Leica B523 manuel d'utilisation

Leica DB LB Research mi croscope and Stu do Lite Imaging so ftware Roo m B523 User Guide Molecular I maging Unit University of Helsinki www .m iu.helsin ki.fi 9.4.2008[...]

1 GENERA L USER INFORMATION .............................................................................. 1 2 SETTINGS FOR TRAN SMITTED LIGHT M ICROSCOPY...................................... 1 2.1 Turning on the microscope .................................................................................................... 1 2.2 XY-stage..........[...]

1 1 General user information Leica DM LB research microscope is onl y for th e Molecular Canc er Biology (MCBP) and Genome-scale Biology (GSB ) r esearch p rograms internal us e. It i s a transmitted light microscope that cannot be used for fluorescence microscop y . Bright-field, polarization contrast, and low-quality d ark-field i llumination are[...]

2 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 th en rotating the stage by ha nd (circle in Figure 2). Re-screw the lock after use! Figure 2. Adjustment wheels for stag e control (arrows) and ro tatio[...]

3 Objective Numerical Aperture (NA) Color ring Coverg lass (c .g.) thickness Position for auxili ary 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 Table 1. [...]

4 The fine focus has two modes: when the left focus wheel is pulled out, each l ine on the wheel equals 1 µ m of movement. When it is pushed in, each line equals 4 µ m of movement. Figure 5. Focusing wheels 2.6 Eye pieces (oculars) Adjust the folding bridge o f the ocul ars until you see, using bot h e y es, onl y one circular field of view throu[...]

5 Figure 7. Dual viewing a ttachment Figure 8. Power source for the light pointer There is a light poin ter i n the viewing att achment that uses an external power sourc e. Thus, y ou need to connect the transformer into a power outlet b efore using the pointer (Figure 8). After use, disconnect the transformer. Notice that y ou can us e the monitor[...]

6 Figure 9. Light pointer controls 2.8 Koehler illumination Koehler ill umination needs to be adjusted in the beginning of ever y imagi ng 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 t he adjustment wheel[...]

7 Instruction View in eyepiece 4. Center the diaphragm i n the field of view by r otating the condenser a djusti ng screws. In other words, you adjust the illuminating path so that it is pr operly aligned along the optical axis of the microscope. 5. Open t he l uminous- field diaphragm by turning t he adjustment whee l counter- clockwise until the [...]

8 2.9 Polarization contr ast Figure 10 . Analyzer for polarization contra st imaging Figure 11 . Polarizer For polarization contrast microscop y , an an alyzer (a polarizi ng filter) is inserted in the light path b y pus hing it all the wa y in (Fi gure 10), and another polarizing filter (polarizer) is placed labeled side upwards i n a holder below[...]

9 • Grun : a green panchromatic filter that was used for contrast enchantment for B/W photography These filters were used for fil m photography and are not useful for digital photography. However, when usin g low intensit y light (yellowish), DLF-filter adjusts the light back to the direction of blue, making the image more pleasant to view. Figur[...]

10 3 Image acquisition 3.1 Accessing the com puter If the computer is turned off, push the power b utton to turn it on. Whe n the lo gin screen appe ars, lo g in using y our hyad lo gin a ccount. C heck that the domain is set to LTDK. 3.2 View Finder applic ation A 10-bit Ol y mpus DP50 color CC D camera is attached on the t op of the microscope. I[...]

11 simply m ultiplied 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 mea sures the optim al expos ure ti me automatically. Change Spot and Exp[...]

12 Figure 16 . Histogram level adj ustment. Red square: levels butt on 3.3.6 Focus tool Focusing tool assi sts you i n focusing. If the focusing toolbar is not visible, enable it in the Options menu. Once activated, two bars appear (Figure 17). The bl ue one shows the curre nt focus v alue, and the red one a history of the best level reached since [...]

13 15). Make sure the stage do es not move at all between the images or image qualit y decreases. 3.4 Capturing the im age 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 scree[...]

14 3.6.2 Scale bars The Studio L ite software does not provide means to insert scale bars. They can be added later on in an y im age editing software such as Adobe Photoshop. R eady -made scale bars for the full resolution images are avail able at http://www.miu.helsinki.fi/instruments/LeicaDMLB/scale_pictures.htm (in P hotoshop format). The page a[...]

15 • Cover the microscope with the red hood 6 T roubleshooting 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 t he lamp chamber? If not, the halogen bulb m ay n eed to be changed - contact MIU 6.2 Light is too dark Check: • whether the polar[...]

16 6.7 Computer login fa ils Check that: • domain is LTDK • use are usi ng y our h yad account user ID and p assword - the same ones you are using for accessing your university e-mail account[...]