MIU User Guide For Zeiss AxioVert 200 Inverted Fluorescence Microscope Molecular Imaging Unit (MIU) University of Helsinki www.miu.helsinki.fi 2.9.
CONTENTS 1. General................................................................................................................. 3 1.1 Instrument .................................................................................................................. 3 1.2 Reservations................................................................................................................ 3 1.3 Billing ......................................................................................................
1. General 1.1 Instrument This user guide covers the use of the Zeiss AxioVert 200 inverted epifluorescence microscope. For more information on each MIU instrument, visit: http://www.miu.helsinki.fi/instruments/index.htm An inverted microscope is usually used for observing living cells on culture dishes and bottles that do not physically fit under an upright microscope.
1.5 Users’ mailing list All registered users are automatically added to the users’ mailing list. Please read those emails to know what is going on! 1.6 User guide This user guide will be continuously updated by the MIU personnel. Check that you have the latest version of the user guide: go to http://www.miu.helsinki.fi/instruments/ and compare the date of the latest update with the date on the first page of your printed user guide.
Figure 1 Adjustment of the light path to a correct port 2 Arrow 1: ocular/camera selection Arrow 2: selection of the front/side port. Red circle: focusing knobs 1 Keep the knob below the oculars turned so that the white line points to the eye (Fig. 2, red arrow). Figure 2 Ocular light path adjustment The lower button on the lower right of the microscope is used to activate/deactivate the halogen (HAL) light pathway (Fig. 3).
2.2 Objectives There are following objectives in the microscope: Position Type Magn. NA Oil/Dry Phase 1 Plan-Neofluar 10x 0.3 Dry Ph1 2 LD Achroplan 20x 0.4 Dry Ph2 3 Plan-Neofluar 40x 0.75 Dry - 4 LD Achroplan 40x 0.6 korr Dry Ph2 5 LD Achroplan 63x 0.75 korr Dry Ph3 Table 1. AxioVert 200 objectives The objectives are changed by manually turning the objective revolver. You can see the chosen objective in the little LCD display on top of the condenser.
2.4 Transmitted (bright-field) light microscopy Transmitted light microscopy is used for specimens that are stained, or have natural pigment so they are able to absorb light. Koehler illumination ensures that illumination is homogeneous and devoid of disturbing scattered light. Thus, adjustment of Koehler illumination is essential every time transmitted light microscopy is done.
- Select a 10x objective and DIC .3-.4 condenser position by rotating manually the condenser wheel - Focus on the specimen - Close luminous field diaphragm until its edges can be seen through oculars (Fig. 6, a wheel in front of the top part of the condenser). - Focus the image of diaphragm edges by moving the condenser up or down (Fig. 6, arrow 2). - Use centering screws to move the diaphragm image to the center of the field of view (Fig. 6, arrows 3).
Pull the analyzer located on the left side of the microscope to the left so that it is in position shown in (Fig. 10). Figure 7 The polarizer for DIC imaging Figure 8 The analyzer for DIC imaging Rotate the condenser to the correct DIC position (Fig. 5). Each DIC position is marked with a range (e.g. ..3-.4). Numerical aperture of the objective must fit into that range. Above the polarizer, there is a slider that is used for adjusting the angle between the polarizing plane of the polarizer and analyzer.
As a last step, adjust the iris diaphragm using the slider above the DIC condenser (Fig. 5). First, fully open it and then, if needed, close it as much as necessary for enhanced contrast. When you are done with DIC imaging, remember to turn the polarizer away from the light path and push the analyzer back in. 3. Imaging 3.1 Zeiss AxioCam HRc 14-bit color CCD camera Turn the camera on by plugging the power cable in (Fig. 12). The green light indicates that the camera is on.
Focusing is done by using the focusing knobs on either side of the microscope. The refresh rate of live image can be adjusted (Fig. 14). Quality of live image depends on the refresh rate. However, this setting has no influence on the actual image that will be taken. There are three options for the refresh rate: slow (good quality) medium (medium quality) fast (poor quality) Figure 12. Refresh rate settings for live image 3.2.2 Adjustment of Camera Settings 3.2.2.
Figure 13 Camera settings in AxioVision software In the image window there is a "French flag" button in the tool bar at the bottom (Fig. 12). This will show under/overexposed areas in blue/red pseudocolor. Pressing the button again removes the pseudocolor. 3.2.2.2 White balance White balance adjustment is not needed in fluorescence imaging because there is only one color at a time. However, inappropriate white balance settings may lead to poor image quality.
The recommended minimum camera resolutions for each objective are: Objective NA Recommended min. resolution 10x 0.3 3900 x 3090 20x 0.4 2600 x 2060 40x 0.75 2600 x 2060 40x 0.6 1300 x 1030 63x 0.75 1300 x 1030 If you are using unnecessarily high camera resolution, the image file size will be high but no added image resolution will be achieved. This is because the resolving power of the objective will become a limiting factor.
BestFit shows the whole range excluding specified percentage of actual minimum and actual maximum values Min/Max shows the whole range from actual minimum to actual maximum (use for automatic adjustment of best contrast and brightness) Linear shows linearly the whole range without detecting actual minimum and maximum values (use for restoring the original values) Figure 15 Display tab With SAVE and RESTORE buttons it is possible to apply the same setting to other images. 3.3.
When you click with the right mouse button and select VIEW ANNOTATIONS, you will remove the scale bar. If you select VIEW ANNOTATIONS again, the scale bar will appear again. The annotations including the scale bar are stored in the meta data on a “.zvi” image. On other image file formats if you want the scale bar in the saved image, you must select BURN-IN ANNOTATIONS option before you save your image (see the following chapter). 4. Saving and transferring images 4.
You can save images temporarily to that folder and later move them into your own computer. The folder will be emptied occasionally (it may happen every week for files older than two weeks). From your own computer, you can access the Images-folder in several ways: Appletalk-network: Biomedicum cancerbio / Mcbserver1 / Images Windows-network: Cancerbio / Snap Server 4400 (Mcbserver1) / Images FTP: ftp://mcbserver1.hi.helsinki.fi/images/ 4.
taken!). There is a 3 GB limit for each user. It is recommended to transfer images somewhere else after your imaging session.
5. Ending your imaging session When you have finished your imaging session you should: - Close the fluorescence light shutter (Fig. 3) Log off from the computer On working days (Mo-Fr) leave the equipment on if your reservation ends before 16:00. If your reservation begins before 16:00 and ends after 16:00 and you are not present at 16:00, please leave AxioVision software on to show that you will continue your imaging session.
The whole image is too bright: Is the transmitted light (halogen bulb) off (p. 5)? Cannot see the whole field of view: Is the fluorescence filter correctly in place? Is the shutter for fluorescence light completely out of the light path (p. 5)? 6.1.2 Transmitted light microscopy You cannot see anything: Is the halogen light on (p. 5)? Is the intensity of the light adjusted properly (p. 5)? The image is too bright and colored: Check that the shutter for fluorescence light is closed (Fig. 3, p. 5).
6.3 Computer It’s not possible to log on to the computer: Use HYAD user name. Check that the domain selected is ATKK. I do not have HYAD-username: You can get it from the user account office. The telephone number is (09) 191 25629 and the email is meilahtiluvat@helsinki.fi. 6.4 AxioVision software Any problem: - Help-command in AxioVision software (F1 or right side of the upper bar) is a useful tool to get detailed information about AxioVision functions.
