Biomedical Engineering Reference
In-Depth Information
5. The
Identify Primary Objects
module is used in the nuclei
channel to mask nuclei as objects.
Otsu Adaptive Thresholding
algorithm works well for nuclei, since all nuclei have approxi-
mately the same intensity and shape. Since the Hoechst 33342
staining is usually strong with little background, a two-class
thresholding can be used with a
Thresholding Correction
Factor
of approximately 1.2. Select
Discard Objects Touching
The Border Of The Image
and
Fill Holes In Identifi ed Objects
(
see
Note 9
).
6. The
Identify Secondary Objects
module is used in the phalloidin
channel to identify the cells around the nuclei. Since the HeLa
Kyoto cells have a rather compact morphology and usually
grow in coherent patches,
Distance—B
can be used as a method
to identify the secondary objects and
Otsu Adaptive
as the
thresholding algorithm. For cells with a heterogeneous mor-
phology,
Propagation
is a better method to identify the sec-
ondary objects. To improve object identifi cation,
Otsu Per
Object Thresholding
can also be used, but will prolong the anal-
ysis. Because of the variable fl uorescence intensity of phalloidin
staining within each cell, it is recommended to use
Three-Class
Thresholding
and assign the middle intensity class to the fore-
ground. The thresholding correction factor can be set below 1,
since secondary objects will only be identifi ed if a primary
object is present. This can be useful when fl uorescence inten-
sity is low.
Fill Holes In Identifi ed Objects
should be selected.
7. The
Measure Object Intensity
module is used to measure the
fl uorescence intensity of one or more channels within the
identifi ed objects, such as the intensity of Tf fl uorescence
within each cell.
8. The
Export To Spreadsheet
module will write the data into a
Microsoft Excel spreadsheet for further data analysis.
Alternatively, the fi nal CellProfi ler output fi le may be analyzed
with MATLAB (MathWorks), from where it can also be
exported to a Microsoft Excel spreadsheet using a correspond-
ing script.
9. From the menu bar, select
Test
and
Start Test Run
. In
Test Run
mode, it is possible to manually cycle through the individual
modules of the pipeline. The settings of the modules can be
adjusted and their effect can be observed directly. When the
settings are satisfactory,
Test Run
mode is ended by choosing
Test
and
Stop Test Run
from the menu bar.
10. To start the analysis, select
Analyze Images
.
11. The resulting output fi le contains all measured parameters
including the total and the mean fl uorescence intensities per
cell, as well as further information acquired during the anal-
ysis (e.g., the number of objects identifi ed in each image).
