VWR Microscope Image Capture and Measurements Standard Operating Procedure
Version 1.0
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Important
Images are not to be saved to the computer. They will be deleted without warning. Use a USB drive or burn a CD. No additional software is too installed on the machine without prior approval by Dr. May or Brian Wajdyk. The computer is on the network/internet. Please be careful of which sites you visit.
Version 1.0
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Figure 1 Digital Micrograph Suite
Figure 2 AxioVision LE
Version 1.0
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Figure 3 Selector Rod
Operating Procedure 1. Preparation 1.1. Find the area of the sample to be imaged in the normal manner. Center the area of interest as much as possible. The field of view is less wide for the camera than seen through the eyepieces.
2. Load the software 2.1. Logon to the computer if necessary. 2.2. Double click the Digital Microscope Suite icon on the desktop. Also “C:\Program Files\EasyOn\Digital Microscope Suite\MicroCam2.exe”
3. Acquire image 3.1. If the sample was in focus through the eyepieces, it will be very close to being in focus for the digital camera. 3.2. Pull out the white‐ringed pushrod to divert the light to the camera port. 3.3. Fine focus while watching the live image. 3.3.1. In the event that you cannot see your sample after changing the selector to camera, go to a lower powered objective. Be VERY careful to not touch your sample to the lens. 3.3.2. Video refresh rate is not quite real‐time. Version 1.0
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4. Saving the image 4.1. Select the Binder icon with the green + to create an album. 4.1.1. No micrographs should be saved to the root of the album list. Beware that this is the default when you click the camera icon. 4.2. To save the image you can select the camera icon located under the image on the left side. If you are unsure where the images are being saved, hover your mouse icon over the image. 4.3. No images are to be saved to the hard drive. Please delete your album by clicking the binder icon with the red X. Images saved to the hard drive will be deleted without notification.
5. Measurements 5.1. Double click the Axiovision Icon on the desktop. Also "C:\Program Files\Carl Zeiss Vision\Axiovision 4\AxioVs40.exe" 5.2. By default, the image is shown at full resolution. This makes it appeared to be zoomed in at a higher magnification. 5.3. You will need to zoom out in order to see the entire image. This can be done by selecting the magnifying glass under the image with the vertical arrows (3rd icon from the left). You can select anything in between by using the magnifying glass with the + to increase the zoom or alternatively the magnifying glass with the – to decrease it. These manipulations do not change the actual image in anyway. Only the way viewed is changed. 5.4. Measurements are very accurate using the software. To measure a feature on an image go to the Workarea in the upper left section of the screen and click on Scalings. 5.5. The available scalings will be shown. Default is Pixels so you will need to select the scaling based on the microscope objective used and the measurement unit desired. For example to measure a feature you obtained an image with the 10x objective in millimeters, you would click on the scaling marked “10x mm”. 5.6. Once you have selected the scaling you can hit the “Activate” button. All further measurements will be made using this scaling until the software is shut down. 5.7. Now you have to apply that scaling to your current image by hitting the “apply to Image” button. 5.8. Now you are able to measure your features. Select the Measure pull down menu to Length. Note: You may want to zoom in on your area of interest if a more accurate measurement is needed. The software won’t be more accurate because it counts pixels, but you might have an easier time selecting the correct pixels at full resolution depending on your sample. 5.9. Click and drag a line then click again to define the area to be measured. The result will show up on the screen. 5.10. The true magnification varies based on screen resolution, display resolution, and scaling when printing. The Scale bar will always be correct relative to the features no matter how the image is displayed. To add the scale bar press the scale bar icon in the top toolbar. 5.11. Click and drag as before. Version 1.0
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Appendix A. Components
Figure 4 Microscope Components
1. Eyepiece (2) 2. Trinocular Body 3. Polarizer 4. Field Iris Diaphragm 5. Field Iris Diaphragm Centering Screws 6. Filter Wheel 7. Aperture Iris Diaphragm 8. Screw of Lamp House 9. Focusing Lens Lever 10. Lamp House 11. Lamp Adjustment Lever 12. Locking Screw 13. Coarse Focusing Knob 14. Fine Focusing Knob 15. Plug for Vertical Illumination 16. Transmitted / Incident Illumination Toggle Switch 17. Lengthwise Knob 18. Cross Knob 19. Collector 20. Field Diaphragm 21. Abbe Condenser and Iris Diaphragm with Filter Tray 22. Condenser Centering Screws 23. Abbe Condenser Locking Screw 24. Stage 25Objectives 26. Tube Holding Screw 27. Analyzer Lever Version 1.0
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Appendix B. Microscope Specifications Body: Optical Body, inclined 30°, able to rotate a full 360°with 53mm to 75mm interpupillary adjustment. Left Eye diopter adjustment of ‐5 to +5. Nosepiece: Quintuple ball‐beating reversed nosepiece with positive click stops and smooth operation. Eyepieces: 10X Plan wide field, Focal Length 25mm, Field Φ18mm. Stage: Integrated Mechanical Stage size 185mm X 140mm with Glass insert 98mm X 77mm. Right–hand Coaxial drop–down X–Y Control Knobs. Range of motion 75mm X 50mm. Focus Mechanism: All stainless steel and brass gear train system Adjustable Tension Control to prohibit drift and Adjustable Up‐Stop to protect slides, specimen and objective lenses. Coaxial coarse and fine adjustments with dial markings at 0.002mm increments. Incident Light System: Condenser, Field and Aperture Iris diaphragm, four color Filter Wheel and Polarizing filter Transmitted Light System: 1.25 N.A., Abbe Condenser with iris diaphragm Rack and Pinion Focusing, Easily centered with two adjustment knobs. Illumination: 20 watt, 6 volt Halogen bulb with electronic dimmer for Incident and Transmitted light systems. Fuse: 0.5 Amp, located in the base Digital Imager: Celestron Digital Microscope Imager. Max resolution 1280x1024 (1.3Megapixel) Version 1.0
