Excel Zrobiony

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Image analysis using PC_Image.

Step 1 Open PC_Image from the Start menu

Step 2

Step 3

Step 4

Open an image using Image > Open. Locate the file (make sure ttype" Windows Bit-map (BMP) is selected. Double click on the imselect the image and click OK.

Resize the open window to fit the full depth of the screen. Use thethe bottom and right hand side of the image so that the whole the s

Calibrate the system by selecting System > Calibrate dimensions >will appear instead of the mouse cursor. Draw a line along the scaleimage and click the left mouse button to finish. Make sure that thein XY" dialogue box matches the value beneath the scale bar (i.e. 1Type microns or micrometers in the "Name for units" dialogue bsystem is now calibrated and should not required subsequent csession (while the program remains open) as each image has bemagnification. Calibration of the system is needed for images (n assessment) that have been taken at different magnifications an

sessions.


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Step 5

Draw a region of interest (ROI) to select the area of the cell. Youthe faint outline of the cell. Select ROI>Draw or Move ROI. In thFreehand then position the cursor at the edge of the cell. A left moto start drawing around the cell. A blue line will show you the ouOnce you have completed the outline a left or right mouse click enmake a mistake just select freehand in the dialogue box againOnce finished select Quit.


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Step 6

Step 7

Increase the contrast between the gold labelled antibody and surroregion of interest by selecting Image>LUT Operations>Square>Quit

Threshold the ROI to binary. Select Image > Threshold Image toscroll bar in the dialogue box to the far right. Move the bottom svalue in the box immediately below the left hand side of the chartthresholding is adjusted, click OK.


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Step 8Dilate the highlighted areas to ensure they are picked up in tselecting Binary>Morphological Operations>Dilate>Quit.


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Step 9

Step 10

Set the acceptance criteria to eliminate any objects that areyou wish to measure by selecting Measures>Set Acceptadialogue box, under the heading Enter criterion for measurethen select OK

Open the select measures using Measures > Select object meabox, deselect everything except the area, as shown in the image b


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Step 11

Measure the cell areas by selecting Measure > Measure objectto save an FFA format file (which will appear as an ".FDT"same FFA file for all the images of cell on the same surfacimages on different surfaces save a separate FFA file. Onc

program will measure the objects that meet the measurementbox will open summarising the measurement results. Click O


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Step 12Save the FFA file as a text file by selecting Measures > Save meastext file. Do this for each image. You can use the same text filesame surface but use a different text file for a different surface.


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Important. When you have finished analysing all the images for one surfaceprogramme 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 N soon as you have thresholded your new ROI this will be eliminated. It will not affect youbothers you then close down the program and start again for each image. Remember toFAA file when measuring the objects on your new image.


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Step 10Save the FFA file as a text file by selecting Measures > Save meastext file. Do this for each image. You can use the same text file

same surface but use a different text file for a different surface.

Important. When you have finished analysing all the images for one surfaceprogramme and open it again in order to analyse the next set of images.


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hat under "List files ofage name to open or

scroll bars to scroll toale bar can be seen.

XYCURSOR. A crossbar at the base of thevalue in the "... pixels00.000) and click OK.

ox and click OK. Thealibrations during thisen taken at the samet those used for this

d for any subsequent


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should be able to seee dialogue box, pressse click will allow you

tline you have drawn.ds the function. If youand proceed to draw.


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unding material in the.

inary Move the toproll bar right until thereads 120. Once the


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e measuring step by


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arger than the labelce Criteria. In theents type OA


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S. You will be askedextension). Use the

e - when analysingthe file is saved thecriteria. A dialogue.


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ures as text. Save theor each image of the


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lose the PC_Image

OT be concerned. Asr measurement. If thisselect the appropriate


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ures as text. Save theor each image of the

lose the PC_Image


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0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

25.5

10.6

34.0

Area of adhesion

Substrate name

Area of adhesion [m 2]


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Descriptive data

Substrate name Substrate name Area of adhesion Standard deviation 1/2

Calcium Phosphate 21.51 Calcium Phosphate 25.5 7.7

Calcium Phosphate 36.19 phosphoric acid 10.6 6.7

Calcium Phosphate 20.02 thermanox 34.0 3.4

Calcium Phosphate 18.71Calcium Phosphate 30.85

phosphoric acid 4.6

phosphoric acid 2.84




Thermanox 35.0760

Thermanox 31.2797

Thermanox 34.6680 Processing image analysis data

Thermanox 30.2874

Thermanox 38.8461 Important!

Step 1

Step 2

Adhesion data for each of the threesubstrates

Area of adhesion m2

This is the simple 'Overview' form of the instructions. The detailed procedures c"Using Excel to summarise data" worksheet.There is also an alternative instructio

are using Excel Office 2003 version

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

Import text files containing cell area data onto the Thermacid worksheets (tabs for these worksheets are at the bworksheet).

The image analysis cell area data should be copied and pasections in the main "Adhesion data" table on the "Summar


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Step 3

Step 4

Step 5

Step 6

Step 7

Step 8

The substrate names should be typed into the relevant s"Adhesion data" table on the "Summarised data sheet"

Functions to calculate the mean area of adhesion and steach of the three substrates should be put into the "DeAdjust the number of decimal places to 1.

Create a vertical bar chart using the mean cell areas for eastandard error to create the error bars.

Format the chart to include the y-error bars, appropriate fonbe large enough that it can be read when the chart is resizethe axes and titles, number of decimal places in the axes acontaining the mean cell areas for each of the substrates.

Click on the edge of the worksheet to select the whole char

copy the chart and paste it into PowerPoint and Word.

Click on the edge of the worksheet to select the whole charcopy the chart and paste it into PowerPoint and Word.

This is the simple 'Overview' form of the instructions. The detailed procedures c"Using Excel to summarise data" worksheet.


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Substrate names Mean cell area

Calcium Phosphat 23.

#REF! #RE

#REF! #RE


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d/2

5.78

#REF!

#REF!


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Detailed Instruction

Proce

Step 1

Procedure

From the menu bar

This opens the Text I

The worksheet should now look sometData: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

Step 2

To: Import text files cacid worksheets (tabworksheet).

Click on worksheet

"DATA" > "GET EXT

Navigate to the appr

Choose 'Delimited' theable to see in the previ'Next' and 'Finish' in th

The data value highliarea of adhesion. Th


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Step 3

Step 4

Procedure

measurement.You willimage.

The substrate namesthe "Summarised data

Insert the relevant Fstandard deviation for

No more than 1 decimcells > right click >andof decimal places from


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for Processing Image Analysis Data and Creating a Chart in Exc

sing image analysis data

Step 1

Procedure

port Wizard Step 1 of 3

Step 2

ing like this:

Step 3

Step 4

ntaining cell area data onto the Thermanox and phosphorics for these worksheets are at the bottom of the current

RNAL DATA" > "FROM TEXT"

priate directory and select the TEXT file to import

n 'Next' and make sure only 'comma' is ticked. You should beiew box that the data entries are split into columns. Choosee following two steps.

hted in yellow (to the right of "Detect. Area") represents thedetect area values should be copied and pasted into the

" "


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Step 5hould be typed into the relevant sections in the main table onsheet"

Note that it is po

nctions to calculate the mean area of adhesion and theeach of the three substrates into the "Descriptive data" table.

al place should be used for the values in the table. Select thechoose 'format cells' or directly select the appropriate numberthe "Number" tab on the control bar


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el for Export into Word or Powerpoint

Creating a chart

First select 'Insert' from the tabs along the top

Then select 'column' and choose the first 2D column

In the box that opens select 'add'

Select an appropriate chart layout and insert title and axis labelsNote - Choose a style without gridlines

Click 'close'

Repeat for the bars in your 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.

In the new control bar that has opened select 'SelectData'

A new box will open- in the series name box type oneof the substrates names- order is unimportant

In the box below labelled 'series values' remove thecontents ( ={1}) and then select the cell with therelevant data with a left mouse click. Repeat the 'add'with the two other data items. Click 'OK' when dataentry is finished.

A number of chart options become available. Click onsome of the Chart layout' options - this allows you topreview your data in that chart layout

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

Select the'Both' direction and 'Fixed value'. Type inthe appropriate standard deviation data value in thebox next to the 'Fixed value' option box thatrepresents the data in the bar you selected in thechart-check the instruction schedule to see whichvalue you should be using.

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 theappropriate number of decimal places for the scale of measurements you

Area of adh


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Click on the edge of the chart to select it. It is now possible to copy the chartand paste it into PowerPoint or Word.

sible to further annotate your chart using the same annotation tools in Word

or Powerpoint after pasting.


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0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

25.5

10.6

34.0

Area of adhesion

25.5 10.6 34.0

Substrate name

sion [m 2]


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Processing image analysis data

Step 1

Procedure

Procedure Click "Next"

Import text files containing cell area data onto thphosphoric acid worksheets (tabs for these workshthe current worksheet).

Click on worksheet

"DATA" > "IMPORT EXTERNAL DATA" > "IMPOR

Select the TEXT file to import.


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Procedure Tick "Comma"

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

Step 2

Step 3

Step 4

Procedure

Click "Finish"

The image analysis information will be presented iexample above. The data highlighted in yellow (to tis the area representing area of adhesion. This dpasted into the relevant sections in the main tablesheet" You will need to scroll down the page for thimage.

The substrate names should be typed into the reletable on the "Summarised data sheet"

Functions to calculate the mean area of adhesion afor each of the three substrates should be put into th

No more than 1 decimal place should be used foSelect the cells > right click > select the "Number" tanumber of decimal places. Alternatively use the dectoolbar at the top of the screen to reduce or increaplaces.


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Procedure

Procedure

Click "Finish"

box for the second time. this will take you back to the

Insert the cell codes for cells containing the s"Category (X) axis lables" text box.

Click "Next"

Use the tabs at the top of the chart wizard to igridlines, remove "legend".

Click "Next".

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

Format the chart to include the y-error bars, approprishould be lar e enou h that it can be read when the


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Step 8

poster) for the axes and titles, number of decimal plabox containing the mean area of adhesion for cells o

Click on the edge of the worksheet to select the wholto copy the chart and paste it into PowerPoint and W


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calcium phosphate andeets are at the bottom of

DATA"


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in a format similar to thee right of "Detect. Area")ta should be copied andon the "Summarised data

data derrived from each

vent sections in the main

nd the standard deviatione "Descriptive data" table.

r the values in the table.> Custom and adjust the

imal place buttons on these the number of decimal


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the "Data for submission"> Copy and Edit > "Pastecimal places displayed.

hesion for each substrate

.

e areas into the "Values", select the range of cellsicon to the right of the text


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ubstrate names into the

put the title, axis labels,

ate font size (the textchart is resized to fit the


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e chart. It is now possibleord


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