Motorized Axio Observer Start-up instructions

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1 Start-up instructions 1. If using fluorescence turn on Fluorescent light source. TL light Source (Hal 100) 2. Turn on microscope using switch on lower left side of the microscope. 3. If imaging, turn on computer and start Axiovision. TL Aperture ) Diaphragm TL Field Diaphragm Condenser FL light source (HBO 100) FL Filter Turret Trouble Shooting: P: The microscope is on but there is no transmitted light. S: Hold a piece of white paper over the Hal 100 light source. If there is no light, the bulb may be burnt out. If there is light, check to make sure the field diaphragm is are open. Next be sure that H is the selected contrast method. Lastly be sure the light is being sent to the eyes. P: The microscope is on but there is not Fluorescent light. S: Be sure the Fl light source is on, the top light on the power supply should be lit, if not you may need to turn the power supply off and on again. If the top light is lit and there is still no light be sure the RL shutter, RL aperture diaphragm and RL field diaphragm are all open. If there is still no light, check to make sure the correct filter spot is selected in the Fl Filter Turret. P: Everything is on but the image in my live window is all black (or white). S: Make sure light is being sent to the camera. Click the measure button in Axio Vision to take an exposure reading and click the linear button to properly display the image on the screen.

How to Set up Koehler Illumination Step 1: The Field Diaphragm 1. Using a low power objective (10x or 20x) focus your specimen 2.

Move the condenser to it s bottom most position. 4. Open the aperture diaphragm all the way. 5.

Focus the image of the condenser by raising or lowering the condenser carrier. 7.

Remove one eyepiece to view the back focal plane of the microscope. 2.

Slightly adjust the position of the aperture diaphragm until desired contrast/resolution is achieved.

2 How to Set up Koehler Illumination Step 1: The Field Diaphragm 1. Using a low power objective (10x or 20x) focus your specimen 2. Correct for color temperature by increasing the light intensity and adding neutral density filters. 3. Move the condenser to it s bottom most position. 4. Open the aperture diaphragm all the way. 5. Close down the field diaphragm until it s edges are visible in the field of view. 6. Focus the image of the condenser by raising or lowering the condenser carrier. 7. Center the image of the field diaphragm with the centering screws located on the sides of the condenser. Step 2: The Aperture Diaphragm 1. Remove one eyepiece to view the back focal plane of the microscope. 2. Close the aperture Diaphragm so it illuminates approx 80% of the field of view. 3. Return the eyepiece and observe your specimen 4. Slightly adjust the position of the aperture diaphragm until desired contrast/resolution is achieved. Note: The above process should be followed every time a different objective is moved into place. Only after this procedure has become second habit, will you know when shortcuts are o.k. Benefits of Koehler Illumination: * Evenly illuminated image * Minimized reflection or glare or effect of dust in the system * Minimum heating of specimen * Field diaphragm becomes a focus reference for easy focusing of specimen * Microscope is all set for employing contrasting techniques (Brightfield, Oblique, Phase, DIC, etc.)

3 Field Diaphragm Neutral density control Condenser focus knob Contrast Technique Control Aperture Diaphragm Light intensity control Specimen focus knob

$It allows the human eye to see the differences in refractive index and thickness of different cell components.$ It does this by using matching optical modulators, a phase stop in the condenser, and a phase ring in the objective.

It does this by using matching optical modulators, a phase stop in the condenser, and a phase ring in the objective.

4 Setting up Phase Contrast Phase contrast is ideal for examining thin, unstained samples, e.g. culture cells. It allows the human eye to see the differences in refractive index and thickness of different cell components. It does this by using matching optical modulators, a phase stop in the condenser, and a phase ring in the objective. NOTE: not all objective contain phase rings objectives with green writing on them are phase objectives and have the phase ring inside. The size of the phase ring will be written on the objective as Ph1 Ph2 or Ph3 Step 1: Matching the Objective to the Condenser In order to work the phase ring in the objective must match the phase stop in the condenser. The matching is simple. If using a Ph1 objective you must select Ph1 in the condenser. If using Ph2 objective, select Ph2 in the condenser, and if using a Ph3 objective select Ph3 in the condenser. Step 2: Aligning the Phase Rings 1 - Observe the back focal plane, either by using a phase telescope or by removing the eyepiece. - Ideal contrast is achieved when the two rings are superimposed (B). If the rings are not superimposed (A) follow the next two steps to fix the problem. - Using 1.5 mm ball drives adjust the alignment of the phase stop by inserting them into spaces 1 & 1 and turning until the images are super imposed. - Replace the eyepiece and observe the specimen. Contrast should be drastically improved. - 1 This step only needs to be done if the contrast is not optimal or if the microscope is being set up for the first time.

Setting up Differential Interference Contrast DIC short for Differential Interference Contrast is a contrasting technique that allows the user to view thin transparent specimens at high resolution as

5 Setting up Differential Interference Contrast DIC short for Differential Interference Contrast is a contrasting technique that allows the user to view thin transparent specimens at high resolution as well as a high level of contrast. It does this using a combination polarizer s and wollaston prisms in the beam path of the microscope. Step 1: Match DIC sliders to objective and condenser position. Each objective has a matching DIC slider (pictured at right). Each DIC slider is optimized for a specific DIC Setting which will be written on the DIC slider as either roman numeral I, II, or III. Step 2: Move the fluorescence filter turret to a position that contains an analyzer. (Usually marked analyzer or DIC ) Step 3: Adjust for Koehler illumination. Step 4: Adjust the final image by turning the small knurled silver knob on the DIC slider above the objective. Key: 1. Nuetral Density 2. Condenser 3. DIC Slider 4. Analyzer Slider (option1) 5. Analyzer Slider (option 2) Important notes: DIC can only be done through glass surfaces. It will NOT work with plastic dishes! If your final image does not look correct check to make sure the analyzer is crossed with the polarizer of the DIC prism in the condenser. You can check this by pulling out the DIC slider, and with no sample present and the light on the image should black.

Setting up Fluorecence Fluorescence: Light generated by a high performance lamp reaches the exciter filter, the filtered light is reflected by a beam splitter (dichroic) on focused on the specimen

Step 1: Switch on the Fl. light source and allow to warm up for approx 15 min. Step 2: On the reflector turret select the desired filter cube.

6 Setting up Fluorecence Fluorescence: Light generated by a high performance lamp reaches the exciter filter, the filtered light is reflected by a beam splitter (dichroic) on focused on the specimen via the objective. The specimen absorbs the light and then emits it s fluorescent signal which is transmitted through the dichroic and then passes through an emission filter before reaching our eye. Step 1: Switch on the Fl. light source and allow to warm up for approx 15 min. Step 2: On the reflector turret select the desired filter cube. (Depending on dye used) Step 3: Close the transmitted light shutter and open the reflected light shutter. Step 4: Focus the specimen. Step 5: Adjust the reflected light field diaphragm to be just barely larger than the objective field of view. Fl Lamp Alignment Occasionally it will be necessary to replace and align the FL light source. In the past this was tricky, now it is really very simple! Step 1: Remove the lamp housing from the microscope and remove the cover. This is done using the red handled 3mm tool with each microscope. Step 2: Remove the old bulb by pushing the clamping lever (7) and pulling straight up on the cooling body (1) Then while holding bulb, push the clamping lever on the cooling body (4) to release bulb. Step 3: Insert the new bulb securing it into place with the clamping levers. Step 4: Properly dispose of old bulb adhering to building safety codes. Step 5: Replace lamp housing cover and secure lamp housing to microscope. Step 6: Align bulb by pulling out the alignment window on the side of the microscope and using the 3mm red handled tool in the two alignment slots on the side of the lamp housing. Once you see the mirror image and the real image side by side and focused, use the control knob for the collector (5) to create a completely even illumination across the field. See figure at right. 1. Cooling Body 2. Fixation screws 3. Lamp mount 4. Clamping lever 5. Control knob for collector 6. Bulb 7. Clamping lever

Navigating the Touch Screen: (aka: the TFT) Your microscope can be controlled in three ways. 1. by manually using the buttons on the stand 2. by using the computer and Axiovision software 3.

The Home Page: This screen displays a snapshot of all the current microscope settings.

7 Navigating the Touch Screen: (aka: the TFT) Your microscope can be controlled in three ways. 1. by manually using the buttons on the stand 2. by using the computer and Axiovision software 3. by using the touch screen to the side of the microscope. The choice is completely up to you. This short instruction page will guide you through the most basic functions of the TFT. 1. The Home Page: This screen displays a snapshot of all the current microscope settings. From this screen you have several control options: - You can also control the shutters for Transmitted Light (Bright field, DIC, or Phase) or Reflected Light (Fluorescence). - By touching the Load Position button you can lower the stage to put your sample on and then return the stage to it s prior position by hitting the work position button. - By touching the Microscope button a new screen will appear. 2. The Microscope Page: From this screen there are three main functions. - Individual component control: by touching the tabs at the top of this screen you can select the individual components of the microscope, such as objective, and reflector cube. Example: by touching 63x the microscope will move the 63x objective into position over the sample. - Contrast Manager: the buttons on the lower portion of this screen allow you to quickly switch between different contrast methods. By clicking DIC the microscope will move all the necessary components for viewing your sample in DIC, you can then touch the FL button and the microscope will move into position for viewing your sample in Fluorescence. - Shutter Control: As on the main page you also have control of the transmitted light shutter and the reflected light shutter..

INSTRUCTIONS FOR COURSE WORK 4 (AxioVert) Instructor: Anne Vaahtokari (MIU) 1. Purpose of the work

INSTRUCTIONS FOR COURSE WORK 4 (AxioVert) Instructor: Anne Vaahtokari (MIU) 1. Purpose of the work In this work, you will get familiar with an inverted epifluorescence microscope. Also, you will learn