Cell Imaging Unit UIC Bárbara Fekete ZEISS AXIOIMAGER MICROSCOPE MANUAL

Transcription

1 Cell Imaging Unit UIC Bárbara Fekete ZEISS AXIOIMAGER MICROSCOPE MANUAL 1

2 TABLE OF CONTENTS ANATOMY OF THE ZEISS AXIOIMAGE MICROSCOPE 3 INITIALISATION PROCEDURE 4 LEFT SIDE OF MICROSCOPE (DETAILED VIEW) 4 RIGHT SIDE OF MICROSCOPE (DETAILED VIEW) 5 AXIOIMAGER T.F.T. DISPLAY SCREEN 5 1. T.F.T. DISPLAY -HOME PAGE 5 2. T.F.T. DISPLAY - MICROSCOPE PAGE 7 SCANNING MODE 8 1. START NEW EXPERIMENT: 8 2. FOCUS ON SPECIMEN 9 3. SET FOCUS-CORRECTION 9 4. ACQUIRING ADJUST BRIGHTNESS/CONTRAST SAVE AS ZVI FILE EXPORT 12 2

3 ANATOMY OF THE ZEISS AXIOIMAGER INVERTED MICROSCOPE Eyepieces 11. Camera 2. Fluorescence module 12. TFT display 3. Mechanical stage 13. TL and RL shutters 4. Polarizer for transmitted light 14. Transmitted light brightness 5. Condenser aperture 15. Control wheel for luminous-field diaphragm 6. Condenser 16. Sliding button for transmitted-light diffusing glass 7. Condenser focus 17. Push-pull rod for camera path deflection 8. Condenser centering 18. ON/OFF switch 9. Coaxial coarse & fine focus Sextuple objective nosepiece 20. 3

4 INITIALISATION PROCEDURE 1. Ensure that both the AXIOIMAGER control box and the mercury arc lamp power supply have been switched on. After switching on the control box the microscope will take a few seconds to initialize. The Mercury lamp should always be first-on and last-off. This prevents any electrical surges caused by ignition damaging other equipment on the same circuit. 2. During microscope initialisation the stage position will be reset and recently used settings will be recalled. After initialisation the microscope status menu will appear on the T.F.T. display screen. LEFT SIDE OF MICROSCOPE (DETAILED VIEW) A B C A B C Reflector Turret controls (back/ for ward) Focus wheel Stage position controls (up/down) 4

5 RIGHT SIDE OF MICROSCOPE (DETAILED VIEW) D F G A B C E A B C D E F G Transmission neutral density filter control (up/down) Field iris diaphragm Transmission neutral density filters Focus wheel Nosepiece control (back/forward) FL Fluorescence light shutter HAL - Halogen light on/off AXIOIMAGER T.F.T. DISPLAY SCREEN 1. T.F.T. DISPLAY - HOME PAGE After switch on, the microscope is initialized. This process takes a few seconds. Then, normally the Home page appears. Displays the following information: You can call all other pages via the buttons on the left navigation button bar. The middle section of the control area displays the detected configuration. 5

6 At the right edge, the following control elements are arranged: o Load Position button. After you pressed this button, the stage moves down to the load position. While the stage is moving, you can stop stage movement by means of the Stop button. When the stage arrived in the load position, the Load Position popup window appears with the following control elements: Stage returns to operating position. Stage moves up towards the operating position as long as you press this button. Stage moves down as long as you press this button (maximally up to stage stop). o L-Shutter/ TL-Shutter buttons. The Close and Open buttons close or open the shutters for reflected light (RL) and transmitted light (TL). o Make it visible button. This button serves to switch the microscope to a basic state: Transmitted-light lamp adjusted to medium intensity (3V) Luminous-field diaphragm opened Aperture diaphragm opened TL Shutter open, RL shutter closed All filter wheels in transmitted light switched to blank aperture (100% directed to eyepieces) Condenser switched to brightfield Reflector turret switched to the nearest HAL position (Halogen = transmitted light) Light path switched to 100% to eyepieces 6

7 2. T.F.T. DISPLAY - MICROSCOPE PAGE The Microscope page is accessible by pressing the Microscope button on the navigation button bar on the Home page. The Microscope page provides access to the Control, Automatic and XYZ pages. These are described separately below. Objectives Press the corresponding button to swing the desired objective into the light path. Objective type Magnification N.A. Immersion DIC prisms N PLAN 5x N PLAN 10x PL APO 20x Yes N PLAN 40x Yes PL APO 63x 1.4 oil - Reflector Press the corresponding button to swing the desired reflector module into the light path. 7

8 SCANNING MODE If you didn t already does it start the Axiovision.software available both as a Desktop shortcut and the Windows start menu. AxioVision s main window is divided into two main areas Workarea & Workflow. The elements of the Workarea and the Workflow are used to select and operate image processing functions and functions for controlling the camera and microscope. 1. START NEW EXPERIMENT 1.1 In the Work Area dialog, load a new experiment template 1.2 Select an experiment Turn off the channels not required by right clicking on their tabs. 8

9 2. FOCUS ON SPECIMEN 2.1 Click on C tab to select Channel controls Activate your brightest channel by selecting the channel s tab and then clicking on Go. 2.2 Adjust camera exposure in AxiocamHR box by clicking on Camera control and selecting Measure with the Automatic box checked 2.3 Open Live window by depressing the Live button on the main toolbar then focus on specimen. 3. SET FOCUS-CORRECTION Each wavelength has a slightly different focal plane due to poor chromatic correction of the objectives and filter block effects. This may be negligible for your specimen and you may not need to correct for it. 9

10 3.1 Click on the Extended Parameters button in the Multi-Channel Acquisition dialog to open the Extended Parameters dialog. 3.2 Click on the Focus offset (µm) button to open the Focussing window: 3.3 Focus the specimen using the microscope focus control. Use the zoom and auto-exposure buttons to help you focus. 10

11 3.4 When the specimen is in focus, click OK and this will enter the focus offset value in to the channel s properties in the Extended Parameters dialog. 3.5 Repeat for all channels. You may need to change these values when you change objectives or specimen. 4. ACQUIRING Click the Start button to start acquisition. 5. ADJUST BRIGHTNESS/CONTRAST 5.1 Open properties dialog: 5.2 Change percentage to 1% and click Best Fit. Repeat for each channel. 11

12 6. SAVE AS ZVI FILE Save as ZVI as this will save many of you imaging parameters. Exporting your data without saving as a ZVI can result in data loss. DON T DO IT! 7. EXPORT 7.1 Select location for files. 7.2 Select all channels. 7.3 Select Generate Merged Image Select TIFF Unselect all other boxes Click Start. 12