Magnify Tool. Used to zoom in or zoom out of images. Use the option key (Macintosh) or alt key (Windows) to toggle in/out control of the zoom.

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1 SEM Image Mode The SEM Image Toolbar SEM Image display and acquisition functions are controlled from the SEM Image Toolbar. If the toolbar is not visible at the top of your screen, selecting Mode SEM Image from the top menu will make it appear: NOTE: Before any images can be acquired with Revolution, the physical connection of the 4pi system to the microscope must be performed, and the microscope must be on and in working order (that is, the microscope must be able to acquire an image to a CRT monitor as it normally does). The tool bar is divided into four sections: SEM Image. Used to access the setup, set the resolution, and acquire or preview images. Options. Used to match Revolution's magnification to the magnification set at the microscope (the software can directly read this information from some microscopes). Also used to specify the beam energy in kvolts (informational only; the software can directly read this information from some microscopes). Tools. See Basic Operation for similar descriptions. Magnify Tool. Used to zoom in or zoom out of images. Use the option key (Macintosh) or alt key (Windows) to toggle in/out control of the zoom. Drag Tool. Used to drag image around in zoomed mode. Selector Tool. Used for selecting/moving any item with a valid handle in an image. Text Tool. Used to create or edit text annotations in an image. Measurement Tool. Used to make measurements in images, or to calibrate when editing magnification (see further below this page). Status. Lists useful information about the current operational state, including the current scan position of the x-pixel, the current scan position of the y-pixel, and current frame number, and the approximate time left for the acquisition to finish. When acquiring, the status will look similar to this: If the cursor is held over a pixel in a live image, the x- and y-position of the cursor will be displayed, with the grayscale value at that pixel. Previewing Images At the beginning of the the setup procedure (in other words, now), and any time after, click on the preview button to open a live preview image on the computer screen. The number of SEM images which appear on screen, and other characteristics of the image acquisition, depend on adjustments described below. At times, you may have to refer to the Scan Generate Prefs, but these are normally already set by your install representative. To launch preview images, click on the preview button When the preview images appear on screen, the status section of the toolbar will inform the user of the current x- and y-pixel positions in the scan, the current frame, and the remaining time for the frame to complete (for faster setups, such as small images with small dwells, the remaining time may be pinned to < 1 second, and the x-pixel position may not update). Holding the cursor over any particular pixel in the image will cause the image datum at that pixel to appear in the status section as well: x- and y-pixel position, and gray-scale value at that pixel. Starting a preview puts the 4pi system in direct control of the microscope's scanning circuitry. At the moment control is actuated, the user should hear a click from the 4pi scan control hardware (Scanning Interface Unit or Universal Spectral Engine - the click is the relay-set being energized) and the microscope's CRT screen should be blanked.

2 If the microscope's CRT screen is not blanked, it is an indication of an incorrect connection or other fault. Immediately click the preview button again to relinquish control back to the microscope. Double-check for incorrect installation. If blanking problems persist, contact 4pi Analysis before proceeding! First Run When the system is first used to preview or acquire an image, a number of controls may not be set correctly. Before accurate images can be acquired, several setup procedures should be followed. These procedures can be repeated at any time, but should not be required more than once: Set Scan Controls Adjust Resolution (Size) and Aspect Ratio Setting the SEM Acquisition Options Setting the Image Acquisition Options Setting the Line Profile Options Setting the Default Save Format Adjusting the Brightness and Contrast "Squaring" the Pixels Scan Adjustment and Artifact Removal First Run - Set Scan Controls Scan controls are set with the Scan Generate Preferences. Normally, these are set by the install technician and should not have to be adjusted further. First Run - Adjusting the Image Resolution (Size) and Aspect Ratio This step is not mandatory, but it may reduce any confusion associated with image acquisition. We recommend that the aspect ratio in Revolution match that of the microscope's CRT. The software's default aspect ratio is 1:1 (square) with a resolution of pixels; however, a number of microscopes have non-square aspect ratios. A common value is 4:3 (horizontally rectangular), but there are notable exceptions (Hitachi, 3:4). Click the toolbar menu to see the available resolutions. Revolution default settings include 5 square (aspect ratio 1:1) and 5 horizontally rectangular (aspect ratio 4:3) resolutions. The aspect ratio and/or image resolution is changed by simply selecting from the menu. New aspect ratios and image resolutions can be created (or deleted) by selecting Edit Size in the menu. A new window appears with several options for setting custom resolutions. A new size can be selected from this window's pop-up menu, as it can be from the image-size menus. Selecting a different size will update the window's image parameters immediately. The Auto Ratio check box simplifies image size definition. When checked, only the horizontal size needs to be supplied. The other sizes and spans will be calculated according to the horizontal and vertical ratios. The ratio entries cannot be decimal fractions, but can each be as high as

3 Therefore, a ratio of x can be created by entering 4151 and 3227 into the respective ratio boxes; however, the spans are necessarily limited to integer values, so that such high precision in selecting the ratios may be meaningless. Aspect ratios of greater than or less than one are supported. With Auto Ratio unchecked, the user is free to select both the horizontal and vertical sizes, as well as the spans: When any available edit field changes, the rest of the fields are recalculated onthe-fly. The software tries to be smart about second-guessing what the user wants, but changing too many parameters back and forth may confuse the algorithm. Before adding/changing a newly defined image, double check that all the numbers are correct. Any number of sizes can be set up and saved independently. After entering the desired values, the user must click on either Change or Add (the user can also Delete any defined image size) before exiting by clicking OK. If this is neglected, the new definitions or deletions will not stick. Clicking OK will automatically sort the sizes in ascending order according to horizontal size, and subsort the sizes in ascending order according to vertical size. N.B. : The spans can be used to restrict the possible DAC values, and should be modified only by experienced users. Note especially that changing the spans can result in non-square pixels unless special adjustments are made to the hardware. Contact 4pi before changing the spans for any defined sizes. The remaining first-run procedures require the SEM setup window. To access this window, hit the Setup button in the middle (Tools) section of the SEM Image Toolbar: The preview window(s) should be open in the background so that you can see the effect of your changes. First Run - Setting the SEM Acquisition Options Frames. This checkbox determines whether the image is collected as single frames, or as a collection of frames. Turning frames on is required for Spatial Frame Locking (real-time drift correction). The number of frames to collect can be specified. Line Sync. This checkbox determines whether the image is collected while syncing the scan to the power line (or custom-defined) frequency (see the Scan Generate Preferences for custom definitions). First Run - Setting the Image Acquisition Options Logical Channel and Enable. Logical channels are numbered Channel 1 - Channel 8 and define which channels appear on the monitor. By default, Channel 1 is enabled and always visible in the setup window; the other channels are selected with the menu: To make a channel appear on the screen, check its Enable box. To make the image go away, uncheck its Enable box. Each of the logical channels has a default physical channel which can be changed in the Scan Generate Preferences. Acquire. This menu selects the type of averaging, if any, and the pixel data depth. Modes include Single, Average, and Dwell (see Dwell

4 below for further explanation). Pixel data depths currently supported are either 8- or 16-bit, unsigned. Acquired data is scaled for display in the image window according to the Display setting, but all ADC, Counter, and SCA data are acquired in 16- bit mode. If 16-bit mode is selected, the data are presented on screen as is (65,536 levels). If 8-bit mode is selected, the data are handled as follows (if all channels are in 8-bit mode, certain transfer economies are leveraged for somewhat faster scanning): ADC: the upper 8 bits of the 16-bit word are retained and displayed. In other words, the number of grey levels in the image are reduced from to 256: black stays black (0-255 map to 1), white stays white ( maps to 255), and grays are rounded down to the nearest grey level. The image should not visibly change unless your display is capable of more than 256 grey levels (i.e., a greater than 24-bit color video card). CNTR or SCA: the lower 8 bits of the 16 bit word are retained and displayed. Choose enough bits to accommodate the expected number of counts per pixel. At 8 bits this is 255 counts any more and the count "wraps around" to zero. So if you have more than 255 counts per pixel in any pixel, choose 16 bit. However, if the number of counts is low relative to then the pixels may be too dark to see if you are using a black background, or too light to see if you are using a white background. If so, choose a different Display setting to make them visible. To determine approximately how many counts you will have per pixel, multiply the count rate from the source by the Dwell time for the channel. Display. In real-time, this setting scales the data displayed in the image window. Note: the actual data are only modified if Save As Displayed is selected. Otherwise, only raw original data are saved to disk. Full Range: The raw data are displayed without any scaling. Be careful here, images with pixel data whose values are relatively close to zero will not be visible against the black background. Max/Min Linear: In real-time the image linearly rescales to the largest and smallest pixel data values acquired thus far. Be careful here also, if the data are not well understood, odd effects may appear. This mode is important for WDS and X-ray mapping, where low count rates can require long dwell times in order to present a signal. This mode allows one to quickly see whether there is a changing signal for long-dwell images or maps. Max/Min Log: Similar to Max/Min Linear except that it scales using the natural logarithm of the data values. Max/Min Sqrt: Similar to Max/Min Linear except that it scales using the square root of the data values. Edit Custom: Allows the user to define arbitrary types of display scaling. Dwell. The dwell edit field varies according to the Acquire mode selected: Dwell: The dwell time is the amount of time that data from the ADC are being logged while the electron beam is at a single pixel position. The minimum dwell time is 1 microsecond. The ADC converts at a rate of 10MHz, so every 1 microsecond of dwell corresponds to 10 ADC samples. For video acquisition, the maximum dwell time is 1600 microseconds (1.6ms or 16,000 readings per pixel). Note that this is not a video frame average. Publication-quality images may demand dwell times on the order of 100 microseconds; while this can create long acquire times, it compares favorably to the requirements for gated-integrator operation. Average: The edit field number sets the number of samples at each pixel. The ADC samples at 10MHz, so averaging 10 samples is equivalent to a dwell of 1 microsecond. This setting can be used to get sub-microsecond dwells. Single: The edit field is not available. The ADC is sampled exactly once (100ns). The fastest operation is achieved in this mode, and is recommended when acquiring multiple frames. If the physical channel is a TTL counter ( CNTR 1 - CNTR 4) instead of the ADC (e.g., WDS maps), then the dwell time is the amount of time that the counters are allowed to increment. As with ADC channels, the minimum CNTR dwell time is 1 microsecond. The maximum dwell time is 16 seconds (enter as 16,000,000 microseconds count your zeros!). We suggest that a back-of-the-envelope calculation be performed to determine if your selected dwell and measured count rate will yield any counts. Multiply the count rate from the source by the dwell time entered here to get an average number of counts per pixel. If this value is not at least one count per pixel then a longer dwell or higher count rate is necessary.

5 First Run - Setting the Line Profile Options Often, brightness and contrast are adjusted by eye. In other words, if it looks good, it is good. However, Revolution supplies waveform monitor tools that allow the image to be adjusted with more precision: Show Line Profile. Checking this box overlays a real-time graph of sequential line profiles on the image, useful for feedback on the scan and video operation. Since this graph is updated in real-time, it may jump around quite a bit because of the sample morphology. For Spectral Engine II systems, the SEM Gain and Offset potentiometers on the 4pi SIU (Scanning Interface Unit) directly control the magnitude (contrast) and vertical offset (brightness) of this signal. For Universal Spectral Engines, the gain (contrast) and offset (brightness) are controlled by software from the Scan Generate Preferences. The top and bottom of this window define the full-scale ADC values. The zero of the signal is displayed at the centerline of the image. The full-scale setting can be set via the View% edit box (described below). Use the microscope Contrast and Brightness controls to set the CRT image to the desired level; for Spectral Engine II systems, the SIU can then be adjusted to set the signal shown in the line profile overlay to the desired level. for Universal Spectral Engine systems, the video signal can be adjusted in software (see Scan Generate Preferences). For Spectral Engine II systems ADC A is adjusted with the SEM Gain and Offset potentiometers on the front panel of the SIU. The gain and offset potentiometers for the ADC B, ADC C, and ADC D are located inside the SIU. For Universal Spectral Engine systems all ADCs are adjusted via software./p> Coincident with the centerline zero level is a draggable reference line. Click your mouse within the vicinity of this line and drag. A new line will appear that can be positioned anywhere within the image. This marks the line in the image which is used for the real-time line profile overlay. In acquired images, the line profile can be displayed or hidden, regardless of the setting of the checkbox before acquisition, using the menu item Options Show Line Plot or Options Hide Line Plot. There is also a contextual menu item (right-click) to control this. Show Every Line. Checking this creates a tick mark indicating the current scan-line to run on the left side of the image, giving real-time feedback on the scan position, and a real-time picture of sequential linescans. Set Color. This button accesses the operating system's color-picking services, and allows the line-profile color to be set. View %. Enter a number between 0 and 100 to set the physical size of the line profile within the image window as a percentage of the halfheight image window size. The default is 95%. First Run - Setting the Default Save Format The default format for saving images can be specified here. mac users have the additional option of specifying the "owner" of the file, primarily for work in pre-osx systems. Save Format Options Format. Allows you to select the type (format) for saved image files, independent of x-ray spectra, x-ray images/maps, or color phase maps. Revolution is a 4pi proprietary format. It allows graphical objects to be embedded in an image. This allows objects (e.g., micron markers and line profiles) to be moved or removed from an image, even after it is acquired, saved, closed, and reopened. TIFF-Mac is TIFF written in big-endian format. TIFF-PC is TIFF written in little-endian format. EPS is Encapsulated Postscript, a vector graphics format. JPEG is the Joint Photographic Experts Group format for compressed images. Recommended for web publishing. The JPEG compression slider allows the user to select the quality of the resulting graphics file. PICT is the native Macintosh graphics format (not available on Revolution for Windows). BMP is the native Windows graphics format (not available on Revolution for Macintosh). Owner (Mac only). Allows you to select the owner for images, independent of x-ray images/maps, color phase maps, or spectra.

6 Windows and Mac OSX use 3-letter extensions to specify the owner; however, since the Microsoft specification is not unique and is set as an option in the operating system, Revolution always shows the owner as "None" for Windows. Pre-OSX Macintosh uses an invisible 4-character resource (the creator) to specify the owner. Revolution is a 4pi-specific creator called ' MBDY'. The equivalent Windows extension is ".mbd". NIH Image sets the creator to ' Imag' for the program NIH Image. The equivalent Windows extension is unknown. Photoshop sets the creator to ' 8BIM' for the program Adobe Photoshop. The equivalent Windows extension is "ps". IPLab sets the creator to ' IPLB' for the program IPLab Spectrum. The equivalent Windows extension is unknown. Save as Displayed. Saves the image or map to disk exactly as it looks on the computer screen. This is useful if you have applied custom scaling to the raw image data and want to preserve the appearance. Warning: this checkbox will rescale the original data to new values - the original data will be lost! First Run - Adjusting the Brightness and Contrast To begin the contrast and brightness adjustment, turn on the Preview, and turn on the Show Line Profile and Show Every Line checkboxes (see above description and figure below). The system takes control of the microscope and displays the image acquisition in real time. The line profile overlay represents the microscope video signal. Complete the adjustment to your taste by adjusting the microscope's brightness and contrast controls, and the 4pi controls, if necessary. First Run - Squaring the Pixels

7 After selecting the desired aspect ratio, the pixels must be set square. In principle, this requirement is met when the image acquired on the computer screen matches the master image on the CRT or polaroid. In practice, the user must make sure that the CRT or polaroid are also displaying square pixels. Many times, they are not! To square the pixels, adjust the size and location of the image that appears in the preview window until it matches the S(T)EM CRT or polaroid image. To adjust, use the front-panel X-Scan and Y-Scan Gain and Offset pots if the system has a Scanning Interface Unit. The Universal Spectral Engine controls for this are set in software. Use the X-scan (horizontal) offset to shift the image left and right. Use the Y-scan (vertical) offset to shift the image up and down. Likewise, use the gain controls to adjust image compression in the horizontal and vertical directions. This is an iterative process, because the gain and offset controls will appear to affect one other. Furthermore, the microscope's CRT may be blanked when the preview mode is active; thus, alternating between preview mode and normal microscope operation is required in order to compare the result to the microscope CRT and make adjustments. When finished, the pixels will be square regardless of the image resolution defined in the software (as long as the size conforms to the defined aspect ratio) and no further adjustment should be necessary. To perform this procedure correctly, one should use a calibration standard. More information on squaring the pixels can be found below in the discussion about horizontal or vertical distortion (below). First Run - Scan Adjustment and Artifact Removal Even after the correct aspect ratio has been selected, a number of artifacts may appear with respect to the final correct rendition of the microscope CRT image (shown below at right). horizontal or vertical shift horizontal or vertical distortion horizontal or vertical overscanning horizontal or vertical mirror image inverted video rotated image 60/50 Hz noise retrace delay distortion nonlinear compression artifact normal and correct A description of each of these artifacts is shown below, with remedies. To make adjustments, the user must continually compare the microscope's CRT image with the acquired computer image. This requires that the preview window be dismissed and restarted repeatedly, since only one scan generator can control the microscope at a time. Shift And Distortion horizontal shift vertical shift horizontal distortion vertical distortion Horizontal and vertical shifts are controlled with the X-scan and Y-scan Offset controls. Adjust both to get the image centered properly. Horizontal and vertical distortions are controlled with the X-scan and Y-scan Gain controls. If the pixels happen to be approximately square, this artifact may be mistaken for a magnification setting error. Adjust both controls to remove image compressions and/or expansions. Virtually all installations require these adjustments to be made in the field. The process is iterative and sometimes tedious, but should only be required once. The final result is an image whose pixels are dimensionally correct in both horizontal and vertical directions (that is, "square"). Overscanning

8 horizontal overscan vertical overscan Horizontal or vertical overscanning occurs when the X-scan and Y-scan Gain is are set too high, causing the beam to be driven outside of its design range. The visual effect is to create a repetition of the horizontal or vertical boundaries of the image. Adjust the relevant Gain down to eliminate overscanning. When overscanning is eliminated, adjustment of the Offset may again be necessary. Miscellaneous Artifacts horizontal mirror image vertical mirror image inverted video rotation Mirror images occur when the DAC drives the beam in a direction opposite to what is required. Likewise, image rotation (swapped definitions of X- and Y-scan directions) can occur for certain microscopes due to their scan generation circuitry, or for example if the X-scan drive cable is accidently swapped with the Y-scan cable during installation. Horizontal and vertical scan definitions can be changed in real time with the mirror checkboxes in the Output Scan Setup. Inverted video (swapped definitions of black and white) can occur for certain microscopes due to their video processing circuitry. Video inversion can be selected independently and in real time for each enabled channel in the Input Channel Setup. Synchronization 60/50 Hz sync off 60/50 Hz sync on 60 or 50 Hz noise is caused by poor isolation of the power mains from the imaging instrumentation. Generally, noise of this sort is either generated by the microscope itself or is due to improper grounding, and is more evident at higher magnifications and at edges with high contrast (see above example). If such interference cannot be eliminated by proper grounding or instrumentation adjustments, it can be minimized by synchronizing the electron beam sweep with the phase of the electrical power sinusoid. To apply this synchronization, select the proper menu item from the Output Scan Setup. Retrace Delay Distortion retrace delay distortion 0 µs retrace delay distortion 100 µs retrace delay distortion 1600 µs

9 A retrace delay distortion is caused by driving a linescan forward too soon after a horizontal retrace. As a result, the program begins acquiring linescan data at the beginning of a new line before the beam has had the chance to retrace and position itself correctly. The distortion manifests itself as a variable number of garbage pixels at the beginning of each linescan. In rare instances, there may also be a vertical retrace delay artifact. The solution to either is to increase the horizontal (or vertical) retrace delay from the Output Scan Setup. In the example above, not even 1600 µs is enough to completely remove the artifact. In such a situation, it may be helpful to use the Park Beam at Zero. Non-linear Compression Artifact The non-linear compression artifact (example images not shown) is related to the retrace delay distortion but is a more extreme example. In this case, there is not enough delay capability in the horizontal retrace delay parameter. In addition, there may be nonlinearities in the scan generator which affect the 4pi scan signal. This artifact is strongly dependent on the type and model of microscope. If this artifact is observed, the Park Beam at Zero control should definitely be turned on, and the horizontal retrace delay set to its maximum value of 1600 µs. If the artifact is still not reduced, in almost all cases it can be eliminated by increasing the amount of time that the software waits after sending a new pixel position to the electron beam and before it starts recording data: the pixel delay. Increasing pixel delay to a large number will significantly reduce the speed of scanning and acquisition. Contact 4pi support for specific information and custom troubleshooting help. Acquiring and Saving Images Once the setup is complete, dismiss the SEM Image Setup window with the OK button. To start an acquisition, click on the acquire button in the toolbar The image will be captured in separate window(s), appearing in real time. When the acquisition is complete, the image will stay on the computer screen, unless other image-saving options have been set in the General Preferences. The default format of an image or map is selected in the SEM Image Setup (see above, this page). The acquired image can be saved to disk by keystroke ( clover-s on Mac, control-s on Windows), selecting File Save (menu), or simply selecting the close box in an acquired image window. This also works for those images that are in process and only partially acquired. The standard dialog appears for the operating system, and has this menu appended to it: The selections available are: Revolution. Save the data as a proprietary file that can be reopened in Revolution. By using this format, the image presentation can be adjusted at any time after the file is saved to disk. TIFF-Mac. Save the data as a TIFF file in Macintosh format. This procedure flattens any layers, and can only be further modified by a 3rd party pixel editor. TIFF-PC. Save the data as a TIFF file in Windows format. This procedure flattens any layers, and can only be further modified by a 3rd party pixel editor. JPEG. Save the image as a JPEG graphic, suitable for web presentation. This procedure flattens any layers, and can only be further modified by a 3rd party pixel editor. PICT (Macintosh) or BMP (Windows). Save the image in the native format of the operating system as a picture that can be pasted into a presentation program. This procedure flattens any layers, and can only be further modified by a 3rd party pixel editor. Micron Marker Calibration The least confusing way to perform the setup of the micron marker is to first acquire a working image. Under the assumption that this has been done, select Edit/Setup from the Mag menu of the toolbar:

10 The [resizable] Magnification Setup and Edit window opens: Select a feature whose dimensions are known. Use the zoom tool to enlarge the feature if necessary. Very important: make sure the mag shown is the one that the microscope is set to currently.

11 Position the cursor, press the mouse button down, and drag the dimension tool across the feature you wish to calibrate against: A green dimension line will appear. The endpoint can be dragged to any location from its starting point. A dimension measure will appear aside the line. For systems that have never been calibrated, the measure will appear as "***". In the Set Calibration section, enter the desired line length and unit (in this example, 25 nm), turn on Make Default, and hit the Set button. The dimension in the image will change to reflect the new measurement. The calibration is now locked in across all magnifications. Micron Marker Options While the edit window is open, you can set prefs for the future behavior of the micron marker and its appearance in images. Mag Menu. Shows the available magnifications; related edit fields or menus are updated when a new mag is selected. Use this menu to Add and Delete mags. Tweak. A multiplicative correction to finely adjust the size of the micron marker for this one magnification only. This parameter can be used to adjust for non-linearities (if any) that occur with different magnifications. The software will remember the tweak correction factor for each defined magnification, thus allowing the greatest possible precision across all magnifications. Override Auto-Sizing. Prevents the software from automatically resizing the micron marker. This feature can be used to create a micron marker of a specific size and unit to match any presentation requirement. When it is turned on, an edit field and dimension menu are highlighted for information entry. Show Marker. Determines whether a marker is shown anywhere in an image. Show Below Image. Places micron marker into the information box below the image. The micron marker can only appear below the image if the Append Below Image checkbox is also selected in General Prefs.

12 Marker Frame. Adds a frame around the marker border for contrast improvement. Reverse Video. Reverses black to white for contrast improvement. Transparent Marker. Removes solid background from around numbers. Not available in Windows. Font Options. The font menu displays all installed fonts. Any can be selected, although few will be suitable. Use the associated size menu and style checkboxes (below the font menu) to modify the font in the usual ways. Make Default. Clicking this checkbox for any subsection makes those settings the default going forward for the program as a whole. When they are turned off, the settings in the window apply only to the image that is displayed in the window. This allows images to be edited for magnification changes without affecting other operations. Save, Open. Exports or imports a straightforward text file that contains the full menu of mag sizes and associated values. This feature is for backing up the mag data in order to transfer the information to another copy of Revolution, or if a file corruption forces the main prefs to be thrown away (after the mags are reset to defaults by throwing away preferences, previously exported mags can be imported to restore extensive additions or modifications). Users of Revolution should export their mags and use the Open button here to insert settings into Revolution 1.6. See notes further below on this page. This feature can also be used to reorder the mags as they appear in the menu. The text files have a sort option built in; with a text editor, the sort can be changed to Ascending (default), Descending, or NoOrder (spelling counts), and the file can be reimported to implement the sort. It is possible to import a different number of menu items than were exported, so the user can construct all menu items by hand independently of the program, if desired. EM Client EM Client is a mechanism for limited communication between Revolution and the microscope. Using it, Revolution is able to import various information, such as the instrument magnification, beam energy, or image size, for automatic inclusion in images. For EM Client to work, the _Config file for your particular microscope must be placed in the Revolution folder (see left). Note: the user must still do the micron marker calibration step described above. If the client is installed for your microscope, EM Client should be set in Revolution's toolbar Mag menu: Relevant information will be automatically collected over the comm link. Micron Marker Usage Notes If the 4pi system is not equipped with an EM Client for your microscope, Revolution must be updated on a continuous basis in order that the micron marker be drawn correctly:

13 In pure manual mode, select the magnification to match that of the microscope, for every image. To be prompted for the mag without adding it to the menu, select Ask Mag. A small dialog will pop up before every acquisition, allowing you to enter a mag and proceed with the acquisition with just a couple of key presses. To be prompted for the mag and add it to the menu, select Ask & Add. A small dialog will pop up before every acquisition, allowing you to enter a mag and proceed with the acquisition with just a couple of key presses. In addition, that magnification will be added to the menu for future use. This setting is useful when the microscope is initially configured with the 4pi system, in order to rapidly populate the menu; however, it is not recommended for continuous use, as the menu will become awkward to handle. From any image on screen, the mag settings described above can be accessed by right-clicking on the image and selecting Edit Magnification: Once the changes have been made, the image on screen will be updated. Upgrading from Revolution v1.5.6: Micron Marker Issues Compared to version 1.5.6, version of Revolution uses a more comprehensive format for storing image information, in particular to hold the information about scope magnification at the time of acquisition, and micron marker calibrations. Unfortunately, this means there is no 1-to-1 transfer of information when a v1.5.6 image is opened in v pi Recommends that a copy of v1.5.6 be kept to open and manipulate images acquired with v There still remains the issue of transferring the calibration from the older version to the newer version. 4pi recommends the following procedure: 1. Make sure a proper backup is performed on all images and all versions of installed Revolution software.

14 2. Temporarily launch Revolution v1.5.6 and export the stored magnifications. Make a note of the name and location of the exported text file. 3. While still running Revolution v1.5.6, open a properly calibrated v1.5.6 image, with a visible micron marker and known EM magnification, and save the image as a TIFF (or JPEG) image, with the micron marker visible and preferably set in the image itself for convenience. Make a note of the name and location of the saved TIFF (or JPEG) image file: 4. Quit Revolution v Launch Revolution v1.6.0 and open the TIFF (or JPEG) image that was saved in step 3. The micron marker should of course be visible. 6. Right-click on the image and select Edit Magnification as described above. The first step is to import the mag settings from version (step 2 above). Use the Open button and browse to the settings file. Select it and all the settings that can be imported from will appear in the dialog window. 7. Using the method described above, set the magnification to that used to make the image, and set to default a micron marker that matches the one already in the image. The final result is shown below:

15 8. An appropriate alternative is to just recalibrate in v1.6.0 using a standard. Since the v1.5.6 installation was already calibrated, the results should be the same as in Launch Revolution v1.6.0 and import the stored magnifications from the previously exported text file. Use the Open button (above) to import the text file's data. 10. While still running Revolution v1.6.0,