Beginning Your Animation: The Direct Fluorescent Antibody Technique: Positive Test

4 3. Select “Insert”, “Timeline”, and “Frame.”

G. Deleting Frames

To delete frames from a timeline layer, use the keyboard command “Shift+F5”, or right-click on the desired frame and select “Remove Frames.”

II. Beginning Your Animation: The Direct Fluorescent Antibody Technique: Positive Test

1. Set the size and background color of the stage to be used in your animation. My demo is set at 5 X 5 inches. Once you have set the size and background color, save the file. 2. Insert the CD-ROM into its drive. 3. If you wish to view the finished Flash document you will be making, load in the Flash file .fla file “posdfa_ASM” from your CD-ROM. If you wish to view the finished Flash document of this animation that is on my web site, load in the Flash file .fla file “dfluaby_gk.”

A. Moving the Animation Title Text On to the Stage

Having text move onto the stage from the left or right is very simple. 1. Change the name of layer 1 to “frame” if you wish to place a border around your animation. To change the name, double-click on the layer you want to rename, type in the new name, and hit enter; see p. 2. 2. Add a new layer by left-clicking on the left-hand “+” icon sheet with curled edge below the layer column see p. 2 3. Change the name of layer 2 to “text 1”. See p. 2. 4. To add a border around your animation, import frame from the CD-ROM into your library. See p.2. 5. Click on frame 1 of the frame layer. Click on the select arrow and drag the frame to the stage to form a box around the stage. 6. Now click on frame 1 of the “text 1” layer. Click on the text tool “A”, click on the stage, and type Direct Fluorescent Antibody Test.” You can use the Properties Box to change font type, font size, font color, etc. Position the text where you want it to end up on the stage. In the “Properties” box, choose Static Text. 7. Use the keyboard command F8 to convert text to a symbol and place it in your library see p. 3. 8. Click frame 36 of the timeline default speed is 12 frames a second so this will represent 3 seconds and convert it to a keyframe using the keyboard command “F6” – or by clicking “Insert”, “Timeline”, and “Keyframe” – or right-clicking on frame 36 and selecting “Insert Keyframe.” See p. 3. 9. Click on frame 1 of the timeline for the “text 1” layer. Hold down the “shift key” and drag the text to the right just off the stage into the gray area. 10. Click on any frame between frame 1 and frame 36 of the “text 1” layer, go down to the properties box and select “Motion” from the drop-down menu next to Tween. 11. Hit “Enter” “Return” on MAC to play the animation. 12. Save the animation. See Fig. 2 to view the final screen. Fig. 2: Moving Text On to Stage

B. Add Unknown Antigen Bacterium to the Animation

1. Add a new layer by left-clicking the left-hand “+” icon sheet with curled edge below the layer column. Change the name of layer 3 to “antigen” see p. 2. 2. Import the “antigen” illustration from the CD-ROM to the library of your new animation see p. 2. Drag the illustration of bacterium01 from the library to the stage and position it in the center. 3. While still on frame 1 of “antigen,” type “unknown antigen” and center it inside the bacterium. See Fig. 3 to view the final screen. 5 Fig. 3: Unknown Antigen

C. Add Known Fluorescent Antibodies to the Animation

1. Add a new layer by left-clicking the left-hand “+” icon sheet with curled edge below the layer column. Change the name of layer 4 to “text 2.” 2. Add 12 more new layers and label them “fluaby1” through “fluaby12.” 3. Click on frame 36 of each of the “text 2” and the 12 new “fluaby” layers and convert to a keyframe by using the keyboard command “F6.” To save time, hold the “shift” key down while you select frame 36 of “fluaby12” and “text2” to select all 13 layers and then use “F6” to convert them all to keyframes. 4. Click on frame 36 of the “text 2” layer. At the bottom of the stage, type in “Known fluorescent antibody is added to the slide.” 5. Import the “fluoraby” illustration from the CD-ROM to the library of your new animation see p. 2. 6. Starting with the “fluaby1” layer, click on frame 36 and drag the illustration of the fluorescent antibody from the library to the stage. Position it in the upper left hand corner and, if necessary, rotate the illustration so that the Fab portion of the antibody is pointing towards the bacterium. The “free transform” tool used for rotation is the icon in the tools menu that appears as a square surrounded by dots and is located just below the pencil tool see Fig. 5. After selecting the rotation tool, move the cursor arrow near the selected illustration of an antibody until you see a curved line with an arrowhead. Move the mouse to rotate the image clockwise or counterclockwise. 7. Working clockwise around the bacterium, repeat for layers “fluaby2” through “fluaby8.” These 8 fluorescent antibodies will eventually bind to the bacterium. 8. Add a fluorescent antibody to each of the four corners of the stage in layers “fluaby9” through “fluaby12.” These 4 fluorescent antibodies will eventually be washed off the slide. 9. Click on frame 72 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” Hold the “shift” key down while you select frame 72 of the topmost and bottom most layers to select all and then use “F6” to convert them all to keyframes. 10. Save the animation 6 See Fig. 4 to view the final screen. Fig. 4: Adding Known Fluorescent Antibody

D. Moving the Fluorescent Antibodies to the Antigen

1. Click on frame 108 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” Hold the “shift” key down while you select frame 108 of the topmost and bottom most layers to select all and then use “F6” to convert all frame 108s to keyframes. 2. Click on frame 108 of the “fluaby1” layer and drag fluaby1 down to the bacterium. Using the “free transform” tool and the arrow keys, rotate the antibody so it fits the epitopes on the bacterium. Magnifying the image with the “magnification” tool will make this easier. 3. Click on a frame between 72 and 108, go down to the properties box and select “Motion” from the drop-down menu next to Tween. 4. Repeat steps 2 and 3 on frame 108 of each of the remaining “fluaby” layers. 5. Do not move “fluaby9” through “fluaby12.” These will be washed off the slide later. 6. Save the animation. The final screen is shown in Fig. 5. 7 Fig. 5: Move Fluorescent Antibodies to Antigen

E. Wash Slide to Remove Unbound Antibodies

1. Click on frame 109 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” Hold the “shift” key down while you select frame 109 of the topmost and bottom most layers to select all and then use “F6” to convert them all to keyframes. 2. Click on frame 109 of the “text 2” layer and delete the text in frame 109. Replace with the following text: “The slide is washed. Any fluorescent antibodies that are bound to the antigen are not washed off the slide.” 3. Go to frame 157 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” Hold the “shift” key down while you select frame 157 of the topmost and bottom most layers to select all and then use “F6” to convert them all to keyframes. This will give a 4 second span to read the text. 4. Go to frame 193 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” This will give a 3 second span for the unbound fluorescent antibodies to “wash off” the slide. 5. Now we will make the unbound fluorescent antibodies “wash off” the slide. Click on frame 193 of the “fluaby9” layer. Now go to the top of the screen and click on “Modify,” then “Transform,” and then “Scale and Rotate.” Type “0” in the scale box and click OK see Fig. 7. 6. Click on any frame between frame 157 and frame 193 of the “fluaby9” layer, go down to the properties box and select “Motion” from the drop-down menu next to Tween. 7. Repeat steps 5 and 6 for layers “fluaby10” through “fluaby12.” 8 Fig. 7: Scaling an Object

F. Showing the Known Antigen Fluorescing

1. Go to frame 194 of each layer in your animation and make it a keyframe by using the keyboard command “F6.” Hold the “shift” key down while you select frame 194 of the topmost and bottom most layers to select all and then use “F6” to convert them all to keyframes. 2. Click on frame 194 of the “text2” layer and delete with the delete key. Type the following in frame 194: “In a positive test, the antigen fluoresces when viewed with a fluorescent microscope.” 3. Go to frame 194 of the antigen, select the bacterium, and delete it with the delete key. Now drag “antigen_flu” from the library to the stage and line it up so the epitopes fit the antibody molecules. This shows the antigen fluorescing once the fluorescent antibodies bind. 4. Save the animation. The final screen is shown in Fig. 6. 9 Fig. 6: Fluorescent Antigen G. Ending the Animation A. Add a new layer by left-clicking the left-hand “+” icon sheet with curled edge below the layer column. Change the name of this layer to “action.” B. Select frame 1 of the “action” layer. At the top of the screen click “window”, “Other Panels,” “Common Libraries,” and “Buttons.” C. Select a playback button and drag it to the upper left-hand corner of the stage. D. Click on frame 194 of the “action” layer and make it a keyframe by using the keyboard command “F6.” E. To stop the animation after it plays, click on the final frame of the animation 194 of the “action” layer. Open the “Action-Frame” window above the screen. Type the following in the right-hand box of the Action-Frame window: stop ; Alternately, click ”Global Functions,” click “Timeline Control,” and drag the command “stop:” into the Actions-frame box box to the right. F. Select the playback button. Open the Behaviors” window on the right-hand side of the screen, click on the “+” sign, and click on “Movieclip,” click on “Goto and Play at frame or label.” In the “Goto and Play at frame or label” window, type “1” in the box at the bottom and click “OK.” See Fig. 8. 10 Fig. 8: Stop : and Goto and Play Commands

H. Publish and Play the Animation

A. Go to frame 1 and hit “Enter” “Return” on MAC to play the animation. B. Publish the animation as html by clicking on the blank screen and selecting “Settings” in the Properties Box, “html” in the drop-down menu, and “Publish” and “OK” in the html menu. C. Hit “Control+Enter” “Command+Enter” on MAC to test the animation. 11

III. Other Bells and Whistles A. Moving an Object Along a Path