Image analysis using PC_Image.

Step 1 Open PC_Image from the Start menu

Open an image using Image > Open. Locate the file (make sure that under "List files
Step 2 type" Windows Bit-map (BMP) is selected. Double click on the image name to open o
select the image and click OK.

Step 3 Resize the open window to fit the full depth of the screen. Use the scroll bars to scroll
the bottom and right hand side of the image so that the whole the scale bar can be seen

Calibrate the system by selecting System > Calibrate dimensions > XYCURSOR. A cros
will appear instead of the mouse cursor. Draw a line along the scale bar at the base of th
image and click the left mouse button to finish. Make sure that the value in the "... pixe
in XY" dialogue box matches the value beneath the scale bar (i.e. 100.000) and click OK
Step 4 Type microns or micrometers in the "Name for units" dialogue box and click OK. Th
system is now calibrated and should not required subsequent calibrations during th
session (while the program remains open) as each image has been taken at the sam
magnification. Calibration of the system is needed for images (not those used for th
assessment) that have been taken at different magnifications and for any subseque
sessions.
 Draw a region of interest (ROI) to select the area of the cell. You should be able to se
the faint outline of the cell. Select ROI>Draw or Move ROI. In the dialogue box, pres
Freehand then position the cursor at the edge of the cell. A left mouse click will allow yo
Step 5 to start drawing around the cell. A blue line will show you the outline you have draw
Once you have completed the outline a left or right mouse click ends the function. If yo
make a mistake just select freehand in the dialogue box again and proceed to draw
Once finished select Quit.
 Increase the contrast between the gold labelled antibody and surrounding material in th
Step 6
region of interest by selecting Image>LUT Operations>Square>Quit.

Threshold the ROI to binary. Select Image > Threshold Image to Binary… Move the to
scroll bar in the dialogue box to the far right. Move the bottom scroll bar right until th
Step 7
value in the box immediately below the left hand side of the chart reads 120. Once th
thresholding is adjusted, click OK.
 Dilate the highlighted areas to ensure they are picked up in the measuring step b
Step 8
selecting Binary>Morphological Operations>Dilate>Quit.
 Set the acceptance criteria to eliminate any objects that are larger than the lab
you wish to measure by selecting Measures>Set Acceptance Criteria. In th
Step 9
dialogue box, under the heading “Enter criterion for measurements” type OA<0.
then select OK

Open the select measures using Measures > Select object measures. In the dialogu
Step 10
box, deselect everything except the area, as shown in the image below. Click OK.
 Measure the cell areas by selecting Measure > Measure objectS. You will be aske
to save an FFA format file (which will appear as an ".FDT" extension). Use th
same FFA file for all the images of cell on the same surface - when analysin
Step 11
images on different surfaces save a separate FFA file. Once the file is saved th
program will measure the objects that meet the measurement criteria. A dialogu
box will open summarising the measurement results. Click OK.
 Save the FFA file as a text file by selecting Measures > Save measures as text. Save th
Step 12 text file. Do this for each image. You can use the same text file for each image of th
same surface but use a different text file for a different surface.
Important. When you have finished analysing all the images for one surface close the PC_Imag
programme and open it again in order to analyse the next set of images.

When a second image is opened, the previous ROI will be superimposed onto it. DO NOT be concerned. A
soon as you have thresholded your new ROI this will be eliminated. It will not affect your measurement. If th
bothers you then close down the program and start again for each image. Remember to select the appropria
FAA file when measuring the objects on your new image.
 Save the FFA file as a text file by selecting Measures > Save measures as text. Save th
Step 10 text file. Do this for each image. You can use the same text file for each image of th
same surface but use a different text file for a different surface.

Important. When you have finished analysing all the images for one surface close the PC_Imag
programme and open it again in order to analyse the next set of images.
make sure that under "List files of
ck on the image name to open or

een. Use the scroll bars to scroll to

whole the scale bar can be seen.

imensions > XYCURSOR. A cross

ong the scale bar at the base of the
sure that the value in the "... pixels
ale bar (i.e. 100.000) and click OK.
" dialogue box and click OK. The
ubsequent calibrations during this
mage has been taken at the same
r images (not those used for this
ifications and for any subsequent
he cell. You should be able to see
e ROI. In the dialogue box, press
ell. A left mouse click will allow you
w you the outline you have drawn.
ouse click ends the function. If you
box again and proceed to draw.
dy and surrounding material in the
Square>Quit.

d Image to Binary… Move the top

he bottom scroll bar right until the
of the chart reads 120. Once the
ked up in the measuring step by
e>Quit.
ts that are larger than the label
et Acceptance Criteria. In the
or measurements” type OA<0.5

object measures. In the dialogue

the image below. Click OK.
asure objectS. You will be asked
an ".FDT" extension). Use the
same surface - when analysing
A file. Once the file is saved the
easurement criteria. A dialogue
s. Click OK.
> Save measures as text. Save the
me text file for each image of the
surface.
e surface close the PC_Image

nto it. DO NOT be concerned. As

ot affect your measurement. If this
emember to select the appropriate
> Save measures as text. Save the
me text file for each image of the
surface.

e surface close the PC_Image

 Area of adhesion
40.0

35.0

30.0

25.0

Area of adhesion [μm 2]

20.0

34.0
15.0

25.5

10.0

5.0 10.6

0.0

Substrate name
 Adhesion data for each of the three
Descriptive data
substrates

Substrate name Area of adhesion µm2 Substrate name Area of adhesion Standard deviation 1/2 standard deviation
Calcium Phosphate 21.51 Calcium Phosphate 25.5 7.7 3.8
Calcium Phosphate 36.19 phosphoric acid 10.6 6.7 3.4
Calcium Phosphate 20.02 thermanox 34.0 3.4 1.7
Calcium Phosphate 18.71
Calcium Phosphate 30.85
phosphoric acid 4.6
phosphoric acid 2.84
phosphoric acid 14.11 This is the simple 'Overview' form of the instructions. The detailed procedures can be found on the
"Using Excel to summarise data" worksheet.There is also an alternative instruction worksheet if you
phosphoric acid 18.98
are using Excel Office 2003 version
phosphoric acid 12.43
Thermanox 35.0760
Thermanox 31.2797
Thermanox 34.6680 Processing image analysis data
Thermanox 30.2874
Thermanox 38.8461 Important!

See detailed instructions in Word doc schedule for importing text documents into Excel Office 2007
particularly if you encounter any difficulties

Step 1
Import text files containing cell area data onto the Thermanox and phosphoric
acid worksheets (tabs for these worksheets are at the bottom of the current
worksheet).

Step 2 The image analysis cell area data should be copied and pasted into the relevant
sections in the main "Adhesion data" table on the "Summarised data sheet"
 The substrate names should be typed into the relevant sections in the main
Step 3
"Adhesion data" table on the "Summarised data sheet"

Functions to calculate the mean area of adhesion and standard deviations for
Step 4 each of the three substrates should be put into the "Descriptive data" table.
Adjust the number of decimal places to 1.

Create a vertical bar chart using the mean cell areas for each substrate and the
Step 5
standard error to create the error bars.

Format the chart to include the y-error bars, appropriate font size (the text should
be large enough that it can be read when the chart is resized to fit the poster) for
Step 6
the axes and titles, number of decimal places in the axes and a text box
containing the mean cell areas for each of the substrates.

Click on the edge of the worksheet to select the whole chart. It is now possible to
Step 7
copy the chart and paste it into PowerPoint and Word.

Click on the edge of the worksheet to select the whole chart. It is now possible to
Step 8
copy the chart and paste it into PowerPoint and Word.

This is the simple 'Overview' form of the instructions. The detailed procedures can be found on the
"Using Excel to summarise data" worksheet.
Substrate names Mean cell areas Standard Deviations
Calcium Phosphate 23.36 11.56
#REF! #REF! #REF!
#REF! #REF! #REF!
St.d/2
5.78
#REF!
#REF!
 Detailed Instructions for Processing Image Analysis D
Processing image analysis data

Step 1
To: Import text files containing cell area data onto the Thermanox a
acid worksheets (tabs for these worksheets are at the bottom
worksheet).

Procedure • Click on worksheet

From the menu bar

• "DATA" > "GET EXTERNAL DATA" > "FROM TEXT"

• Navigate to the appropriate directory and s

This opens the Text Import Wizard Step 1 of 3

Choose 'Delimited' then 'Next' and make sure only 'comma' is ticked.
able to see in the preview box that the data entries are split into colum
'Next' and 'Finish' in the following two steps.

The worksheet should now look something like this:

Data:PC_ImageFilename:
C:\PC_IMAGE\IMAGES\CALC.FDT
Thursday 11 of October
Calibration units: microns
X calibration: 0.06100
Y calibration: 0.06100

FIELD MEASURES
Field Index 1
Object No 369

Field Area 259.25

Detect.area 21.51
Field Total 21.75
Area percent 8.3

The data value highlighted in yellow (to the right of "Detect. Area")
Step 2 area of adhesion. The detect area values should be copied and p
relevant sections in the main table on the "Summarised data s
measurement.You will need to scroll down the page for the data der
image.
 The data value highlighted in yellow (to the right of "Detect. Area")
Step 2 area of adhesion. The detect area values should be copied and p
relevant sections in the main table on the "Summarised data s
measurement.You will need to scroll down the page for the data der
image.

The substrate names should be typed into the relevant sections in the
Step 3
the "Summarised data sheet"

Insert the relevant Functions to calculate the mean area of adh

Step 4
standard deviation for each of the three substrates into the "Descriptiv

Procedure
No more than 1 decimal place should be used for the values in the ta
cells > right click >and choose 'format cells' or directly select the appr
of decimal places from the "Number" tab on the control bar
 tructions for Processing Image Analysis Data and Creating a Chart in Excel for Export into Word
Processing image analysis data Creating a

Step 1

text files containing cell area data onto the Thermanox and phosphoric
sheets (tabs for these worksheets are at the bottom of the current Procedure
).

worksheet
menu bar

"GET EXTERNAL DATA" > "FROM TEXT"

to the appropriate directory and select the TEXT file to import

s the Text Import Wizard Step 1 of 3

elimited' then 'Next' and make sure only 'comma' is ticked. You should be
e in the preview box that the data entries are split into columns. Choose
Finish' in the following two steps. Step 2

ook something like this:

Step 3

Step 4
value highlighted in yellow (to the right of "Detect. Area") represents the
dhesion. The detect area values should be copied and pasted into the
ections in the main table on the "Summarised data sheet" for each
ent.You will need to scroll down the page for the data derived from each
 ate names should be typed into the relevant sections in the main table on
Step 5
marised data sheet"
Note that it is possible to further annotate your ch
or Powerpoint a
relevant Functions to calculate the mean area of adhesion and the
eviation for each of the three substrates into the "Descriptive data" table.

han 1 decimal place should be used for the values in the table. Select the
t click >and choose 'format cells' or directly select the appropriate number
places from the "Number" tab on the control bar
rt in Excel for Export into Word or Powerpoint
Creating a chart

To: Create a vertical bar chart using the mean area of adhesion for each
substrate and the standard error to create the error bars.
Are
40.0

First select 'Insert' from the tabs along the top

35.0
Then select 'column' and choose the first 2D column
In the new control bar that has opened select 'Select
Data'
In the box that opens select 'add' 30.0
A new box will open- in the series name box type one
of the substrates names- order is unimportant
In the box below labelled 'series values' remove the 25.0
contents ( ={1}) and then select the cell with the
relevant data with a left mouse click. Repeat the 'add'
with the two other data items. Click 'OK' when data
entry is finished. Area of adhesion [μm 2]
20.0

A number of chart options become available. Click on

15.0
some of the Chart layout' options - this allows you to
preview your data in that chart layout

Select an appropriate chart layout and insert title and axis labels
10.0
Note - Choose a style without gridlines

5.0

Select one of the bars on your chart, then select 'layout tab' at the top of the
screen. In 'Analysis' select 'Error bars'. A list will appear-select the 'more
error bars options'. Format the chart to include the y-error bars, appropriate 0.0
font size (the text should be large enough that it can be read when the chart
is resized to fit the poster) for the axes and titles, number of decimal places
in the axes and a text box containing the mean area of adhesion for cells on
each of substrate.

Select the'Both' direction and 'Fixed value'. Type in

the appropriate standard deviation data value in the
box next to the 'Fixed value' option box that
represents the data in the bar you selected in the
chart-check the instruction schedule to see which
value you should be using.

Click 'close'
Repeat for the bars in your chart.

Before copying your chart to go into your work for submission ensure that
you have a title and axis headings and UNITS where relevant for your chart.
Ensure that the level of precision is appropriate -are the values to the
appropriate number of decimal places for the scale of measurements you
have taken?
 Before copying your chart to go into your work for submission ensure that
you have a title and axis headings and UNITS where relevant for your chart.
Ensure that the level of precision is appropriate -are the values to the
appropriate number of decimal places for the scale of measurements you
have taken?

Click on the edge of the chart to select it. It is now possible to copy the chart
and paste it into PowerPoint or Word.
that it is possible to further annotate your chart using the same annotation tools in Word
or Powerpoint after pasting.
 Area of adhesion
40.0

35.0 34.0

30.0

25.5
25.0

ea of adhesion [μm 2]
20.0 25.5 10.6 34.0

15.0

10.6
10.0

5.0

0.0

Substrate name
 Processing image analysis data

Step 1
Import text files containing cell area data onto the calcium phosphate and
phosphoric acid worksheets (tabs for these worksheets are at the bottom of
the current worksheet).

Procedure • Click on worksheet

• "DATA" > "IMPORT EXTERNAL DATA" > "IMPORT DATA"

• Select the TEXT file to import.

Procedure • Click "Next"

 Procedure • Tick "Comma"

• Click "Finish"

Data:PC_ImageFilename:
C:\PC_IMAGE\IMAGES\CALC.FDT
Thursday 11 of October
Calibration units: microns
X calibration: 0.06100
Y calibration: 0.06100

FIELD MEASURES
Field Index 1
Object No 369
Field Area 259.25
Detect.area 21.51
Field Total 21.75
Area percent 8.3

The image analysis information will be presented in a format similar to the

Step 2 example above. The data highlighted in yellow (to the right of "Detect. Area")
is the area representing area of adhesion. This data should be copied and
pasted into the relevant sections in the main table on the "Summarised data
sheet" You will need to scroll down the page for the data derrived from each
image.

The substrate names should be typed into the relevent sections in the main
Step 3
table on the "Summarised data sheet"

Functions to calculate the mean area of adhesion and the standard deviation
Step 4
for each of the three substrates should be put into the "Descriptive data" table.

Procedure
No more than 1 decimal place should be used for the values in the table.
Select the cells > right click > select the "Number" tab > Custom and adjust the
number of decimal places. Alternatively use the decimal place buttons on the
toolbar at the top of the screen to reduce or increase the number of decimal
places.
 Copy the results from the "Descriptive data" table to the "Data for submission"
Step 5 table by selecting the relevant cells and using Edit > Copy and Edit > "Paste
special" > "Values". Do not reduce the number of decimal places displayed.

Create a vertical bar chart using the mean area of adhesion for each substrate
Step 6
and the standard error to create the error bars.

Procedure • Click on the chart wizard icon or INSERT > CHART

• Select the "clustered column" histogram type chart

• Click "Next"

Procedure • Select the "Series" tab at the top of the dialgue box.

• Click "Add"

• Insert the cell codes for cells containing the average areas into the "Values"
text box (click on the icon to the right of the text box, select the range of cells
containing the mean adhesion area and click on the icon to the right of the text
box for the second time. this will take you back to the main dialogue box).
 • Insert the cell codes for cells containing the average areas into the "Values"
text box (click on the icon to the right of the text box, select the range of cells
containing the mean adhesion area and click on the icon to the right of the text
box for the second time. this will take you back to the main dialogue box).

• Insert the cell codes for cells containing the substrate names into the
"Category (X) axis lables" text box.

• Click "Next"

Procedure • Use the tabs at the top of the chart wizard to input the title, axis labels,
gridlines, remove "legend".

• Click "Next".

Procedure • Select "As new sheet" and type in chart title.

• Click "Finish"

Format the chart to include the y-error bars, appropriate font size (the text
Step 7 should be large enough that it can be read when the chart is resized to fit the
poster) for the axes and titles, number of decimal places in the axes and a text
box containing the mean area of adhesion for cells on each of substrate.
 Format the chart to include the y-error bars, appropriate font size (the text
Step 7 should be large enough that it can be read when the chart is resized to fit the
poster) for the axes and titles, number of decimal places in the axes and a text
box containing the mean area of adhesion for cells on each of substrate.

Click on the edge of the worksheet to select the whole chart. It is now possible
Step 8
to copy the chart and paste it into PowerPoint and Word
ta

ata onto the calcium phosphate and

hese worksheets are at the bottom of

" > "IMPORT DATA"

 presented in a format similar to the
yellow (to the right of "Detect. Area")
sion. This data should be copied and
main table on the "Summarised data
page for the data derrived from each

into the relevent sections in the main

f adhesion and the standard deviation

be put into the "Descriptive data" table.

be used for the values in the table.

"Number" tab > Custom and adjust the
use the decimal place buttons on the
ce or increase the number of decimal
 ata" table to the "Data for submission"
d using Edit > Copy and Edit > "Paste
umber of decimal places displayed.

an area of adhesion for each substrate

r bars.

RT > CHART

m type chart

dialgue box.

ng the average areas into the "Values"

the text box, select the range of cells
click on the icon to the right of the text
u back to the main dialogue box).
aining the substrate names into the

wizard to input the title, axis labels,

t title.

ars, appropriate font size (the text

ad when the chart is resized to fit the
f decimal places in the axes and a text
on for cells on each of substrate.
lect the whole chart. It is now possible
Point and Word