Olympus BX-60 upright Fluorescence Microscope with a Ludl Focus Motor and an Evolution Digital CCD Camera

The system setup in 406F allows for acquisition of high resolution images using wide field fluorescence or bright field microscopy. Since, the driver for the CCD camera is a integral part of ImagePro Plus software, the users can proceed immediately after capturing their images to image enhancement and analysis. There is a large variety of options for image enhancement either on-line or off-line using the interface. For example using the Evolution interface, white balance can be adjusted manually to match the best your bright field image, or gain and offset can be modulated for better fit of your image data into the dynamic scale. Thus, the user can obtain images that fit the entire dynamic range, providing not only smoothness but also reliability of scientific data. ImagePro Plus also offers an easy interface for extracting and merging channels from same field, making it possible with single click to produce merge images taken with different filter cubes. 3-D reconstruction module available with version 5.1 of ImagePro Plus would let the users to generate 3D rendering from series of images.

Short guide to basic operations using Evolution MP CCD Camera System

(developed by Dr. Robyn Rufner)

1. With the Focus Motor and Microscope Workstation PC turned ON, Turn ON the BX-60 power switch at the back of the microscope (3) and depress the green Preset Button to adjust the brightness of the halogen lamp to 4 (2 green bars).

2. Insure the Lamp Selector switch (4) is ß for the halogen lamp and the Camera Shutter is Closed (Pushed-In).

3. If you will be using fluorescence filter cubes, Turn ON the power switch to the Mercury Lamp Burner (5). Wait at least 5 minutes for the mercury-arc lamp to stabilize before using a fluorescence filter cube and leave it ON for at least 1 hour! Record the Start and Stop “Lamp Time” in the Log Book.  Proceed to Step #

4. Place the CMIA Test Slide on the microscope stage and Select the 20x objective. With the Filter Block ring on “O” for Bright Field, focus the section using the Left Focus Knob or the Right Joystick Knob. Insure all stage filters are “ON” with the white dot next to the gray circle. If the field of view is too bright, press the green Preset button and press the light-intensity slide-bar control ß until only P is illuminated since the camera is very sensitive!!!

5. Focus your specimen. Insure both Oculars are correctly focused for your eyes so the Objectives will be parfocal! Close/cover your Left eye and use the Left Focus Knob or Right Joystick Knob to view a sharp image with your Right eye. Close/cover your Right eye and move the Left ocular Diopter Adjustment Ring until you visualize a sharp image. This is essential!

6.  Check Alignment of the microscope via Koehler Illumination.

• Turn ON the halogen lamp using “Bright Field” settings and Select the Objective you will be using, such as 20X.
• Place the “Test” slide” on the microscope stage and focus the image.
• Close the Field Aperture (Iris) located under the condenser until only 1/4^th of the field of view is illuminated.
• Focus the edge of the image of the Field Aperture using the Condenser Focus Control knob.
• If the Field Aperture is not centered, gently move the image to the center of the field of view with the 2 Condenser Centering Screws.
• Open the Field Aperture so that it is slightly larger than the field of view. If you subsequently select a higher objective, such as 40X, it may be necessary to increase the opening of the Field Aperture.

7.  At the Microscope Workstation PC, Open Image Pro-Plus 5.1 by double-clicking on the Image Pro-Plus 5.1 icon Þ Acquire Þ Capture Options Þ Setup Acquire. The Evolution MP camera menu will appear.

8. For BRIGHT FIELD IMAGING, Scroll to the appropriate Camera Setting/ Microscope Objective, such as “Bright field 20x” on the Setup menu. Center an Area of Interest (AOI) on your slide, Confirm the halogen lamp light level is not too bright (on 4 or P) and Gently Pull-out the Camera Shutter to the ½ position. Click on “Start Preview” and Focus using the Left Focus Knob or Right Joystick Knob while viewing the Monitor. A focus bar on the bottom of the image has been provided for your convenience. Note: The live Preview Image format is 640 x 480 pixels and will usually appear darker than the high resolution (2560 x 1920) Acquired Image. If the Hue/Color of your stain on the Monitor does not seem reasonable, proceed to step # 9. Click “Snap” to capture the image. Gently Close the Camera Shutter and Click “Stop Preview”. File Þ Save as (Tiff format) Þ to C:\My Temp Files.  Adjust Brightness & Contrast using Image-Pro Plus (Enhance Þ Contrast Enhancement Þ Apply) or Adobe Photoshop (Image Þ Brightness Contrast). Always CLOSE the Camera Shutter before changing objectives or slides or when the camera is not in use!

9. COLOR ADJUSTMENT. If the Hue/Color of your stain on the Monitor does not seem reasonable, move the stage controls so at least half of the Preview Image contains a white portion on your slide. Click on the “Acquire” page of the Evolution MP menu Þ Camera Features Þ Auto White Balance Þ put a Ö in the Auto White Balance. Select the “Set AOI” box and select an area of interest of white background on your preview image by dragging the left mouse button Þ OK Þ “White Balance” Þ OK.  Note: Auto While Balance is only available for Bright Field imaging with a full-size, color image with binning = 1.

10. BRIGHTNESS. For minor brightness adjustments, Select Preview on the Evolution MP menu and change the Exposure Time. For example, change 24 millisec to 20 for a darker image or 24 millisec to 28 for a brighter image. You can also use Acquire Þ Camera Features Þ  “Exposure Options” Þ “Set AOI” Þ “Compute Auto Exposure”.  Note suggested Exposures per Objective using the “Test Slide”:

11. For FLUORESCENCE IMAGING, Place your slide on the stage, Select the appropriate Filter Block/Holder for your fluorochrome and move the Lamp Selector switch (4) on the BX-60 up Ý for the mercury arc lamp. Select the appropriate Camera Setting for your fluorochrome/objective on the Evolution MP menu. Turn-off the room lights and Gently Push the Hg Lamp Shutter (upper right). Once you can visualize fluorescence from your sample via the oculars, Gently Pull-Out the Camera light-path bar to the ½ position. Click on “Start Preview” and Focus on the Monitor. Since auto-exposure is not available for low-light levels, it may be necessary to change the Exposure Time for both the Preview and Acquire pages. Note suggested Exposures per Objective using the “Fluorescence Test Slide” with a Preview Resolution of 640x480 (4) and an Acquire Resolution of 1280x969 (2)

Click “Snap” to Capture the image. Gently Pullout the Hg Lamp Shutter, Close the Camera Shutter and Click “Stop Preview”. File Þ Save as (Tiff format) Þ C:\My Temp Files and later to your Zip Disk (D:\) or to a CD (E:). Adjust Brightness & Contrast using Image-Pro Plus (Enhance Þ Contrast Enhancement Þ Apply) or Adobe Photoshop (Image Þ Brightness Contrast). Always CLOSE the Camera Shutter before changing objectives or slides and when the camera is not in use! When finished, return all settings to the Bright Field mode = Filter Block ring on “O”, Lamp Selector switch ß, Mercury Lamp Shutter Out.

• Before you leave: Close all Image Files and Close Image-Pro Plus menus and software.
• Turn OFF the BX-60 Microscope and the Mercury Arc Lamp (after I hour), Insure BX-60 settings are for the “Bright Field” Mode and DO NOT put the Dust Cover over the camera!
• Copy your files from C:\My Temp Files Þ Removable Disk (D:) or to a CD (E:) or USB Flash Card (F:) and delete your files from C:\My Temp Files. Empty the Recycle Bin and Sign the Log Book.

 

 

Combinations of fluorescence filter cube sets available with Olympus BX-60 microscope:

Filter Cube Holder # 1	Filter Cube Holder # 2
0 = No Filter	0 = No Filter
NIB = 480 ± 20 (FITC, Bodipy)	U = 330-385 (DAPI)
WIG = 520-550 (Texas Red)	G = 470 ± 20 (EN GFP)
Y = 620-660 (Cy5)	R = 535 ± 25 (Rhodamine)
Filter Cube Holder # 3	Filter Cube Holder # 4
0 = No Filter	0 = No Filter
MF = FITC + Texas Red	UV = 400-410 (Indo-1)
WU = FITC + Rhodamine	No Filter
CY = FITC + Cy3 + Cy5	No Filter