|1||Create your first graph|
|2||Edit the graph using the spreadsheet|
|3||Changing the layout of the graph|
|4||Fitting a function to a curve|
|6||Creating a graph using functions|
|7||A few last details|
|8||Deleting objects and dissociating a graph|
|9||Creating a bar plot with error bars|
|10||Spreadsheet slidebar demo|
|11||Subtract background from two b&w images and merge|
|12||Get cell properties|
1 Create your first graph: We will draw a parabola f(x) = (x-5)**2
|step 1.1||Start the program.|
|step 1.2||From the File menu (it is the only available right now), choose new.|
|step 1.3||In the "Select File Type" dialogue window, choose "spreadsheet" and push "OK". A blank spreadsheet "document1" is created.|
|step 1.4||Click in the spreadsheet cell at column 1, row 1.|
|Then type (without the quote marks): "mV" followed by <RETURN>, then type "0" <RETURN>, "1" <RETURN> etc. until "10" <RETURN>. If you type a wrong character, use the <BACKSPACE> key.|
|step 1.5||Click in the spreadsheet cell at column 2, row 1.|
|Then type (without the quote marks): "mA" followed by <RETURN>, then type "25" , "16" , "9" , "4" , "1" , "0" , "1" , "4" , "9" , "16" , "25", where each number is followed by a <RETURN>|
|The spreadsheet should now look like:|
|step 1.6||Click on "column" key number 1 and, while keeping the left mouse button depressed, move onto column key 2 and release the mouse button. Now the two columns 1 & 2 are selected:|
|step 1.7||From the Modify/Stats menu select Line Plot.|
|step 1.8||In the "Set Columns" dialogue window check "X". This means that column 1 will furnish the X-coordinate.|
|step 1.9||Advance one column by pushing the . button. As "Y" is the default, nothing needs to be done here. Hence, column 2 will furnish the Y-coordinate.|
|step 1.10||Push the "OK" button. A new drawing window "document2" is created containing the graph:|
|step 1.11||From the File menu choose save (document2 needs to be the active window. If you have clicked on document1, reactivate document2 by clicking on its title bar before going to the File menu).|
In the "Save" dialogue window type "step1" as filename. The title bar now reads "step1.his" (the extension is added automatically).
|Note that the column keys 1 & 2 in the spreadsheet now contain a "°" character. This means that these columns are linked with a graph on a drawing sheet. The use of these links is the subject of the next exercise.|
2 Edit the graph using the spreadsheet
|If you just carried out step 1, leave the program and restart it. (It is not necessary to save document1).|
|step 2.1||From the File menu choose open and load "step1.his" that was created in step 1. A drawing window is created containing the parabola.|
|step 2.2||Click on one of the elements of the graph. Eight little boxes appear around the graph. It is now selected:|
|step 2.3||Push the "copy" icon in the program window icon bar. The graph is now copied to the clipboard.|
|step 2.4||From the File menu choose new.|
|step 2.5||In the "Select File Type" dialogue window, choose "spreadsheet" and push "OK". A blank spreadsheet "document1" is created.|
|step 2.6||Click in the spreadsheet cell at column 1, row 1.|
|step 2.7||Push the "paste" icon in the program window icon bar. The graph data are pasted onto columns 1 & 2. Note that the column keys 1 & 2 in the spreadsheet contain a "°" character. This means that these columns are linked with the graph on the drawing sheet.|
|step 2.8||Move the spreadsheet such that both graph and columns are visible.|
|step 2.9||Click in the cell at column 2 and row 7. It contains "0". Then type "10" followed by <RETURN>. The contents of the cell are replaced by "10" and the curve in the graph is updated:|
|step 2.10||Select undo from the Edit menu. The original curve is restored. Cell [2,7] reads "0" again.|
|step 2.11||Click on the row key number 2, thereby selecting row 2:|
|step 2.12||Push the "delete" icon in the program icon bar. The row is deleted and the curve in the graph modified.|
|step 2.13||Close all windows without saving.|
3 Changing the layout of the graph.
|step 3.1||Open file step1.his (as in step 2).|
|step 3.2||Double-click on one of the axes (and hence not on the curve). The "Axes" dialogue window appears.|
|step 3.3||Under "Y-axis" on the right, replace "To" "30" by "To" "25".|
Set "Major tick every" "5".
|step 3.4||Double-click on the curve. The "Curve properties" dialogue window appears.|
|step 3.5||Uncheck the "connect points with line" checkbox. Push the "OK" button. The graph should now look like:|
|step 3.6||Select save as from the File menu and give "step3" as new file name (the extension .his will be added automatically).|
4 Fitting a function to a curve.
|step 4.1||Open file step3.his, created in step 3.|
|step 4.2||Double-click on one of the data points (open circles). The "Curve properties" dialogue window appears. Move it such that the graph on the drawing sheet becomes visible.|
|step 4.3||Push the "Function" button. The "Fit" dialogue window appears on top of the previous one. If necessary move it to uncover the graph.|
|step 4.4||From the list of functions select '*parabola" and push the "<<" button. The text in the edit box now reads: "a+b*(X-x0)^2", the definition of a parabola. Push the "OK" button.|
|step 4.5||In the "Curve properties" dialogue window that now reappears, push the "Do Fit" button.|
The "Fit" dialogue box reappears and the fitted curve is plotted onto the graph (in red). The estimated parameters are: a=0 (or very close to it), b=1 and x0=5. Push the "OK" button.
|step 4.7||Push the "OK" button in the "Curve properties" dialogue window. The graph should now look like:|
|While the curve in step 1.10 had its data points interconnected by line segments, this graph has its data points interconnected by a smooth function.|
|step 4.8||Save the drawing sheet as "step4.his".|
5a Students t-test
|step 5.1||Create a new spreadsheet and enter data as in step 1:|
|step 5.2||Select columns 1 & 2 (as in step 1.6).|
|step 5.3||From the "Modify/Stats" menu choose "Get column stats". The following dialogue window pops up:|
It shows means, standard deviations and standard errors of the means for the two columns. Supposing that the data are paired (e.g. each pair of data in a row has been obtained before and after some manipulation), then the probability (P) that the two samples of data in columns 1 and 2 belong to the same parent population is 2.3%.
|step 5.4||Push the "OK" button. The dialogue window disappears.|
|step 5.5||Click on the spreadsheet cell at column 4, row 1 and push the paste icon in the program icon bar. The statistical data are now available on the spreadsheet for future manipulation:|
5b Fishers F-test
|step 5.6||Start a fresh spreadsheet and enter the following data in three columns:|
|step 5.7||Select columns 1,2 & 3 (as in step 1.6).|
|step 5.8||From the "Modify/Stats" menu choose "Get column stats". The following dialogue window pops up:|
Because the t-test is not valid if one disposes of more than 2 sets of data, Fishers F-test (or one-way ANOVA) is carried out. The small P indicates that the three columns of data do not come from the same population. In order to know which is (are) the data set(s) that deviate, Tukey's multiple comparison test (q-test) is carried out next. As the number of data sets may be large (256 columns is the maximum), the result is not shown in the dialogue box. Push the "OK" button and paste the clipboard onto the spreadsheet:
In Tukey's algorithm, the means are sorted in ascending order, which in this case happens to be column 1,2 and 3. Then the q-test is carried out between every possible pair of columns. It can be concluded here that the data in columns 2 and 3 do not come from different populations, while the data in column 1 is significantly different from the other two.
6 Creating a graph using functions: we'll make a sine wave
|step 6.1||Create a new spreadsheet (see step 1)|
|step 6.2||In column 1, row 1 enter "s". In column 2, row 1 enter "V" (see step 1)|
|step 6.3||Click in cell column 1, row 2 and then click in the spreadsheet edit box:|
|step 6.4||Type (without the quote marks) "=(lin-2)*0.01" followed by <RETURN>|
|step 6.5||Click in cell column 2, row 2 and then click in the spreadsheet edit box|
|step 6.6||Type (without the quote marks) "=sin(2*pi*2*" (N.B. no <return> !).|
|step 6.7||RIGHT mouse click in cell column 1, row 2. The following should show:|
|step 6.8||Type ")", followed by <RETURN>. The result is:|
We've now entered two formulas and the (not too exciting) result is shown.
Click in the cell at column 1, row 2, and while keeping the left mouse button depressed move the mouse pointer downwards, moving it over the bottom window border. The window will scroll. Release the mouse button when you point in the spreadsheet cell at column 2, row 102.
|Now all cells in the range from column 1, line 2 to column 2, line 102 are selected.|
From the Fill menu choose down copy. Now all formula will be copied downwards
|step 6.11||Push the "home" key: .|
|In order to plot the two columns, we could proceed as in step 1.6 through 1.11. Here we'll follow an alternative method.|
|step 6.12||Click the column key number 1:|
|step 6.13||From the Modify/Stats menu select Set as X-column.|
|step 6.14||Click the column key number 2 and from the Modify/Stats menu select Set as Y-column.|
|The X and Y columns are now indicated on the column keys.|
|step 6.15||From the Modify/Stats menu select Line plot. A new drawing sheet is created with the following graph:|
|We will combine this graph with the result of step 4.|
|step 6.16||Click on one of the elements of the graph. Eight little boxes will indicate that the graph is selected.|
|step 6.17||Push the copy icon in the program icon bar.|
|step 6.18||Open the file "step4.his".|
|step 6.19||Enlarge the window to show an empty place where you'd like to paste the sine wave and click once on this spot. That is where the centre of the sine wave graph will be.|
|step 6.20||Push the paste icon in the program icon bar:|
|step 6.21||Finally push the "save" icon to save the modified step4.his file..|
7 A few last details: We'll make figure "step4" ready for publication.
|step 7.1||If it is not open yet, open file "step4.his". Size the window such that we have room to work.|
|step 7.2||Select the parabola graph by clicking once on it. Depress the <SHIFT> key and while keeping it depressed click once on the sine wave graph. Release the shift key. Now both graphs are selected.|
|step 7.3||Click on either graph, and while maintaining the left mouse button depressed, move it to the right and slightly to the bottom. The two graphs move. Release the mouse button when there is a reasonable left margin.|
|step 7.4||Unselect the graphs by clicking anywhere on the sheet.|
From the "drawing tool box" click the text tool:
|step 7.6||Click left of the parabola and type "A".|
Click left of the sine wave and type "B".
Click underneath the parabola and type "a parabola".
Click underneath the sine and type "a sine".
Then select the "arrow" tool from the drawing tool box (just left of the text tool).
|step 7.7||Select both the "A" and the "B" text objects by clicking on them and using the shift key as in step 7.2.|
|step 7.8||Click on the "font tool and change the font size to "16". The result is now like:|
|step 7.9||Click the "align tool"
in the drawing tool box. A dialogue window pops up. Choose the following settings and push the "OK" button:|
|step 7.10||Select the two graphs, click the align tool and push the "OK" button in the dialogue window.|
|step 7.11||Similarly bottom-align the two text objects "a parabola" and "a sine".|
|step 7.12||Select the "a parabola" text object and move the object to the left or the right using the horizontal arrow keys on your keyboard. Do the same with the "a sine" object.|
|step 7.13||Save the file. The result may resemble:|
8 Deleting objects and dissociating a graph.
|step 8.1||Load file "step4.his" that was modified and saved in step 7.|
Click above and left of the "A" on the drawing sheet. While keeping the left mouse button depressed move mouse downward and to the right until you reached a point just below and to the right of the text "a sine":
|step 8.3||Release the mouse button and push the "delete" icon from the program icon bar. The five selected objects will be deleted. The sine wave graph remains.|
|step 8.4||Click on the sinewave graph and choose Dissociate from the Edit menu. The graph is now dissociated into several elements. Click anywhere on the drawing sheet to unselect the objects, select the sine wave and then drag it to the left:|
|step 8.5||Double-click on the sine wave. Then RIGHT mouse click until the scissors cursor appears.|
|step 8.6||Move the cursor as in the figure and left-mouse click to cut the sine wave in two.|
|step 8.7||Unselect by clicking anywhere. Select the left sine period and drag it to the left:|
9 Creating a bar plot with error bars.
|step 9.1||Start the program.|
|step 9.2||From the File menu (it is the only one available right now), choose new.|
|step 9.3||In the "Select File Type" dialogue window, choose "spreadsheet" and push "OK". A blank spreadsheet "document1" is created|
|step 9.4||Click in the spreadsheet cell at column 1, row 1.|
|Then type (without the quote marks): "50" followed by <RETURN>, then type "72" <RETURN>,and "66" <RETURN>. If you type a wrong character, use the <BACKSPACE> key|
Click in the spreadsheet cell at column 2, row 1.
Click in the spreadsheet cell at column 3, row 1.
Click in the spreadsheet cell at column 4, row 1.
|step 9.8||Select the columns 1 through 4 (see step 1) and from the Modify/Stats menu select Bar Plot. The "Set Columns" dialogue window pops up:|
|step 9.9||n the "Set Columns" dialogue window check "Y" (the default). This means that column 1 will be the Y-coordinate.|
|step 9.10||Advance one column by pushing the button. Check "SEM". Hence, column 2 will furnish the Error bar.|
Advance again, check "Y", advance again one column (we are now at column 4) and check "SEM".
Push "OK". The following graph will be created:
|step 9.11||In The dialogue window that pops up, carry out the indicated modifications (fill colour, show lower half) and push the "OK" button.|
|This results in:|
|The representation of the white columns may be changed similarly after double clicking on one of the white bars.|
10 Spreadsheet slidebar demo
|step 10.1||From the File menu choose open and load the spreadsheet file "sbardemo.txt" that should reside in your Program's directory. The spreadsheet should look like:|
|Column 3 contains a cosine that is a function of the line index (lin) and column 4 contains a sine function.|
|step 10.2||Select columns 3 and 4 (as in step 1.6) and choose line plot from the Modify/Stats menu. After having set column 3 as the "x" column and column 4 as the "y" column in the dialogue window that pops up, a new drawing sheet is created with a graph containing a circle as shown to the right. As can be seen from the formula in the spreadsheet edit box: "=sin([C2:L$4]*(lin-2)*0.03141)", the frequency of the sinewave is determined by the contents of the cell at column 2 and line 4. This cell contains a slidebar. After selecting this cell, the spreadsheet edit box will show: "=sbar(1,4,11)", meaning that the sinewave frequency may vary between 1 and 11 depending on the position of the cursor, which here is 4. The frequency of the cosine at column 3 similarly depends on the setting of the slidebar at column 2 and line 2.|
|step 10.3||In the slidebar at column 2 and line 4, click on the space between the central cursor and the gadget.
The sinewave frequency changes from 4 to 5 (it is incremented by 10% of the difference between the slidebar's minimum (1) and maximum (11) settings). The data in column 4 change and the graph, which is linked to columns 3 and 4, changes accordingly into:
|11 Subtract background from two b&w images and merge.|
|step 11.1||Download cell.his, containing a microscope field with cells that were stained blue (DNA) and green (GFP). The images were taken by a black&white camera.|
Since the images are black and white and bacause we're going to draw onto them, the pen colour needs to be different from black, red for example. To do so, push the outline colour gadget and choose red as in the figure.
Then select both the image and the rectangle and choose Get mean background from the Bitmap menu. A dialogue window appears showing 10,10,10 for the RGB background values.Push OK.
|step 11.4||Select the bitmap only and issue the command Subtract background from the Bitmap menu. Another dialogue box pops up confirming the background values to be subtracted as well as some other options that are explained elsewhere in detail. For now, push OK and you're done. Repeat 11.3 - 11.4 with the second image.|
Select Anaglyph/Merge from the Bitmap menu. In the 'Set R-G-B bitmaps' dialogue box push the 'blue' button for the 'blue' image and the 'green' button for the 'green image', changing image with the button labelled ''next image'. Then push 'RGB Merge'. A new two-colour bitmap is created.
|12 Get cell properties Determine properties of cells in an image|
All cell nuclei in the previous image are stained with blue dye and some of those are also coloured green. We'll use the blue colour to deliniate the nuclei and then measure their 'green contents'. Select Object properties from the Bitmap menu. A first dialogue comes up (more will follow). Push 'Nucleus colour' and change it to pink (or anything other than green and blue). Uncheck the R and G boxes beside the threshold and nucleus colours. Then move the red and pink lines in the upper panel until the following image is more or less obtained.
Check the 'create tagged bitmap' and the 'total green' boxes and push 'OK'.
A new image and a spreadsheet are created. The image shows all cells labelled with a number. If this image is selected and you choose the pipette tool from the toolbox, a dialogue box pops up that shows the data corresponding to the cell that the pipette is pointing to (cell 19 in this example). The spreadsheet contains a list of 'greenness' of the tagged cells.
If you have second thoughts and would have liked additional properties, not all is lost. Push the 'Choose properties' button on the 'Properties' dialogue box. This results in the appearance of yet another dialogue box.
Now select column 4 labelled 'Green' and create a histogram by choosing Frequency histogram from the Modify menu. Then fill in the dialogue box as shown. Push 'OK' and the histogram to the right is created.