1 Zeiss SteREO Lumar V12 User guide Trouble shooting 28.9.2010 Markku Saari
2 Microscope control... 3 Starting... 3 Basic microscope usage... 3 Camera control and imaging... 4 Camera activation... 4 Camera control... 4 Shading correction... 5 Scalings and scale bar... 5 Data Saving... 5 Ending... 5 Information... 6 Objectives... 6 Filters... 6 Microinjector... 6 Trouble shooting... 6 Drive L:\ mounting... 6 Microscope: focusing doesn't work... 6 Microscope: focus work, but can't give sharp focus level... 6 Microscope: can't find filters... 7 Camera - no image available... 7
3 Microscope control Starting 1. Turn on microscope 2. Turn on the light source you need COMMON SPECIMEN: transmitted light (KL2500 unit, halogen lamp) FLUORESCENCE DYES: HBO-burner (HBO100 unit) - Lamp ignition takes about 5 min - Remember 30 minutes: - When HBO lamp is turned on, keep it on at least 30minutes - When burner is turned off, it has to be cooled down 30minutes before switching on again. 3. Turn on computer - Log in: Username: Lumar Password: stereo 4. Open Axiovision software: shortcut Axiovision Rel. Basic microscope usage 1. Choose a light source a.) Transmitted light (halogen light): Black buttons on remote control - Double click: on/off - Pressing button down: light power adjustment (0-100%) Illumination adjustments in the microscopy body (on the right down) - Bright field, dark field, oblique light b.) HBO for fluorescence samples: Control from touch pad: - Fluorescence filters choose filter and open/close shutter 2. Find a proper magnification Magnification can be changed with 1.) Joystick -, or 2.) Microscope control pedal * Magnification (working distance) can be locked with control pedal * When making images, write always down, a) do you have 1.2x or 0.8x objective, b.) which magnification and camera was used.
4 3. Focus your sample - Focusing can be done with a) Joystick -, or b) Microscope control Camera control and imaging Camera activation 1. Choose lightpath for the camera - Colour camera, Cc3: adjustment locates in the right side of microscope body - Black and white camera, HRm: adjustment locates on the left side * Use b/w camera for fluorescence images! 2. AXIOVISION: Activate camera AND open camera control window 3. Open LIVE -window Camera control 1. Adjust exposure time - Manually: adjusting on the exposure bar - Automatically: Automatic -button * The best result is achieved by manual adjusting 2. Focus image - Focus microscope again to fit the camera - Slow focus speed can be activated by pressing joystick button down 3. Adjust white balance (only with colour camera) - Automatically: automatic - Manually by choosing white reference point from image (with interactive tool) - Manually: adjusting the colour temperature bar (warmer/cooler) * When white balance is adjusted, all colours in the image are represented equally compared to original source. 4. Choose contrast mode (LIVE -window) - Gamma correction for colour camera G0.45 - Linear for b/w camera 5. Take an image - SNAP
5 Shading correction Digital background shading correction is available (in addition to illumination adjustment by mirrors.). In the process of shading correction a reference image has to be taken, which gives a base for correction: images illumination will be corrected according to this reference situation. Camera control window: general shading correction Scalings and scale bar Take an image and then choose a tool: SCALE BAR - Check whether the right scaling calibration is activated: measures scalings - In addition to this, it is good to ensure scaling validity (put normal scale bar under microscope, take an image, add scale bar and compare differences) * In microscopy it is always a good habit to take a reference image with a real scale bar. Then scale bars can be added to images afterwards with any photo processing software. This is possible only, if scale bar image has taken with same magnification (same camera, lens and zoom). Data Saving Open your own data folder (create if needed) D:\USERS\"Lastname Firstname" Create folder for your images always according to the date (inverse!) For example, when date is 21.10.2010 create folder 101021 Transfer your images from computer to safe - Trasfers can be done via USB or internet. - CIC is not in responsible for data storage. Ending 1. Sign your usage on RESERVATION BOOK - Don't turn HBO off, if you know that someone will need it - 2. Shut down: I. Microscope, II. Light source(s), III. computer Power button (kuva) 3. Safe microscope from dust with cover cloth Be careful with HBO unit - it can burn the cover! 4. Clean working surfaces
6 Information Objectives SteREO Lumar has two Zeiss NeoLumar objectives: 0.8x 1.2x * Contact CIC personnel when objective change is needed. Filters SteREO Lumar has two filter sets: 1. UV, GFP, DsRed 2. YFP, CFP Chasing filter sets: Pull filter set straight out of microscopy body and put another one back (filter set recognition is automatic) Microinjector Contact CIC personnel in questions concerning microinjector. Trouble shooting Here is described some common problems. If these tips do not offer an answer, please take contact to CIC personnel. Drive L:\ mounting Connects computer to network drive L:\. In L-drive locates LSM510 META imaging files. 1. Choose: My Documents tools map networkdrive 2. Specify the path: DRIVE: L:\\cicfiler\cicdata FOLDER: cicfiler\cicdata Username: btk\cicuser Password: turku05 Microscope: focusing doesn't work Check if the focus speed is slow instead of fast speed (read "Microscope control") Microscope: focus work, but can't give sharp focus level Check that the lightpath is totally open: (KUVA) * Slow focus speed may help to find focus level
7 Microscope: can't find filters Take a filter set out and put it back. Microscope should recognize sets automatically. Camera - no image available Everything what you can find with microscope oculars you should find also with camera. 1. Check light path on microscope - Is it open? - Which camera? 2. Onko riittävästi valoa - Exposure time? - Lamp on? - Transmitted light: lamp power? - Fluorescence: shutter open, right filter? 3. If this doesn't help - turn off computer and microscope - turn on again