameters

Multi-parameter analysis

Field measurements include:

• Wavelength-specic count and percentage of
negative and positive cells

• Scoring prole counts

Cell-by-cell measurements include:

• Wavelength-specic stained area, and integ­rated and average intensities

• Scoring prole

Powerful data export capabilities

Accurate segmentation

Detection of cells uses Adaptive Background
Correction and displays interactive graphics on
the original images indicating scoring for each
wavelength for immediate verication of accu­racy. The module also generates a segmenta­tion image.

Customization

Wavelength and prole naming, graphics
colors and measurement parameters can all
be customized to match the biology of your
experiment. All settings can be saved for future
experiments.

Interactive data display

Once the analysis is run, the Cellular Results ta­ble allows you to interactively view individual
cells’ data. Clicking one or multiple cells in the
image highlights the data for the selected cell(s)
in the table.

Customization through journaling

Journals are sophisticated and powerful macros
that record and perform a series of tasks without
the need for a programming language. The mo­dules can be incorporated into a Leica MM AF
journal to increase the customization and auto­mation of your analysis.

All measurements can be directly exported to
a text le or Microsoft

®

Excel® for further ana-

lysis.

“MetaMorph® is a Registered Trademark of MDS Analyti-

cal Technologies”

www.leica-microsystems.com