2 How to operate the microscope/obtain an image

Transcription

1 Morgagni Operating Instructions ow to operate the microscope/obtain an image 2.1 Starting the microscope Starting the microscope with several manually-operated steps 1. Turn on the water. 2. Switch on the main power switch. 3. Press the "POWER ON" button (on the right hand side control panel). 4. After the program starts press the "VACUUM ON" button. This function can be set to an automatic mode in the Configuration control window of the Morgagni User Interface (UI). 5. If the cooling device or Cold trap is needed, check the liquid nitrogen level in the dewar vessel. It is important that the Vacuum is ready before the cooling device is used! Microscope ready for filament saturation to obtain emission current 1. Press the igh Tension (T) button on the right hand side control panel. 2. Select a igh Tension in the igh Tension control window of the UI and an Emission bias (Step) in the Filament control window. 3. If Autoheating is selected, the filament is automatically heated till the saturation point (if available). To determine the saturation point, Autosaturation can be used (see chapter 1.3.4). 4. Alternatively, manual saturation can be performed by turning the filament knob clockwise or by using the arrow heads of the filament scroll bar in the UI (in the meantime observe the filament pattern on the projection screen till it has completely disappeared from the beam). When this procedure is completed, the saturation point may be fixed by pressing the Filament Limit button in the UI. 2.2 Mounting a specimen in the holder Make sure the specimen holder is clean and remains clean throughout the entire microscopy procedure. A clean specimen holder contributes to the vacuum of the microscope and thus indirect its lifetime! To maintain a clean status of the holder, never touch the front part of the holder (i.e. that part which is located behind the O-ring). For detailed information about maintenance and cleaning see chapter 3, section 3.7. The specimen holder is designed for accepting 3.05 mm diameter gridsupported or disc-shaped specimens with edge thickness up to 0.34 mm. The holder is equipped with a clamping device to fix the specimen (or grid) in its correct position, and a holder carrier to insert the holder into the microscope. Morgagni Operating Instructions

2 Morgagni Operating Instructions Check that the clamping device is clean and dry. Rest the holder on its support (11) with the spring clamp (2) horizontal and uppermost. Fit the tool (supplied by the manufacturer) into the hole at one side of the clamp (2), then lift the clamp to its fullest extent. Carefully place the specimen in the specimen site (3). Check with a magnifying glass to make sure that it is correctly seated. Carefully lower the clamp onto the specimen (or grid) making sure that the specimen remains correctly in position. 2.3 Exchanging Specimen holders Five individual holders and a holder support are supplied with the microscope. These holders can be loaded with different samples in advance and specimens may be exchanged simply by exchanging corresponding holders onto the specimen holder carrier. The specimen holder is released by depressing the push-button (8) on the carrier, and placed into one of the holes in the specimen holder support ( 2-3, 2-4). Another specimen holder can be collected by firstly depressing the pushbutton (8) of the carrier and fitting the injector end (5) of the carrier onto the pin (4) of the specimen holder. Ensure that these two parts are engaged properly, then release the push-button (8) and carry the complete holder to the next step (inserting a specimen holder into the microscope)( 2-3, 2-4) Description of the parts indicated in 2-1 to Specimen older 2. The hole for inserting the tool 3. Clamping device 4. The pin of the specimen holder 5. Injector of the Specimen holder carrier 6. Specimen holder carrier 7. The pin of the specimen holder carrier 8. The push-button of the specimen holder carrier 9. O-ring 10. Carrier support 11. Specimen holder support 12. Airlock 13. The opening of the airlock 14. LED light of the airlock Morgagni Operating Instructions

3 Morgagni Operating Instructions Fig. 2-1 Specimen holder (closed) Fig. 2-2 Specimen holder (open) Morgagni Operating Instructions

4 Morgagni Operating Instructions Fig. 2-3 Specimen holders and injector of the specimen holder carrier Fig. 2-4 Specimen holder including specimen holder carrier Morgagni Operating Instructions

5 Morgagni Operating Instructions Fig. 2-5 Specimen holder support table + holder Fig. 2-6 Specimen holder support table + holder Morgagni Operating Instructions

6 Morgagni Operating Instructions 2.4 Inserting a specimen holder into the microscope The holder is carried by a specimen holder carrier and inserted through a prepumped airlock which ensures that air introduced by the holder is pumped away before the air lock is opened to the microscope column. It is thus not necessary to switch off the filament or high tension when exchanging specimens. Care must, however, be taken that the intensity on the new specimen is not so high as to damage it. old the holder carrier so that the pin (9) points upwards and rotate the carrier approximately 20 degrees clockwise. Insert the carrier into the airlock so that pin 9 engages in the opening (12) of the airlock; the rotary pump is now started (indicator LED lights) (Figure 2-7). Wait (7 to 10 seconds) until LED light is extinguished. Turn the carrier counter-clockwise (approx. 20 degrees) until a stop is encountered and push the carrier in slightly (a few mm) until it stops. Turn the carrier fully counter-clockwise until a firm stop is encountered (half a turn). Push the carrier gently into the microscope until it stops (Figure 2-8). Fully depress the push-button (8) at the end of the carrier. Keep this push-button depressed and pull the carrier out as far as it will go. Release the push-button (8). Turn the carrier clockwise until a firm stop is encountered (half a turn). Condenser apertures Objective apertures Fig. 2-7 Microscope column with apertures and goniometer Morgagni Operating Instructions

7 Morgagni Operating Instructions I Fig. 2-8 Insertion of the specimen holder 2.5 Obtaining an image Use LM to find the specimen on the grid. Select an area of interest and turn to a higher magnification. Select a condenser aperture and re-check its centering. Select an objective aperture and re-check its centering. Focus the image. 2.6 Focusing the image There are two essential steps for completing the focusing procedure: Step 1 Adjust the combination of the small projection screen and binocular. Introduce the small screen fully into the beam. Adjust the inter-ocular distance for maximum comfort. Insert the beam stop and focus the binocular using the ocular adjustment controls. When the shadow of the beam stop is sharp for each eye separately, the binocular is focused and the beam stop can be removed. Morgagni Operating Instructions

8 Morgagni Operating Instructions Fig. 2-9 The beam stop Step 2 Use the amplitude of the wobbler effect to focus the image Insert a specimen and focus the image Press Diffraction push-button. Select a camera length of 350 mm. Focus the illumination using the INTENSITY knob. Ensure that the objective aperture is correctly centered. Press the WOBBLER push-button the right hand side control panel or in the Wobbler control window of the UI): two spots should appear within the area of the objective aperture. If not, using MULTI X and Y to lower the WOBBLER amplitude until they do. The WOBBLER amplitude and azimuth values are displayed (in Multifunction X and Y coordinates) on the Computer monitor. Press DIFFRACTION push-button again to exit diffraction mode, overfocus the spot using INTENSITY knob and focus the image to minimum blur. When finished, press the WOBBLER button once again to switch if off. 2.7 Correction of the astigmatism Astigmatism is an aberration which is present in all electromagnetic lenses. It is caused by asymmetry of the lens field which can result from inherent asymmetries, or from asymmetrical charges on regions close to the beam. There are three possible astigmatism corrections: Spot or Condensor astigmatism for the illumination system, Image or Objective astigmatism for the objective lens and when in Diffraction mode Diffraction astigmatism Astigmatism Correction for the Illumination System The Condenser or Spot stigmator is used to correct astigmatism of the beam. It can be set up for each SPOTSIZE separately and stays the same for all imaging modes Morgagni Operating Instructions

9 Morgagni Operating Instructions Step-by-step procedure (see also chapter 1.3.7): Remove the specimen and the objective aperture from the beam. Push the Spot button on the left hand side control panel (yellow LED on) or the Condensor button in the Stigmator control window of the UI. Astigmatism is corrected when the focused beam remains as circular as possible when going through beam focus (INTENSITY knob). Adjust this using the MULTI X and Y knobs or by using the mouse controlled blue box in the UI (see chapter ). Alternatively, the filament can be undersaturated until structure is visible in the focused beam. Astigmatism is corrected when this structure is as sharp as possible (adjust INTENSITY, MULTI X, Y) Astigmatism of the Objective Lens (Image) The objective lens stigmator corrects image astigmatism. The objective astigmatism can be corrected at four different sensitivities (Figure 2-10). Value 1 and 2 are for igh Magnifications, value 3 is for Diffraction mode and value 4 for Low Magnification and Shadow Projection. Fig Stigmators Step-by-step procedure (Figure 2-11): Obtain a TEM Bright-Field image of the test specimen at high magnification (around 100kx). Press Image stigmator push button (LED on) or activate the Objective button in the Stigmator control window of the UI. Select a very small hole on the specimen (if available). Adjust the FOCUS until the entire hole is slightly overfocused, yet close enough to focus for the fringe asymmetry to be visible (black fringe inside Morgagni Operating Instructions

10 Morgagni Operating Instructions the hole, Figure 2-11A). Change of focus (lower excitation of the objective lens) gives rise to the images in Figure 2-11B and Figure 2-11C. Turn the MULTI X, Y knobs or the mouse controlled blue box in the UI so that the Fresnel fringe is symmetrical when the objective lens is very slightly overfocused. To carry out this procedure, rotate the MULTI X, Y knobs until the setting for minimum astigmatism is obtained (best symmetry for overfocused image). Repeat the same procedure at higher magnifications until the astigmatism is adequately corrected (Figure 2-11D). The criterion for this is that the fringes of uniform width can be seen at one or two steps position overfocus of the finest step size and change of focus (lower excitation of the objective lens produce images as in 2-11E and 2-11F) Image Astigmatism Correction in the LM mode Insert a specimen and centre a structure of interest. Set MAGNIFICATION to the highest LM value (180X). Press IMAGE push-button (LED on). Press the WOBBLER push button. Focus the image so that the blurred structures are as nearly coincident as possible. Adjust the MULTI X, Y knobs until the structures completely overlap. Repeat the last two steps Diffraction pattern Astigmatism Correction Obtain a TEM Bright Field image of a specimen in the M range. Remove specimen and objective aperture from the beam. Set SPOTSIZE knob to position 4. Press DIFFRACTION button on the right hand side control panel. Adjust the MAGNIFICATION knob to obtain the required camera length. Turn INTENSITY knob clockwise until a low intensity of illumination is obtained on the screen. Adjust the FOCUS knob until the diffraction crossover image is obtained. Press the Diffraction button in the UI. Adjust the MULTI X, Y knobs until a symmetrical 3-pointed image (comparable with the Daimler Benz trade sign) is obtained. Alternatively, the mouse controlled blue box in the stigmator control window can be used Morgagni Operating Instructions

11 Morgagni Operating Instructions A D B E C F Fig Image astigmatism correction (small screen + binocular) Morgagni Operating Instructions

12 Morgagni Operating Instructions 2.8 Photography The recording of plate or 35 mm film negatives is operated and controlled by the Camera control window of the UI (see chapter and ) Step-by step procedure a. Select the exposure mode in the UI (Single, Double, Series, Exp. Select). b. If Exp. Select is chosen, make sure that positions are selected in the Stage control window of the UI (see chapter ). c. If Series are selected, select also: The number of exposures in the series. Focus step by STEPSIZE knob. d. Select the exposure time (Auto or Manual); If the Manual mode is selected, ensure that the correct time has been defined; If Auto mode is selected, the measurement can be made either on the main or on the small projection screen. e. Select the film size (6 x 9 cm; 3.25 x 4 inch) of the camera that is used (plate). f. Check the following data: File data Exp. No. Emulsion value Data Int Stock User code Date g. Insert 35 mm camera, if necessary (this can not be done if the CCD camera is mounted at the wide angle (35 mm) port). h. Press the EXPOSURE push button in the UI or the left hand side control panel. The main screen will be automatically raised and lowered. i. During the plate transport plate and exposure phases the illumination of the panel and spotlight as well as the monitor screen intensity are dimmed. j. When the micrograph has been taken, the illumination is restored. k. When the first exposure in DOUBLE mode is made, the screen will display a message: "First exposure finished". After this the main screen will be lowered, the computer will restore the microscope to the viewing condition and a second exposure on the same photo can be made starting from step f. l. Lower the screen (the main screen is lowered automatically after the last exposure has been taken; the small screen has to be lowered manually) or withdraw the 35 mm camera to restore the microscope to the normal viewing condition, if no more exposures are required Morgagni Operating Instructions

13 Morgagni Operating Instructions Procedures for Double and Series Exposures, Dark Field Images and Diffraction Patterns are described in chapter 3, section Acquisition of Digital Images For acquisition of digital images the Megaview II CCD camera is used. This camera can be mounted both at the wide angle port (35 mm port) or at the post column position and is continuously exchangeable (see chapter 3.7). Image acquisition and analysis is controlled through the AnalySIS software. The software is displayed at the inner part of the Morgagni User Interface and can be started up by activating the AnalySIS icon in the microscope Start up window (Figure 2-12). Fig Start-Up page The following paragraph displays an introduction into the use of the CCD camera. For more detailed information about the image analysis software, please see the attached user s guide manual of AnalySIS. Morgagni Operating Instructions

14 Morgagni Operating Instructions Image Acquisition Once the Analysis icon has been activated the AnalySIS User Interface appears (Figure 2-13). Fig Analysis For image acquisition the CCD must be first inserted into the proper position. This can be done by pushing the green lit button of the external control box (green button-insertion / red button-retraction) or by pressing the I button in the Analysis UI (Figure 2-14). If the CCD is mounted at the post-column position, the main screen must be lifted as well before image acquisition Fig In / Out After insertion, a live image or a single snapshot can be made. Clicking on the camera button (Figure 2-15A) results in a live image that can be subsequently frozen by clicking on the snapshot button (Figure 2-15B). The acquisition speed of the live mode depends on the exposure time of the camera and can be set in the camera control window which appears after pressing the corresponding button (Figure 2-15C). Fig Live (A), Snapshot (B), Camera Control (C) With the + and the - buttons in the camera control window (Figure 2-16), the speed of image acquisition can be enhanced (-) or decreased (+). It is important to note that the intensity must be adapted to the changes in acquisition. A shorter exposure time requires a higher intensity and vice versa Morgagni Operating Instructions

15 Morgagni Operating Instructions Fig Camera Control For various applications it may be useful to have different modes of acquisition. For screening of samples, focusing and astigmatism correction a high acquisition speed at a somewhat lower image resolution may be sufficient, whereas for a final snapshot image resolution is more important than speed of acquisition. It is possible to define three image acquisition modes with different parameters (Figure 2-17). Fig Three acquisition modes For configuration of the acquisition modes, the Configure Input window must be activated. To do so, select in the taskbar: Image, Configure Input (Figure 2-18). ere, settings can be chosen which are related to the image format (image resolution and binning), automatic gain display, appearance of an on-line intensity histogram or FFT, the exposure time, gain and dark reference, remote control (essential for the correct magnification read-out and stitching) etc. Fig Configure Input After image acquisition it is possible to perform various analyses on the image. Quantification, changing of contrast, brightness, changing of geometry, applying imaging filters, addition of text and symbols etc. For all these jobs, see the AnalySIS instruction manual. Morgagni Operating Instructions

16 Morgagni Operating Instructions Multiple Image Alignment Multiple single images can be digitally stitched to one image. This so called Multiple Image Alignment (MIA) has the advantage of an increased field of view at a simultaneously enhanced pixel resolution. For example: two 1k x 1k digital images can be stitched horizontally and vertically into an almost 2k x 2k image. To perform stitching, a calibration must be carried out for each magnification that is used. To do so, press the Calibrate icon (Figure 2-19: second button on the left). Fig Stitching module When the Calibration button is pressed, the specimen is slightly shifted in both the x and y orientation. A specified image feature (not visible during the procedure) must be recognized throughout the shift. After the calibration, the choice can be made whether to choose a stitching of 2x2 (icon 2), 3x3 (icon 3), 4x4 (icon 4) or 5x5 (icon 5) images (Figure 2-19). The captured images are automatically aligned to the stitched high resolution image. Fig Arrange Multiple Images As an alternative to the prefered automated alignment of the acquired images, it is possible to arrange the multiple images in a manual way. To do so, press the Arrange icon (Figure 2-19: most left button) and the following control window is displayed (Figure 2-20). The way in which the images can be stitched Morgagni Operating Instructions

17 Morgagni Operating Instructions (horizontally, vertically, meander, comb) as well as the number of images for stitching can be defined. The quality value should be preferably at 1. Press the OK button to execute the stitching. In the next control window it is possible to move the position of the indivual images (marked by a red frame) with the left mouse button in order to find the optimal overlap (if required) Removing the specimen holder from the microscope If the injector rod has been removed from the microscope after inserting the specimen, repeat the first five steps as described in chapter 2, section 2.3. Fully depress the push-button (8) on the carrier (Figure 2-8). Keeping the push-button depressed, push the carrier gently into the microscope until it stops. Release the push-button (8). Pull the carrier out as far as possible. Turn the carrier clockwise until a firm stop is encountered (half a turn). Withdraw the carrier from the airlock so that pin 9 comes out of slot 12 (Figure 2-7). The specimen may now be removed from the holder and a new specimen can be inserted Removing a specimen from the holder Rest the specimen holder on its support. Lift the clamping device. Remove the specimen by inverting the holder over a petri dish. Use a tweezer to put the grid into a gridbox. Store the holder with the specimen-securing clamp closed. Morgagni Operating Instructions