Olympus Microscope Instructions Terminology: 1. Brightfield. It is to observe the light reflected directly from the sample. The light from the lamp is vertically guided through objectives and incident on the sample. It is the most common way of observing specimens. 2. Darkfield. It is to observe the scattered or diffracted light from the sample. The light from the lamp travels under an angle thus enhancing the scratches or flaws on the surface of the sample. It is suitable for examining flaws on ‘mirror’ surfaces. 3. DIC (Differential Interference Contrast), also called Nomarski. It is a microscopic observation technique where a very small height difference on the surface of the sample (not visible with brightfield) becomes a ‘three-dimensional’ image with improved contrast. This is achieved by using a combination of an analyzer, polarizer, and DIC prism. 4. Working Distance. It is the distance between the bottom edge of the objective and the surface of the sample when the sample is in focus. An objective with long working distance is much farther away from the sample than the objective with a regular working distance, thus minimizing the possibility of a ‘collision’ between the objective lens and the sample. The working distance of the 100X objective is the smallest and it is ~ 1 mm. Do not ever bring your sample closer than 1 mm.

Microscope features

Variable magnifier Nose piece with six objectives

On-off switch for the microscope light

Sample stage motion control

Focusing knob

Adjustment of the microscope light intensity

1

Yellow filter – cuts the UV light.

LBD (Light Blue Daylight) filter – modifies the lamp spectrum to resemble that of the sun light.

Variable magnifier (1X, 1.25X, 1.6X, and 2X)

Knob to switch between brightfield (BF) and darkfield (DF). This knob determines whether the light goes to the camera or the observer’s eye. Push in for the microscope observation, pull out for the camera.

Field stop – by pulling the upper knob out you can adjust the field of view. Do not touch the bottom knob. Aperture stop – Do not touch these two knobs.

Analyzer Polarizer DIC prism

How to use the DIC prism?

2

How to use DIC? Inserted DIC prism

Loosen (counterclockwise) and tighten (clockwise) this knob every time you move the DIC prism. Whether being used or not, the DIC prism must be always tightened. That prevents it from accidental falling out (it costs ~ $3,000).

Pull this knob out if you are using 50X or 100X objectives. Push in for all other objectives.

Rotate this knob gently in any direction to achieve the best contrast.

DIC prism not inserted

If the DIC prism is used, both the analyzer and the polarizer must be inserted. If you would like to view your sample in the polarized light, pull out the DIC prism and leave the analyzer and polarizer inserted. To view it with white light, pull out the analyzer, polarizer, and the DIC prism. Since they cut most of the light, the light intensity will increase substantially. Reduce it. Remember, the analyzer, polarizer, and DIC prism can be used in brightfield only.

3

1. Always leave the microscope in brightfield (BF) with the yellow filer, LBD filter, analyzer, polarizer, and DIC prism inserted. Make sure that the prism is tightened. Check that the upper knob of the Field Stop (FS) and the Aperture Stop (AS) is pushed in and so is the knob that controls the light going to the camera. Turn the light switch on. 2. To view the sample with white light, pull out the analyzer, polarizer, and the DIC prism. Tighten the DIC prism after you pull it out. 3. To view the sample with darkfield, pull out the analyzer, polarizer, and the DIC prism, and move the knob from BF to DF position. You need to increase the light intensity. 4. After you are done, return everything back as described in step1 and turn off the microscope light.

Instructions for the digital camera (DP Manager) 1. Logon using your username and password. 2. Double-click the “DPManager” icon on the desktop. From the top menu select “Capture” and “Start DPController”.

3. Set up the microscope and focus on the sample. When you are ready to take picture, pull out the knob on the upper right-hand side of the microscope. The camera can see your sample now. Click “Live Image” under “Capture” Select the resolution. The This allows you to menu found at the bottom of the screen. true resolution of the camera integrate or average Your sample will appear on the screen. chip is 1360 x 1024 pixels. images.

Choose “Auto” for brightfield and “SFL Auto” for darkfield.

Choose the spot size. You may move the spot. The software uses the area of the spot to determine the brightness, etc., in the “auto” mode. 4

4. To adjust the color balance, select “Color balance” menu. Then select “Manual” and “One Touch”. When using brightfield, use buttons on the left side (White Balance). (When using darkfield, use buttons on the right side (Black Balance)). Point the mouse to the area in the image that should be white (black) and click.

5. Adding scale to the picture. Select “Scale” menu. Then select “Show Scale” and deselect “Free size”. A yellow scale will appear. Click and drag the scale to resize it. If you would like to include the scale in the image, select “Include in Image”. Otherwise, the scale will be on the screen but not saved in the image. Select the type of the scale bar and the number style. Next, choose the objective by clicking one of the six possible options. Finally, select the proper “Adaptor Lens”. The choice of the Adaptor Lens depends on the value of the variable magnifier. Use the following table to find the proper value: Variable magnifier Adaptor lens 1 0.5 1.25 0.625 1.6 0.8 2 1

When selecting an objective, click here. Do not click on the “…” window next to it. That will start the recalibration of the scale.

5

6. Other functions.

White balance

Black balance

Add scale to Color / gray scale the still image

Rotate/ flip

Different ways to view the image on the screen. The fourth icon from the left gives 1360 x 1024 full screen image. Hit “Esc” to exit.

Add text to the image Crop

Help

Sharpening tools

7. Image capture. Click this icon to capture image.

8. Saving images. Up to 10 images can be saved in the buffer. To save images permanently, create your own folder under C:\Documents and Settings\Your-user-name\My Documents. Use jpg format for saving images. Pictures may be transferred via CD or USB memory stick. 9. Printing images. High-quality pictures can be printed using a dye-sublimation printer attached to the computer. The printer requires a special paper and the cost of each print is ~ $5.00. To prevent frivolous printing, you need to see us to get the paper. Do not feed regular paper into the printer. Always use “Print preview” to verify that the image fits on one page. If needed, reduce it. Most images do not fit on a single page. 10. Log off and turn off the microscope after you are done.

6

Alternate software for the digital camera (MicroSuite) The main difference between “MicroSuite” and the “DPManager” is that the MicroSuite allows to print multiple images, to add objects / text to the image, to perform and save measurements, and log and save text files associated with the images in a searchable database. 1. Logon using your username and password. 2. Double-click on the “OLYMPUS MicroSuite” icon on the desktop. The main screen consists of several sections: Menu Bar View Manager

Image Buffer (can be shown as “gallery” or as a “list”)

3. Set up the microscope and focus on the sample. When you are ready to take picture, pull out the knob on the upper right-hand side of the microscope. The camera controls are found on the upper right-hand side of the computer screen:

Set Input Set Magnification Live 680x512 Camera (select the (select the magn. or Control variable of the objective) Live 1360x1024 magnifier) (activates the live image)

Snapshot (with different resolution) SQ = 680 x 512 pixels HQ = 1360 x 1024 pixels SHQ = 2040 x 1536 pixels UHQ = 4080 x 3072 pixels

First, click on “Set Input” and select the proper value for the variable magnifier. Next, click on the “Set Magnification” and select the objective. Click on “Live 1360x1024” to display

7

the live image on the screen. Click on “Camera Control” to open the camera control panel. Inside the panel you will find controls that are similar to the “DPManager” software. However, the controls are arranged differently. Adjust the image. Mode selection (select “Auto” for brightfield and “SFL-Auto” for darkfield.)

Spot Size controls (the software uses the area of the spot to determine the brightness, etc., in the “Auto” mode.)

White Balance Sharpness Black Balance Monochrome

Full Screen View (hit “Esc” to cancel) When you are ready to take the picture, click on any of the “Snapshot” buttons. The image will appear in the Image Buffer. 4. Adding scale to the picture. In the upper main menu, go to “Image” → “Scale Bar” → “Show in Viewport” to display the scale on the screen. To add the scale to the picture permanently, go to “Image” → “Scale Bar” → “Burn into Image”. 5. Saving images. The images are temporarily saved in the buffer. To save an image permanently, first select it in the buffer section by clicking on it. Then right-click the mouse and select “Image Information…”. Type the name of the image. Close the window. Now click again on the image in the buffer section, and drag it onto the “Save” icon located at the bottom of the buffer section. Create your own folder under C:\Documents and Settings\ Your-user-name\My Documents. Pictures may be transferred via CD or USB memory stick. 6. Other functions. Image Protect Image Cropping Overlay Button Bar Delete Overlay Information (locks the image tools (to insert objects, (used when deleting in the buffer) text into picture) objects from the picture)

8

One may draw simple objects or add text to the picture by clicking on “Overlay Button Bar” and selecting the appropriate function. To delete added objects click on “Delete Overlay”. 7. Printing images. High-quality pictures can be printed using a dye-sublimation printer attached to the computer. The printer requires a special paper and the cost of each print is ~ $5.00. Do not feed regular paper into the printer. The software allows you to arrange and print several images on one page. Any image that you wish to print, must be first loaded in the buffer section. Select “Multiple Images” and click on “Layout”. You may set the image size and gaps between images (in cm), add header and footer information, etc. 8. Deleting images from the buffer. To delete particular image, select it with the mouse, then click and drag it into the “Trash” located at the bottom of the image buffer. To delete all images, press “Ctrl”+“A” on the keyboard, then hit “Delete”. 9. Loading old images into buffer. “Open” the folder with the stored images. Click on the required image file name and drag it into the buffer. 10. Measurements. Several things can be measured (see the menu). To erase the objects drawn during the measurement, click on “Delete Overlay” icon.

11. Database tools. The program also offers extensive database tools found under “Database”. One may log text information with any image. The database is searchable. 12. Log out and turn off the microscope after you are done.

9