Leica DB LB Research microscope and Studo Lite Imaging software Room B523 User Guide Molecular Imaging Unit University of Helsinki www.miu.helsinki.fi 9.4.
1 2 GENERAL USER INFORMATION .............................................................................. 1 SETTINGS FOR TRANSMITTED LIGHT MICROSCOPY...................................... 1 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 3 IMAGE ACQUISITION ................................................................................................ 10 3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 3.4 3.5 3.6 3.6.1 3.6.2 4 5 6 Turning on the microscope ............................
1 General user information Leica DM LB research microscope is only for the Molecular Cancer Biology (MCBP) and Genome-scale Biology (GSB) research programs internal use. It is a transmitted light microscope that cannot be used for fluorescence microscopy. Bright-field, polarization contrast, and low-quality dark-field illumination are available. There are no oil objectives available, thus the resolution of the objectives is lower than that of the Axioplans.
movement in y-direction (back and forth), lower in x-direction (side to side). If you need to rotate the stage to get the image in any wanted angle, open the rotation lock by unscrewing it and then rotating the stage by hand (circle in Figure 2). Re-screw the lock after use! Figure 2. Adjustment wheels for stage control (arrows) and rotation lock (circle) 2.3 Placing the sample Place your sample on the specimen stage by manually widening and releasing the spring-operated object holder (Figure 3). 2.
Objective Numerical Aperture (NA) Color ring Coverglass (c.g.) Position for thickness condensor lens N Plan 2.5x 0.07 Brown Any/without c.g. off N Plan 5x 0.12 Red Any/without c.g. off N Plan 10x 0.25 Yellow Any/without c.g. on N Plan 20x 0.4 Green 0.17 mm on N Plan 40x 0.65 Blue 0.17 mm on C Plan 63x 0.75 Black 0.17 mm on auxiliary Table 1. Objectives in Leica DM LB N Plan stands for planachromat and C Plan for achromat objectives.
The fine focus has two modes: when the left focus wheel is pulled out, each line on the wheel equals 1 µm of movement. When it is pushed in, each line equals 4 µm of movement. Figure 5. Focusing wheels 2.6 Eye pieces (oculars) Adjust the folding bridge of the oculars until you see, using both eyes, only one circular field of view through the oculars. Use the diopter adjustment on the oculars to match your vision (Figure 6, part B).
Figure 7. Dual viewing attachment Figure 8. Power source for the light pointer There is a light pointer in the viewing attachment that uses an external power source. Thus, you need to connect the transformer into a power outlet before using the pointer (Figure 8). After use, disconnect the transformer. Notice that you can use the monitor instead of the dual viewing attachment. On the back side of the viewing attachment, there are two control knobs for the light pointer (Figure 9).
Figure 9. Light pointer controls 2.8 Koehler illumination Koehler illumination needs to be adjusted in the beginning of every imaging session for optimal illumination. Proceed as follows: Instruction View in eyepiece 1. Use 10x objective and front lens. Focus on the sample. 2. Close the luminous-field diaphragm by turning the adjustment wheel clockwise. 3. Move the condenser up or down so that the diaphragm edges are in focus.
Instruction View in eyepiece 4. Center the diaphragm in the field of view by rotating the condenser adjusting screws. In other words, you adjust the illuminating path so that it is properly aligned along the optical axis of the microscope. 5. Open the luminous-field diaphragm by turning the adjustment wheel counterclockwise until the edges just disappear from the field of view. This step ensures that you only illuminate the fieldof-view. 6. Pull out one ocular and look through the eyepiece tube.
2.9 Polarization contrast Figure 10. Analyzer for polarization contrast imaging Figure 11. Polarizer For polarization contrast microscopy, an analyzer (a polarizing filter) is inserted in the light path by pushing it all the way in (Figure 10), and another polarizing filter (polarizer) is placed labeled side upwards in a holder below the condenser (Figure 11). The round polarizer is on the shelf above the microscope.
• Grun: a green panchromatic filter that was used for contrast enchantment for B/W photography These filters were used for film photography and are not useful for digital photography. However, when using low intensity light (yellowish), DLF-filter adjusts the light back to the direction of blue, making the image more pleasant to view. Figure 12. Filter controls marked with red arrows 2.
3 Image acquisition 3.1 Accessing the computer If the computer is turned off, push the power button to turn it on. When the login screen appears, log in using your hyad login account. Check that the domain is set to LTDK. 3.2 View Finder application A 10-bit Olympus DP50 color CCD camera is attached on the top of the microscope. It is controlled by a computer and Studio Lite software. To capture an image, open the Pixera Corporation Studio Lite program by doubleclicking its icon on the desktop (Figure 14).
simply multiplied values, thus the same effect can be added later on in image editing software. Therefore, changing the value from 100 is not recommended. 3.3.3 Exposure Mode In the Exposure Mode box, select either Automatic or Manual: • Automatic (AE): The software measures the optimal exposure time automatically. Change Spot and Exposure Adjust if needed (see below). To lock the exposure time, click AE lock button (Figure 15, upper toolbar, rightmost button).
Figure 16. Histogram level adjustment. Red square: levels button 3.3.6 Focus tool Focusing tool assists you in focusing. If the focusing toolbar is not visible, enable it in the Options menu. Once activated, two bars appear (Figure 17). The blue one shows the current focus value, and the red one a history of the best level reached since last reset. When you focus up and down, you should try to hit the highest level (match the blue and red bars).
). Make sure the stage does not move at all between the images or image quality decreases. 3.4 Capturing the image Press the Camera button next to the Live button to take an image. After acquisition, the image will appear in Studio Lite. 3.5 Viewing images To zoom in or out, select the zoom tool in the toolbar at the right edge of the screen (Figure 18, middle button). When zoomed in, you can pan around the view with the panning tool (Figure 18, bottom button). Figure 18.
3.6.2 Scale bars The Studio Lite software does not provide means to insert scale bars. They can be added later on in any image editing software such as Adobe Photoshop. Ready-made scale bars for the full resolution images are available at http://www.miu.helsinki.fi/instruments/LeicaDMLB/scale_pictures.htm (in Photoshop format). The page also contains the scaling information (i.e. how many micrometers each pixel represents), so you can draw the scale bars yourself.
• Cover the microscope with the red hood 6 Troubleshooting 6.1 There is no light • Microscope is not turned on (see Section 2.1) • Light intensity set too low (see 2.1 on page 1) • Do you see any light in the lamp chamber? If not, the halogen bulb may need to be changed - contact MIU 6.2 Light is too dark Check: • whether the polarizer or other filters are on the light path (Section 2.9 on page 8) • that aperture diaphragm is not too closed (Section 2.8 on page 6) 6.
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