The PC3D PROGRAM 

The PC3D program allows you to digitize tracings of rat brain sections, perform primitive (EGA resolution) 3D reconstructions and calculate volume measurements. When sectioning, it is important that the sections be evenly spaced (take every section, or every 2^nd, or every 6^th ...). You should also be careful when collecting sections on microscope slides that you know which hemisphere is which. Once the sections are stained and coverslipped follow the following steps:

1. Microprojector Tracing
2. Input Tracefiles
3. Describe Tracefiles
4. Edit Tracefiles
5. Create 3-D Images
6. Volume Determination
7. Plot Image

Tracing Sections with the Microprojector

• Tracings should be evenly spaced and numbered. (Number should be distance in microns from the first tracing.)
• Orient all tracings the same way on the page.
• Draw only the structures that you intend to digitize.
• Trace all the structures as closed figures.
• Include a scale on one of the tracings to determine the magnification.

Input Tracefiles

1. Select 1. Input Tracefiles from the PC3D menu
2. Place your first drawing under the acetate on the digitizing tablet. Align the tracing so that it is centered under the vertical line on the acetate.
3. Enter "g" on the keyboard.
4. Using the "puck", enter the the Reference Point, Lower Left Point, and Upper Right Point when prompted by placing the crosshairs of the puck over the appropriate marking on the acetate and depressing the "0" button on the puck. A rectangle should appear on the monitor.
5. Trace the 1^st structure. You will hear a beep when the structure is closed. Before the structure is closed, you can erase it by pressing the "E" button on the puck.
6. When the computer beeps, you are prompted to enter a label for the structure on the alpha-numeric keypad of the puck. Each structure will be labelled with a different character 0->F. You must label all structures consistantly within and across drawings. (After the computer beeps, a keyboard entry will erase your most recent tracing.)
7. Continue until all structures have been traced and labelled.
8. When all structures have been traced and labelled, press the "D" button on the puck. You will be prompted to enter a filename on the keyboard.
9. All tracings of a set must have a common filename with the exception of the filename extension. The file name cannot exceed 8 characters. The filename extension specifies the location of the section Use the number assigned to the tracing. If any of these numbers exceeds 3 digits, strip the last digit for the filename extension. ^e.g. if the tracings are numbered 0,480,960,& 1440, the file names might be brain.0, brain.48, brain.96, and brain.144.
10. Return to step 2 until all tracings are digitized, then press "Q" to return to the PC3D menu.

Describe Tracefiles

1. Select 2.Describe Tracefiles from the PC3D menu.
2. You will be prompted to enter a Tracefile generic. This means the filename without the extension.
3. First Tracefile is the extension with the lowest number.
4. Last Tracefile is the extension with the highest number.
5. Tracefile increment is the arithmetic difference between successive extensions.
6. Type "n" to the query about creating a new tracefile set.
7. Enter the picture magnification detemined during the traceing step.
8. Enter the true distance between tracings in microns.
9. Come up with a name for your reconstruction. This name can be different than the filename.
10. For each character that appears on the screen, type the name of the structure assigned to that character. If no structure is assigned to the character, hit the enter key.

EditTracefiles

The amount of editing that you can do is extremely limited. The Edit program is used only to assign mislabelled structures the correct alphanumeric label.

1. Tracefile generic is the filename without extension.
2. Increment is the difference between consecutive extensions.
3. File forma: Enter "b" for binary.
4. Image number: Image number(extension) to edit.
5. Use the up, down, left, and right arrow keys to toggle your way through the different structures on your tracing.
6. Correct any errors by prssing the correct number on the keyboard when the mislabelled structure is highlighted.
7. Save corrections by using the "F5" key.
8. Use the "F3" & "F4" keys to move from drawing to drawing.
9. When done, the "F10" key woll return you to the PC3D menu.

Create 3-D Images

The Create 3-D Images program is used to manipulate your set of tracings in space. It can also be used to emphasize and de-emphasize individual structures for hightlighting.

1. Select 4. Create 3-D Images from the PC3D Menu. You will use the "i" & "r" commands from the create menu.
2. Start with the "i" command. You will be prompted for a generic which is the trace filename without the extension. A screen something like the one at the right will appear. The image at right shows the screen for a series of 41 CAT Scan images: abd.0, abd.10, abd,20, ..., abd.400.
3. Begin by assigning a COLor and an INCrement to each structure. When the cursor is in the color column a list of colours will appear at the bottom of the screen. Choose transparent (-ve) colors to start. (Note If you will be producing hard copy of the 3D reconstruction on a printer rather than a plotter,you will be printing a negative of the image which appears on the monitor. Bear this in mind when selecting colors. Assign Increments equal to the LEVELS AVAILABLE INCR to begin with.
4. Leave the Manual Scaling set to "N"
5. Don't bother entering a screen save file name or plot save file name.
6. Enter a transform. There are two types of commands that can be used:




Command	Action	Syntax	Example
s	scale	s(#axis)	s(4z)
r	rotate	r(#axis)	r(120y)



• Rotation: # = °CCW looking from the positive end of the axis towards the origin.
• Scaling: # = Scaling factor. s(2x) will make the image twice as wide. s(10y) will stretch the image 10 time on vertical axis. s(3z) will triple the spacing between sections.


7. When entering multiple transform commands they must be separated by commas. ^e.g. s(5z),r(25x),r(30y) Commands will be executed sequentially from L-->R.
8. Press the "F10" key to generate an image. If you have not specified a screen save file, the image will remain on the monitor until you press the Enter Key. If you did enter a screen file name, the image will appear momentarily and disappear. To view the image, press "ESC" key then type "r filename".
9. While your image is displayed on the monitor, hitting the PrintScrn Key will save the image to the clipboard from which it can be pasted into another program.

Highlighting Tips

• Begin by choosing a transform using the s and r commands which displays the area of interest to its best advantage
• Experiment with colour: remember the ciolour can be set as transparent or non-transparent. Some use of non-transparent colour will help promote a 3-D effect.
• Individual structures can be emphasized or de-emphasized by changing the increment setting on the Levels to Plot line and/or the INCR for an individual structure.
• Example
• Seven images at 100µm intervals are digitized: brain.0, brain.100, brain.200, ... , brain.600. When you are creating 3-D images, you can't change the images available, but you can change the levels to plot.

	first	Last	Incr
Levels Available	0	600	100
Levels to Plot	0	600	???

• You can change the ??? to some factor of the original value such as 50 or 25. If you change the ??? to 25, the program will generate extra copies of images so that you can plot in increments of 25 rather than 100.
• For each structure, the INCR should be set ato a multiple of the Levels to Plot INCR. ^e.g. If ??? is set to 25 and ...



Polygon Type	COL	INCR
0 Outline	-1	100
1 C Callosum	-2	200
2 Hippocampus	4	25



• Outline is neither emphasized nor de-emphasized. The original 7 tracings are displayed (0,100,200,...,600). The Corpus Callosum is de-emphasized. Only 4 tracings will be displaye (0,200,400,600). Hippocampus will be emphasized, 25 tracings will be displayed (0,25,50,75,...,600).
• Caution: Be careful how you use this highlighting technique. If the Hippocampus INCR had been set to 75, the structure would still be emphasized in the sense that 9 images would be displayed instead of the original 7. The images plotted would be 0,75,150,225,300,375,450,525,600. The problem is that 6 copies (underlined) have been added at the expense of 4 original tracings (100,200,400, & 500. Substituting copies for originals is not good practice.
• When you have created a 3-D reconstruction that you are happy with, fill in the Screen Save File (myimage.pic) and the Plot Save File (myimage.plt) and press the "F10" key to write the image to these files.

Volume Determination

The volume of each polygon type is calculated by measuring the cross sectional area of the polygons of that type by the section thickness and summing across all sections.

Volume is only calculated for objects composed entirely of closed polygons. Volume accuracy depends on the accuracy of the picture magnification and the section thickness which you provide.

1. Select 6. Volume Detemination from the PC3D Menu.
2. Enter the tracefile generic.
3. Accept the default value for the 1^st tracefile in the sequence.
4. Accept the default value for the last tracefile in the sequence.
5. Accept the default value for the tracefile increment.
6. The tracings will be processed and a summary table of the volumes of each polygon type will appear on the screen.

Plot Image

The PC3D program can generate a hard copy of your image but only via an HP Plotter. To plot an image using the plotter follow these steps:

1. Exit to DOS and type the following command at the prompt: mode com2:9600,n.8,1,p and press the Enter key. (This assumes that the plotter is connected to com2. If the plotter is connected to com1, substitute com1 for com2 in the command line.
2. Type PC3D to get back to the PC3D menu.
3. Select 7. Plot to HP Plotter from the PC3D menu.
4. Feed a sheeet of paper into the plotter
5. Type "w", a space, and then com1 or com2 to specify the output port.
6. Type "v", a space, and then a number from 1 to 40 to set plotting speed. 40 is fastest.
7. Type "p", a space, and then the plotfile name (myimage.plt).
8. Enter 25 for the desired width in cm.
9. Respond (y/n) to the pen assignment query.
10. Press ENTER key to start plotting.

It is becoming extremely difficult to find replacement colour pens for the plotter. It is more likely that a colour printer will be used to generate hard copy. PC3D cannot send output to a printer. You must follow these steps to generate a file for a printer:

1. When you create a 3-D image that you like, save it to the clipboard by pressing the PrintScrn key.
2. Open your preferred image editing program (Paint, Paint Shop Pro, Photoshop, ...) and paste the image from the clipboard into your image editor.
3. Perform a negative transform on the image changing the background from lack to white. (All the other colours will be changed as well).
4. Print the image.