Ditabis - Model 25 - Imaging Plate Scanner Micron - Manual

The High Resolution Imaging Plate Scanner Software Manual Edition 3.0: Dez. 2002 © DITABIS Digital Biomedical Imaging Systems AG, Pforzheim 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 2 Index Chapter 1: Scanning Plates .................................................................................................. 3 1.1 Loading Plates to the scanner................................................................................... 3 1.2 The scanning Dialog box ......................................................................................... 3 1.3 Details on Scanning Parameters............................................................................... 6 1.4 Details on Scanning Formats ................................................................................... 8 1.5 Import Microscope Data .......................................................................................... 8 1.6 Importing Data from Data Files ............................................................................. 10 1.7 Unloading Plates .................................................................................................... 11 Chapter 2: Viewing Images................................................................................................ 12 2.1 Opening Images and Previews............................................................................... 12 2.2 Look Up Tables...................................................................................................... 13 2.3 Zooming Images .................................................................................................... 14 2.4 Saving and Exporting Images ................................................................................ 14 2.5 Printing................................................................................................................... 15 2.6 Toolbar................................................................................................................... 16 Chapter 3: Images Manipulation........................................................................................ 17 3.1 Image Filters .......................................................................................................... 17 3.2 Rotating Images ..................................................................................................... 18 3.3 Aligning Images..................................................................................................... 20 3.4 Image Arithmetic ................................................................................................... 20 3.5 Region of Interest (ROI) ........................................................................................ 25 3.6 Undo....................................................................................................................... 26 Chapter 4: Image Analysis ................................................................................................. 27 4.1 Calibrating Images ................................................................................................. 27 4.2 Point Functions ...................................................................................................... 27 4.3 Line Plot................................................................................................................. 31 4.4 Area Functions ....................................................................................................... 33 4.5 Peak Find ............................................................................................................... 37 4.6 3-Dimensional Plot ................................................................................................ 38 4.7 2D-FFT .................................................................................................................. 40 Chapter 5: Image Conversion Tools .................................................................................. 43 5.1 Merge ..................................................................................................................... 43 5.2 Binning................................................................................................................... 44 5.3 Convert................................................................................................................... 44 Chapter 6: Other Functions................................................................................................ 45 6.1 Emergency Functions:............................................................................................ 45 6.2 Transport ................................................................................................................ 46 6.3 Options................................................................................................................... 47 6.4 Configuration Functions ........................................................................................ 49 6.5 Help System........................................................................................................... 49 Chapter 7: Troubleshooting ............................................................................................... 50 7.1 Clearing Plate Jams................................................................................................ 50 7.2 Configuration Problems......................................................................................... 54 7.3 Retrieving data after computer crash ..................................................................... 55 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 3 Chapter 1: Scanning Plates 1.1 Loading Plates to the scanner The process of loading plates into the scanner is invoked by selecting Load Plates from the Scan menu or the Toolbar. The Scanner will then initialise the loading process and prompt the user to insert plates, when ready. Open the scanner door to access the four loading ports. Four Plates can be loaded simultaneously to the loading slots. Insert the plates to the slots only when prompted, and make sure, that the back side of the plate is in touch with the step in the loading slot, by dragging the plate back after inserting. When all plates are in position, press the left button at the scanner, indicated with Continue in the Display. The scanner will now automatically load the plates to the scanner drum. Do not interfere by touching or removing plates, while the scanner is working When the scanner has loaded the plates, the user will be prompted to inset the next set of four. This process can be continued five times, until all twenty plates are loaded to the scanner. The scanner then automatically will invoke the scanning dialog, to start the scan. As always 5 plates will be scanned at once, it is advantageous to use only parts of the loading slots, if less then twenty plates shall be loaded. Up to 5 plates shall be loaded only to the first (rightmost ) slot, for up to 10 plates use the first two slots, for up to 15 plates use the first three slots only. When the scanner prompts for loading, but no more plates shall be loaded, press the right button at the scanner indicated Abort to complete the loading process. 1.2 The scanning Dialog box This Dialog box controls the scanning process. The box is automatically opened after loading, and can be opened manually with Scan on the Scan menu. Scanner settings contain all the various scanner operating parameters. These are combined to setting files (Extension .SET), that can be selected from this Dialog. With the Edit button the settings can be changed. For a detailed discussion on scanner parameters, see below. For standard applications use STANDARD.SET. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 4 Formats are also selected from format files (Extension .FMT) and define the scanable Area of the plate. For a detailed discussion on Formats, see below. For standard applications use STANDARD.FMT. The Filename specifies a wildcard Filename, that will be used for all loaded plates, and added a running number (_1, _2 etc). The filenames can be edited later. The Path specifies the location, where the data files will be stored. Start the Scan with the Start Scan Button. The Scan Control box will remain open while scanning. The Status shows the scan progress. The pixel size of one image is given in X and Y, the percentage of the scan and the scan number. The percentage shown refers to the actual scan of one row of plates. Up to four scans, one for every drum position are needed to scan the complete load of the drum. The Start Scan button then leads to the Common Properties dialog box. This dialog displays the common filename for the images, and allows a comment to be entered, that is valid for all images, i. g. A description of the experiment or microscope session. This comment can later be edited and complemented for the individual images. Abort will end this box, Continue leads to the Scan Map Dialog Box: 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 5 The Scan Map shows the layout of the images on the drum. All loaded plates can be checked, and the filename and comment of this image will be displayed and can be altered. To select images just click on the position in the Scan Map or press Next Image. When all comments and filenames are correct, press Continue and the scan will start. Cancel will end this box without scanning. Note: As the imaging plate is not sensitive to visible light, the imprint of microscope settings is not possible with imaging plates. Care has to be taken, not to confuse the order of the plates when taking them out of the microscope and feeding them into the scanner. Then the information, noted during the microscope session can be entered into the comment boxes as described above. If the microscope supports export of the information that would be imprinted to the negative to a diskette, this file can be read into the above boxes by use the Import Data Button. (See Import Microscope Data below) 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 6 1.3 Details on Scanning Parameters The Scan Parameter box is opened when selecting the Edit button in the Scan Control dialog. If the current parameter set is set to read-only the button reads Copy and edits a copy of the data set. Gain: The gain of the detection unit in the scanner that determines how many counts are created at a given electron dose. As a rule of the thumb, a Gain of 10000 will create about 10000 counts in the image when illuminated with a dose of 1e-/µm2. This is true for a Laser setting of 100% in standard mode. 20% Laser will create about half the counts. The counts that will result at a given Exposition depend on the gain, the laser setting and the electron energy. A Setting of 10000 will be suitable for most applications, but can be reduced for high dose expositions, not to saturate the scanner. Laser Setting: The laser energy can be controlled. Full laser power with 100% will give most signal out of the plate, giving the best Detection Efficiency (DQE) and lowest noise for low dose applications. For standard applications a laser setting of 20 to 50% give better resolution and image quality. The reflected light correction works better at lower laser settings. Resolution and Binning Factor: The resolution can be selected from the box. In case of a fixed resolution instrument only this choice is selectable. The resulting size of the image will be displayed. This display depends on the selected scanning size (see scanning format) With binning pixels can be grouped together to reduce the resolution. There is also the possibility to reduce the resolution later, see the Tools section. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 7 Data Channel: The micron scanner features two simultaneous data channels, each with 16 bit dynamic resolution. In Standard mode, only one data channel is used, the plates are scanned with 16 bit dynamic resolution, only one data file with the extension .IPL will be generated. For applications with High Dynamic range like diffraction patterns the second channel can be used to view the same data set with a different gain setting. The second cannel then views the data with about 33 fold gain. Two images are created, one with the Extension .IPL and the second with .IPH (Higher Gain Image). This two images can also be combined to one image as described below. The second cannel can also be used to simultaneously record the Reflected Light, that is used for gain normalisation and reflected light corrections. The reflected Light can also be used to record Markers and numbers, written to the margin of the plate for identification and for optical scanning. Be aware, that using two data channels will double the amount of data created Combine and higher Data Depth: The high dynamic mode creates 2 digital images of every plate. These two images can be automatically combined to one high dynamic image, then with the extension .IPC by checking Combine High Dynamic. The data depth of the resolution image can be checked from 16 to 32 bit. The 16 bit setting is special, because the data then will be divided by 30 so there is no increased dynamic range but some noise reduction. The 24 and 32 bit settings create data sets in the micron format (See appendix A for details). Use of this formats is recommended when your image processing and evaluation software can handle and import this data depth. The micron software of course can handle it. The 24 bit format is less common, but saves disk space. Black Level Correction: The digital value that refers to no radiation is the black level. In the detector this value is predefined and may drift with temperature. The scanner internally controlled this level and sets it to a given value, when Black Level Correction is checked. In principle, a value of 0 is adequate for this level, and for most imaging applications this is the best setting. However when parts of the image are close to black noise will create negative values that are truncated to zero thus distorting image information. Setting the level to a adequate positive number (i.g.100) will avoid this. When quantifying the data this level has to be subtracted. Save As: The Settings can be stored to a file to recall it later. Checking read-only will set the file to read-only, so the setting cannot be changed afterwards. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 8 1.4 Details on Scanning Formats To change format press the Edit button on the right side. The following dialog appears: If your microscope does not expose the whole plate format, scanning time and disk space can be saved by restricting the scan able area. Margins can be defined around the plate, positions refer to the loading position of the plate. The Settings can be saved to named files to create sets for different applications (e.g. different Microscopes). Marking the set as read-only will prevent unwanted changes to a set. 1.5 Import Microscope Data (Annotate) If the microscope supports export of the information that would be imprinted to the negative to a diskette, this file can be read into the above boxes by use the Import Data Button. Otherwise it is good practice to keep a log of all exposures made with the microscope during the session. As the Imaging Plate is not sensitive to light, the imprinting on the negative cannot be seen. The information however can be added to the data files prior to scanning by pressing the Data Import Button in the Scan Map Dialog Box. The following Box appears: 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 9 There are several options to import exposure data: 1. If the microscope supports a data output, this file can be imported. This is possible with the newer LEO microscopes (902, 910, 912, 922), Philips (CM Series + Tecnai) and some JEOL instruments. In this case you simply select the appropriate import filter for your microscope (if needed, we can create a filter for your microscope type) and select the microscope data file. 2. If not, you may create manually the data file on your microscope’s computer using a text editor, spreadsheet or database program. If you do not have a Computer on your microscope desk, use a notebook computer or even a palmtop. There is a generic import filter for text files TEXTFILE.IFT. 3. There is also a program available from DITABIS for recording logs of your microscope data. Please ask us for details. Data Format of Text File The file is an ASCII text file, where the records of one microscope session are entered. The records are separated by an identifier string. Every record starts with that string that is EXPOSURE: by default (the colon is required without a space character) . The identifier string usually is followed by a running number. There is no need to enter it in upper case characters. However the string may not appear in the rest of the record. All the text that follows is simply taken to the comment field so there may appear whatever you find useful. The Word MAGNIFICATION followed by a number will be evaluated and directly used for image calibration, if present. Example File : EXPOSURE: 3376 MAGNIFICATION 30000 Text entries for this Exposure EXPOSURE: 3377 Text entries for this Exposure EXPOSURE: 3378 Text entries for this Exposure And so on The identifier string can be changed by an entry in the config.mac file. The entry must be: ImportHeaderString = "your String"; 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 10 1.6 Importing Data from Data Files Import Filter: If the above dialog box is present, select the appropriate import filter for your microscope or the generic textfile filter. Data File: Press the browse button and select the file containing the data. This file may also reside on a network drive, if your computers are linked or on a floppy disk. Order: Determines the order you access the plate stack. If the first plate you load to the scanner was the first exposed in the microscope, select Standard Order. If you first load the plate that was the last exposure in the microscope select Inverted Order. Start Import At Number: This refers to the record number in the data file. You usually create one data file for one microscope session or film magazine. As the scanner loads 20 plates at a time, you may use the data file for several scans, until all the plates are processed. At the first scan, enter 1. At the second scan, enter 21, then 41 etc. The Number does not refer to the running number entered in the data set but the record number in the file Press Import and the file data will be imported. The text field in the Scan Map dialog box now holds the information. You may check the first one to confirm the information. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 11 The data File can be selected with the browse button. The Number to start import refers to the running number of image in the set, to start with the first, select 1. The Standard order takes the date in the order in the file to the order of loading, from right to left in the scanner. Inverted order implies that the plates have been reversed by unloading from the microscope, the last set in the data file will be the first loaded to the scanner. The Import filter specifies the data format output from the microscope. The import function can be repeated in case of error. 1.7 Unloading Plates Once the plates are scanned, they shall be unloaded. Do not let the plates in the scanner for a long time (over night or over days) because they will become used to the bend on the drum and need some time to recover. If the Plates are used to the bend, also the unloading process may show problems unloading the plates. To invoke the unloading process, check Unload Plates from the Scan menu or hit the toolbar icon. The scanner then will go into unloading the plates to the loading slots. As soon as the first set of plates is ready, the scanner prompts the operator to remove the plates. Never touch or remove plates before prompted The scanner sensors will miss the plate and be confused After removing the plates, press the left button at the scanner, indicated with Continue. The next set of plates will be unloaded. Repeat this process unless all plates are unloaded. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 12 Chapter 2: Viewing Images 2.1 Opening Images and Previews Images are stored to disk in a proprietary format, containing a file header, the image data and a zoomed image (preview or thumb-nail). As the data is composed several megabytes this preview of the original image can be handled much quicker and easier. To open a preview, select Open Preview from the File menu or use the toolbar icon. Select a file and the preview will be displayed on screen. To open the full image select Open Full Image from the File menu. In this case be aware of the image size, that may easily extend 20 MB and is slow in performance. From the preview there is an easy way to look at a part of the image in full detail. Select Zoom Detail from the View menu when the focus is on a preview. A cursor marked wit an R will be displayed to specify the detail to be loaded. Click with the left mouse button, drag the box and click again. A new frame will be opened and the detail loaded to that frame in full resolution. To manage multiple images with a data base function there is an Image Viewer function. By selecting Viewer from the File menu or hit the toolbar icon, a dialog box is presented: 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 13 This box lists a selection of files within a file path. With Browse Path the path to search can be selected. The Input fields Filename specifies wildcard specification of the filename. In the Comment field, the wildcard .* or blank represents any comment, a input of words here may restrict the search to images, containing this words in the comment input. This allows easy selection of images from a certain experiment, specimen or condition. With New Search the search starts, the matching images with their comment is listed in the field below. The search can be cancelled with the escape key. The View File Info opens the info box that displays all header information in the image data file. Pressing the button refreshed this box with the info of the selected image file. A Preview located right of the Dialog box always shows the preview of the selected image and is closed again with the dialog box. Use the Open Preview and Open Image Button to open a preview or full image of the selected file 2.2 Look Up Tables Look-Up Tables are used to set the contrast and brightness for viewing images. This is important, because imaging plate images contain much more grey values than can be displayed on a computer screen at a time, printed on paper or can be recognised by the eye. With the look-Up table function the user interactively selects that part of the contrast range, that contain the valuable information. The Look-Up Table dialog box appears when selecting Look-Up Table from the Image menu or from the toolbar. There are several shapes for Look-Up Tables available: Contrast/Brightness is the most common selection for images. It works like the controls of a TV. The sliders control the steepness of the ramp and the offset. Ramp is the mostly used shape for High dynamic images. The start and end point can be selected with the sliders. Inverse Ramp shows the data in negative representation, as would be done on a film negative. Gamma is a non-linear function, that condenses or enlarges the contrast range. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 14 The sliders can be operated in 3 Ways: 1 Moving the Slider with the mouse. The display will be updated when the button is released. 2 Clicking to the arrow. The slider will be moved one increment and display is updated. Holding down the button will create a continuous move and update. 3 Clicking inside the scroll bar. The slider will be moved a big increment and display is updated. Holding down the button will create a continuous fast move and update. Apply to Pixels will manipulate the image data according to the chosen look up table setting. The data will be manipulated and the LUT linearised. This is important, when the image shall be printed or exported (see also the description of that functions). If the Ramp is very steep viewing only a small part of the dynamic range, Applying to Pixel gives a better representation on screen and easier adjustment of the sliders. Note that the Data will be changed in the computer memory and does not affect the file, unless you save it. The Histogram Function calculates the histogram of the viewed image and displays it in the LUT Window to ease LUT setting. Cancel ends the dialog box and undoes pixel data manipulation. (if Undo is enabled) OK ends the dialog. 2.3 Zooming Images The images can be zoomed on screen. To enlarge or reduce size, select Zoom In or Zoom Out from the View menu or hit the toolbar icon. To set the Zoom factor directly, select the desired zoom level from the Zoom submenu. Zoom Factor and State of the Image (Preview or Image) will be indicated in the status bar when the image is selected. Attempting to zoom in a preview, you will be prompted to load a detail. 2.4 Saving and Exporting Images To save images in the micron format, select Save from the File menu or from the toolbar. If A ROI is set only the defined frame will be saved. If the frame is in a preview image, not the preview but the original data, corresponding to the frame will be written to the file. As the next step, the comment information will be displayed in a box. The comment, saved with the original file will be preset and can be edited. After entering the appropriate comment press Continue. Now the file box is presented to enter the destination filename and path. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 15 To save an image in an other then the internal format, use the Export function in the File menu or the toolbar to invoke the export dialog box. When exporting images, you have to be aware of the data depth of file formats. The micron format is a 16 to 32 bit format, representing each pixel with 16 to 32 bit of information. Most export formats represent each pixel only with 8 bit, so there are only 256 grey levels that can be represented. This is enough for images to view or print, so this formats are called Desk-Top Publishing (DTP) formats. When exporting to DTP-Formats thus the information content of the images is reduced. The user must be aware of this and control this process. The way to do so is to use the Look-Up Table function and optimally set up the display. Then press Apply to Pixels to modify the image information according to the LUT. The exported image will then look like the image on screen. A shortcut to the Apply to Pixels is to check the Use LUT setting in the export box. There are also two formats that support 16 bit per pixel. You can use this formats to transfer data to other image analysis software packages. See also the Convert function below. Note: To export an image it is not necessary to open it to full resolution. Exporting from a preview will also save the image data. To restrict the Area to export, create a Region of Interest. (Image menu, Region of Interest / Create). 2.5 Printing The printing function (File/Print or Toolbar) sends the actual image to the windows printer. The image will be enlarged to fit the paper width. If a region of interest is set, only this frame will be printed. To get best print results, use the Look-Up-Table function as described above. Image overlays as ROIs, measurement objects and the micron bar are printed as well. To avoid this, select full screen ROI and delete objects before printing. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 16 2.6 Toolbar A scalable toolbar can be displayed using the View/Toolbar/Show Command. The toolbar can be positioned to the top, bottom, left an right by dragging it with the mouse. With the toolbar the following functions can be executed: Open Images and Image Viewer Save Image, Export and Print Set a rectangular Region of Interest and set Region of Interest to Full Frame. Look Up Table Loading and Unloading Plates Calibrate Analyse Functions: Point, Line, Areas and Peaks Zoom Functions: Zoom in and out, Zoom Detail from Preview 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 17 Chapter 3: Images Manipulation 3.1 Image Filters Filters on Images can be invoked from the Image/Filter submenu. A collection of the most used filters can be executed direct from the menu. For more advanced filtering application there is a filter dialog box executed with Image/Filter/more. Generally, filters are designed to enhance features or remove noise. the micron software filter operations affect only the selected region of interest. Spatial filters fall into three general categories: noise reduction, edge enhancement, and sharpening (high pass). Noise reduction filters strip extraneous data. Edge enhancement filters intensify the transition between luminance values to emphasise gradients. Sharpening filters enhance high-frequency information by emphasising local pixel transitions and effectively sharpen contrast and edges. In addition to these, you can design your own custom filters. This dialog provides a list of useful filters. You can create new filters and add them to this list using the Add command found in the dialog's Edit menu. You can use the Remove command to remove filters that you added. Occasionally, a filter you apply will not produce the effect you desire; use the Undo command (Image menu) to remove the effect of the last filter operation. Edit menu Add Displays a blank Custom Convolution dialog so you can add a new custom convolution filter to the list of filters. Refer ahead for instructions. Edit Displays the Custom Convolution dialog for the highlighted custom convolution filter, so you can edit it. Remove Removes the highlighted custom convolution filter from the list. Apply Applies the highlighted filter on the list to the active image’s ROI. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 18 3.2 Rotating Images The Rotate command on the Image menu provides extensive tools for image and ROI rotations and manipulation. The image data within the ROI may be rotated to any angle, either clockwise or counter-clockwise. You can specify the centre of rotation to be any point within the active image window by manually marking the point or by supplying its spatial co-ordinates. Additional tools for flipping or reflecting the ROI about horizontal, vertical, or diagonal axes are also provided. To display the Rotate Image dialog box, select Rotate in the Image menu. Buttons, options, and information fields: Angle of rotation: This is the exact angle by which the image within the ROI will be rotated. It may be specified either by typing the desired angle of rotation within the edit box or by the scroll bar directly next to the angle edit box. To calculate a desired angle, use the Line-Plot function. Rotate Right This button causes the image within the ROI to be rotated to the right (clockwise) by the specified angle. Rotate Left This button causes the image within the ROI to be rotated to the left (counter-clockwise) by the specified angle. Flip Horizontal This button causes the image to be reflected about its central horizontal axis. Note that this function is not available for irregular ROIs. Flip Vertical This button causes the image to be reflected about its central vertical axis. Note that this function is not available for irregular ROIs. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 19 Reflect Diagonal This button causes the image to be reflected about an axis running from the lower left of the ROI through the centre point of the ROI to the upper right of the ROI. Note that this function is not available for irregular ROIs. Center of rotation X: The X axis spatial co-ordinate of the center of rotation. Y: The Y axis spatial co-ordinate of the center of rotation. Specify point This button enables the Mark and Edit buttons, which let you mark a point to be used as the center of rotation. Center of ROI This button automatically selects the center of the current ROI as the center of rotation. Reference point This button automatically selects the selected reference point (if one has been designated) as the center of rotation. Coord. Syst. Origin This button automatically selects the origin of the image co-ordinate system as the center of rotation. The co-ordinate system origin, by default, is the lower left corner of the image. Mark Invokes the point marking function, enabling you to manually mark a point on the image to define the center of rotation. The image will be rotated around this point. This button is enabled only if the Specify point radio button is selected. When you click this button, you are placed in point marking mode. Click the primary mouse button to mark the desired center of rotation. Click the secondary mouse button to exit point marking mode and return to the Rotate Image dialog. Note: if you mark more than one point only the last point is considered. Edit Lets you edit the current point(s). 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 20 3.3 Aligning Images The Align command opens Align Image dialog box. This dialog lets you align images to a target based upon a set of fiducial points. This Function is useful to align images of the same sample area that have been taken while the sample was moved or drifted. Load the image that is base of the alignment before opening the align box. The Box will only work with that image (To align more images reopen the box). To align images first select the desired operation (Rotation, Magnification, Parallax. Shift is always corrected) Use Mark Points to mark the first set of fiducial points in that image. Then load the image that shall be aligned with Load. Mark the fiducial points in that image. Now the function Align calculates the target image in a way that it fits to the source image. Use Restore to undo the function if the result is not optimal. You may want to change the box size and retry. Use Ok to close the box and keep the changes. Use Cancel to close the box and undo the changes. 3.4 Image Arithmetic The Image Arithmetic command on the Image opens a dialog box that provides controls for combining images arithmetically. You can combine an operand image with the region of interest on the video monitor using a variety of arithmetic and logical operations. THE MICRON SOFTWARE provides many basic and complex operators. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 21 How to combine images The Arithmetic Operations dialog box employs an image list. To perform an operation with an image, you must add the image to the list. The image list can hold many images. It also contains a temporary storage area (a buffer) that can hold one image. When you combine an image from the list with the region of interest of the image monitor using an arithmetic operation, the result of the operation replaces the region of interest. When using arithmetic operators, the images are dealt with on a pixel by pixel basis. If the images are different sizes, the image in the image list is scaled or clipped to match the selected region of interest before the operation is performed. After the operation, the result is copied to the region of interest. Arithmetic operations apply the selected operator (add, divide, etc.) to the two selected images. For example, the Add operator will add the value of a pixel in the region of interest to the corresponding pixel in the image list and copy the result to the region of interest. (Note: The maximum value for the resulting pixel in this example is the maximum for the selected image bit depth.) This operation is performed on each pixel in the image. Using the arithmetic operations Averaging several images can reduce image noise or correct uneven lighting conditions. Image subtraction is useful for subtracting the background from an image. In addition to these classical operations, the micron software offers many more arithmetic and logical operators. You may wish to experiment with these operators and with combinations of filters and arithmetic operations. For example, by subtracting a low-pass version of an image from the original image, you can achieve a subtle edge detection (unsharp masking). Buttons, options, and information fields: ROI to Buffer Copies the currently selected region of interest to the image called Buffer. If Buffer already is on the list, it is replaced. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 22 ROI to List Adds the contents of the current ROI to the Source Images list using the name that appears in the text box beside Rename. File to List Adds a file you specify to the Source Images list. The micron standard format however is not supported, so the easier way to do is ROI to list. Files can be used with this function that are exported using the Export function. Scalar to List Use this button to place a scalar value into the Arithmetic Operations source list. It creates, in the source list, a name of the form “#N” where N is the scalar value you specify. This value can then be merged back onto the frame buffer using various arithmetic operations. Delete Removes the selected image from the Source Images list. Info Displays information about the selected image. Source Images: Lists the images available for arithmetic operations. By default, this list includes all current images and all images from the Undo stack when Arithmetic Operations is first invoked. Rename: Renames the selected image file with the name you supply in the text field. Apply: Applies the selected operation to the specified images, adds the current ROI to the Source Images list as an Undo image, and places the result of the operation in the ROI. Function: The arithmetic operator you select from this list is performed when you click the Apply button. Refer to Arithmetic Operators for a description of each. Scale Factor: A numerical value employed by the Multiply and Divide operations. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 23 Alignment: Options for defining how the result of an operation will be fit to the destination ROI if they are dissimilar in size or shape. Do not cast range Select this option to prevent the micron software from automatically recasting pixel luminance range to force conformity between images. By default, if the source image is of a different pixel type than the current ROI the source is converted, or cast, to the destination pixel type before the arithmetic operation is performed. Use Calibration's Aspect Ratio not applicable. Alignment (Image Scaling Options) Clip Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. Any portions of the image that exceeds the ROI or window maximum bounds to the right or to the bottom is simply clipped. Center Anchors the center of the image at the center of the ROI or active window. Any portions of the image that exceeds the ROI or window maximum bounds both to the sides or to the top and bottom is simply clipped. Scale to Fill Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. The image is then scaled uniformly in both the X and the Y directions in order to completely fill the ROI or window. This will usually result in either the right side or the bottom of the image being clipped to some degree. Scale to Keep Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. The image is then scaled up until the image fills either the width or the length of the ROI or window, whichever is encountered first. This keeps the entire source image visible within the destination. This often results in an unfilled area at either the right or bottom of the region. Scale to X Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. The image is then scaled up until the image fills the horizontal extent of the ROI or window. This will normally result in either an unfilled area at the bottom of the region or a portion of bottom of the image clipped. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 24 Scale to Y Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. The image is then scaled up until the image fills the vertical extent of the ROI or window. This will normally result in either an unfilled area at the right side of the region or part of the right side of the image being clipped. Scale X and Y Anchors the upper left hand corner of the image at the upper left hand corner of the ROI or active window. The image is then scaled up until the image fills the entire extent of the ROI or window. This ordinarily requires differential scaling of the X and Y dimensions of the image, thus distorting the aspect ratio To perform an arithmetic operation: 1. Choose Image Arithmetic from the Image menu. The Arithmetic Operations dialog box appears. 2. Send the first image to the Source Images list or the temporary storage (buffer). 3. Open the second image. You will combine this image with the one on the image list. 4. Select the first image from the list. If you stored the image in the temporary buffer, click on Buffer in the image list (if Buffer is already selected, you may skip this step). If you stored the image on the image list, click on the name of the image. 5. Select a region of interest (Image/Region of Interest/Create). The first image will be combined with the second image only in the region of interest. 6. Select the operator you want to use from the operator list. 7. Click the Apply button. An hourglass icon appears as the image from the list is being combined with the image on the video monitor. The result replaces the region of interest. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 25 3.5 Region of Interest (ROI) Region of Interest is a mask on the image that restricts image operations to that area. Simple ROIs are square fields (select toolbar icon or Image/Region of Interest /Create to create a ROI). The ROI can be moved and reset to the full image frame. ROIs can be saved to files and loaded again. The micron Software is capable of handling multiple regions of interest and arbitrarily shaped regions of interest. The Complex ROI command on the Image/Region of Interest menu opens a dialog that lets you control this capability. Additional ROI and image manipulation options are also accessible with this command. The Buttons allow the creation of Circles, Rectangles and Freehand ROIs. To move ROIs press the Move button. When multiple ROIs is not activated the ROI moves with the cursor and can be placed with the left mouse button. When multiple ROIS are activated, first the desired RIO has to be selected with the left mouse button, then pressing the right mouse button, the ROI can be moved. Place the ROI with pressing again the left mouse button. Delete one of the multiple ROIs with the Del. button. First select the ROI with the left button, then delete it with the right mouse button. Clear deletes all ROIS. The default operational mode is the creation of a single region of interest. If a new ROI is created in this mode, the old ROI is deleted. If you enable the Activate multiple ROIs check box, the micron software will permit you to create an arbitrary number of active ROIs on your image. In this mode, when a new ROI is defined, the old ROI or ROIs remain present and active. Any image manipulation operation will then be performed on all ROIs in the active image frame. The default mode in the micron software is to perform image processing or analysis actions at areas inside the boundaries of the region of interest. However, the micron software also provides you with the flexibility to exclude the area inside a defined ROI from any processing steps. This capability is accessed with the Process inside the ROI check box. If multiple ROIs in the image overlap, the micron software also has the flexibility either to perform any image processing or manipulation actions in the total combined ROI areas or to exclude any overlap areas from such actions. The default mode is to exclude overlap areas from processing. This default may be changed using the Process overlapping check box. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 26 3.6 Undo This command on the Image menu reverses most operations that cause a change in the pixel data of the image. Undo permits you to reverse operations because the micron software saves the current image before it makes any changes you request. When you select Undo, the micron software retrieves the previous version of the image and re displays it. Note: The list of saved Undo images appears on the source list in the Image Arithmetic dialog. This means you can perform a processing operation on an image, then combine the original image and the result through an arithmetic operation. You may configure the micron software to provide multiple levels or steps of Undo, thus permitting you to back up several steps in a process. The default setting for this parameter is 1. The number of Undo steps is set in the Options dialog accessed from the Extras menu. The possible number of Undo steps is dependent upon your image size and your available system memory. Selecting zero as Undo steps disables Undo and speeds up Operations with large images. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 27 Chapter 4: Image Analysis 4.1 Calibrating Images For image analysis spatial calibration is necessary. The standard calibration of the image is based on the actual size of the imaging plate and the scanning pixel size. It is possible however to enter the microscope magnification to calibrate to specimen dimensions. To open the Calibrate Dialog Box select Calibrate from the Image menu or from the toolbar. The filename of the selected image is shown. The calibration information is stored in the header of the image file and can be changed with the Change button. Enter the primary magnification and the preferred display unit, then press Change. The following analyse functions will give out their data according to this calibration. Calibration will be automatically set when the keyword MAGNIFICATION with a following value is present in the image comment when scanning. The box also allows to set a micron bar to the image. Enter the desired length of the bar in the field Micron Bar. Chose White or Black for the overlay color. Then press Place to position the bar to the image. Use Move to adjust position. 4.2 Point Functions The command on the Analyse menu and the toolbar opens the Point Morphometry dialog box. The Point Morphometry allows you to quickly collect data about features that can be characterised by a point or set of points. Point Morphometry extracts information about a point’s position, luminance (intensity) values, and distance from a reference point. Point Morphometry can be used for such tasks as shape analysis, densitometry, landmark identification, and dimension inspection. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 28 The following measurements are collected: Cartesian (x,y) co-ordinates of the position of each point, distance from a reference point, and grey value. You can save the data to a data file for use in another application (such as a statistical analysis or word processing program). Buttons, options, and information fields: Mark Points This button lets you manually mark points on the image using the mouse. Each click of the primary mouse button creates a single point. Points are represented on screen with single marks. Edit Points This allows you to edit the points presently shown on the image. When you click this button the pointer becomes an edit tool. Click the secondary mouse button to exit the edit mode and return to the dialog. Draw Last Points This redisplays the last set of points created. The data set associated with the point set also is re-extracted and displayed. Show Reference This controls whether the reference point appears on the image. The reference point appears as a small sunburst symbol. Move Reference This allows you to move the reference point to a new location on the screen. Clear This clears all objects from the screen image and removes all data from the data fields. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 29 Save Data This button writes the existing data displayed in the dialog’s data fields to the selected data file. If data for the current point set has already been saved the button is disabled (grey) until new data is extracted. This precludes your saving a duplicate set of data and invalidating your work. If no file is selected the button is disabled and no data can be saved. Data Information Fields X/Y The Cartesian (x,y) co-ordinates shown in calibrated units. Dist The distance of the point from the reference point. Grey The grey value of the point. File Menu Open Data File Choose this command to select or create and open an ASCII file in which your data sets will be stored. After you select a filename a special dialog box opens to allow you to supply up to three text strings as data file headers. The Header Information dialog box appears below. Exit This command closes the Point Morphometry dialog. Setting up a data file You can save your Point Morphometry data directly to a data file you specify. Set up a new data file or choose an existing data file. The function also permits you to write headers, notes, and delimiters to the data file while you are collecting data. Measurement data are saved in tab-delimited ASCII format. A header is attached to each data file. Opening a data file: 1. Select Point Morphometry from the Data menu. Select the Data File command from the dialog’s File menu. The Open File dialog box appears. 2. Type the name of the new data file in the File name box. The data file name must adhere to standard DOS file naming conventions. By default the micron software assigns the extension TXT to data files. Select an existing file if you want to append data to the file. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 30 3. Click Open. The Point Morphometry dialog box title now includes your data file name and the Header Information dialog box appears, giving you the option of creating a header for the data file. 4. Enter text that describes your work session. Note that you can change the titles of each field in the data heading as well as the specimen information associated with each field. After you enter text in an edit box you must tab out of the box for the information to be accepted. 5. Click OK. The Software adds the header information to the data file and returns you to the Point Morphometry dialog box. 6. Click the Save Data button. Each time you click the Save Data button, the current data set is saved to the selected data file. Collecting point data: 1. Select Point Morphometry from the Data menu to open the Point Morphometry dialog box. 2. Click the Mark Points button. The dialog closes and the pointer cursor is labelled P to indicate you are in point creation mode. 3. Move the arrow cursor by moving the mouse. To mark a point, move the arrow to the location you want to mark. 4. When the cursor is positioned correctly, click the primary mouse button. A point marker appears on the image. You can continue marking points by moving the cursor to the next position you want to mark and clicking the primary mouse button. Each click of the primary mouse button creates a point. 5. To stop marking points, click the secondary mouse button. 6. When you click the secondary mouse button to return to the dialog box, the measurements are automatically gathered from each of the points you marked. This data is displayed in the results fields at the right of the dialog. 7. Click the Save Data button to save the data to the specified data file. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 31 4.3 Line Plot The command Line on the Analyse menu and the toolbar opens the Line Plot dialog box. The Line Plot function allows you to quickly collect data about features that can be characterised by a line. Line Plot extracts information about a line’s position, intensity profile, and angle from reference. The following measurements are collected: Cartesian (x,y) co-ordinates of each vertex, length, angle from the reference point, and luminance profile. You can save the data to a data file for use in another application (such as a statistical analysis or word processing program). Buttons, options, and information fields: Draw Line This button lets you manually draw a line on the image using the mouse. To end the line click the secondary mouse button. You are returned to the Line Morphometry dialog box. Move Line This allows you to move the line you’ve drawn to a new location on the screen. Clear This clears all objects from the screen image and removes all data from the data fields. BOX Displays the Plot in a scalable box. The Box can be scaled and copied to the clipboard using the Edit/Copy menu function. The View menu allows for other representation of the data, as for ASCII data, that also can be copied. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 32 Export This button writes the existing data displayed in the dialog’s data fields to a data file. Line Width The line can be integrated laterally to reduce noise. Line Width is given in Pixel. Zero Based When checked the floor of the data is zero, otherwise the lower value in the data set. Data Information Fields Length: The overall line length in calibrated units, as given with the Calibrate function. Angle The angle as calculated from endpoint to endpoint with respect to the reference point. Beg., End. x/y coords The Cartesian co-ordinates of the two endpoints of the line in calibrated units. The beginning and ending points are defined by the way you draw the line. For example, if you draw a line from right to left the beginning point is the right end of the line and the ending point is the left end of the line. Conversely, if you draw a line from left to right the beginning point is the left end of the line. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 33 4.4 Area Functions The command Areas on the Analyse menu and the toolbar opens the Area Morphometry dialog box. The Area Morphometry dialog allows you to quickly collect data about features that can be characterised by an area. There is also a function to create the areas automatically, based on a set threshold. Area Morphometry extracts information about an area’s size, shape, position, and intensity. Area Morphometry can be used to analyse shape, surface texture or particle size. It can also be used for cellular analysis, object densitometry, and measurement of histological sections. The Area Morphometry dialog reports a set of measurements including the area, perimeter, centroid (centre point) position, circularity, longest axis length, shortest axis length, and the mean, standard deviation, and variance of the intensity values inside the object. Buttons, options, and information fields: Draw Area This button lets you manually draw an area on the image using the mouse. Click the primary mouse button to anchor your starting point, then move the mouse to draw the area boundary. If you hold the primary mouse button as you draw you will be able to draw “freehand” any shape area. If you click and release, you will draw straight lines which form the area boundary. In this case, each click creates a vertex. In either case, click the secondary mouse button to return to the dialog and collect data. Multiple Mode This allows you to create objects automatically based on a threshold setting and the present sampling parameters. Set Threshold Opens the Threshold control box to manually set a threshold. Set the Threshold in a way, that features that shall be created as areas are coloured completely and the background is not. Sometimes it is useful to apply a filter (average, median)before creating areas. Use Undo to remove the filter for measuring. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 34 Geometric Area This opens another dialog that lets you specify precise dimensions and positions for rectangular and circular areas from which to extract measurements. Edit Area This allows you to edit the area presently shown on the image. Draw Last Area This redisplays the last area(s) created. Move Object This allows you to move the area object you’ve drawn to a new location on the screen. Clear This clears all objects from the screen image and removes all data from the data fields. Save Data This button writes the existing data displayed in the dialog’s data fields to the selected data file. If data for the current area has already been saved the button is disabled (grey) until new data is extracted. This precludes your saving a duplicate set of data and invalidating your work. If no file is selected the button is disabled and no data can be saved. Data Information Fields These fields display data for the first area found. Use the scroll bars at the bottom of the results box to view data for other areas if you’re working in multiple mode. Number of Areas Displays the total number of areas found within the region of interest. Highlighted Area In the case of multiple areas, indicates to which area the data displayed pertains. Area The total area of the object in calibration units. Perimeter The total length of the perimeter of the area object in calibration units. X Position: The x position of the area centroid. Y Position: The y position of the area centroid. Circularity: The ratio of the area perimeter length squared divided by the area’s area. The minimum value of four pi is achieved only by true circles. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 35 Longest Axis: The length of the longest axis expressed in calibrated units. Width The greatest width of the area measured perpendicularly from the longest axis and expressed in calibrated units. Mean Grey: The mean grey value of all pixels enclosed by the area object. Std. Dev. Grey: The standard deviation among grey values within the area object. Var. of Grey: The total degree of variance between the highest and lowest grey values observed within the area object. File Menu Open Data File This command lets you create and open an ASCII file in which your data sets will be stored. After you select a filename a special dialog box opens to allow you to supply up to three text strings as data file headers. Exit This command closes the Area Morphometry dialog box. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 36 Opening a data file: 1. Choose Area Morphometry from the Data menu. Select the Data File command from the dialog’s File menu. The Open File dialog box appears. 2. Type the name of the new data file in the File name box. The data file name must adhere to standard DOS file naming conventions. By default the micron software assigns the extension .TXT to data files. Select an existing file if you want to append data to the file. 3. Click Open. The Area Morphometry dialog box title now includes your data file name and the Header Information dialog box appears so you can attach a header to the data file. 4. Enter text that describes your work session. Note that you can change the titles of each field in the data heading as well as the specimen information associated with each field. After you enter text in an edit box you must tab out of the box for the information to be accepted. 5. Click OK. The micron software adds the header information to the data file and returns you to the Area Morphometry dialog box. 6. Click the Save Data button. Each time you click the Save Data button, the micron software saves the current data set to the selected data file. Collecting area data: 1. Select Area Morphometry from the Data menu. 2. Click the Draw Area button. (If you want to create multiple areas, first click the Multiple Mode check box, then Create Areas.) When you click on Draw Area the pointer cursor is labelled A to indicate you are in area creation mode. 3. Move the mouse to move the cursor to where you want the area boundary to start. 4. Click the primary mouse button to anchor the beginning point of the area boundary. An anchor point appears on the video monitor. 5. Create an area boundary using one of the following three methods: a. To connect rubber lines around the area, move the mouse around the area and then click the primary mouse button to anchor each vertex of the area boundary. Continue placing vertices to "connect the dots" around the area. When the area is marked, click the secondary mouse button to exit the area marking mode. b. To sketch the area boundary, click and hold the primary mouse button, then drag the mouse around the area. When you are finished, click the secondary mouse button to exit the line marking mode. While you are sketching, you can erase a line by holding down the Shift key while holding down the mouse button, then backing across the line you created. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 37 c. To automatically trace an area on a screen image, manually trace a small segment of the boundary. The segment indicates the edge and the direction you want to trace. The micron software will use this segment as a hint to begin automatically tracing a line along the specified edge. The image must contain a luminance gradient of some kind for the micron software to automatically track along an edge. If you were able to identify and trace a line segment along an edge, hold the Control key, and click the primary mouse button to start the automatic tracing. You can increase the tracking speed by holding down the Control key. To stop the tracking cursor, press any keyboard key. To backup (erase), press the Shift key. To restart the cursor or to give the cursor a directional hint while it is moving, click the primary mouse button. When the boundary is completed, click the secondary mouse button to exit the auto-trace mode. Click the secondary mouse button again to exit area marking mode. When you exit the area marking mode, the cursor on the video monitor will disappear and the cursor on your computer monitor will change to a Pointer icon. You are returned to the Area Morphometry dialog, which displays the data extracted from the area(s). Click the Save Data button to save the data to the specified data file. 4.5 Peak Find This Analyze Function is determined to identify and quantify peaks of diffraction patterns. You may open it from the analyze menu or the toolbar. First use Define Center to specify the center peak. You will get a selection Cursor to draw a Box that broadly specifies the range of the peak. The maximum magnitude and the minimum will be given as Magnitude and Background. The Find Peak Function works similar, but also gives the distance to the center peak in calibrated units (standard is mm on the plate) and the angle in degrees. A circular averaged peak profile is obtained by Peak Profile. A view-box is opened with the data, clicking into the box allows measurement in the box. If the box is enlarged it also shows the axis dimensions. The length of the line is specified by the box width. Export Data will write the data to a text file, that can be imported to Excel. At the first time a filename must be selected, then the data is appended to that file every time Export Data will be pressed. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 38 4.6 3-Dimensional Plot The command 3D-Plot on the Analyse menu opens the 3D Luminance dialog box which displays a three dimensional illustration of the surface of a selected region of interest. The three-dimensional representation of the ROI surface is based on the luminance values contained in the ROI. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 39 Buttons, options, and information fields: Select ROI This button allows you to manually select the desired region of interest. View This button begins the process of displaying the 3D ROI. Zero based Selecting this option sets the micron software to take the luminance value based on zero as an absolute pixel luminance value. Deselecting this option sets the micron software to take the luminance value based on the lowest pixel value in the ROI. Rotation, XY The values in this box determine the degrees of the rotation angle for the luminance display. Sampling, XY The values in this box determine the number of luminance values sampled and displayed. The default setting, 32, should be adequate. To increase detail, type in a higher integer value. The best range for sampling values is between 32 and 64. (The maximum value is 64.) Viewpoint This value represents the viewpoint from above the X, Y plane. The default setting for the value in this box is 2000.0. You may adjust this value to any number of your choosing for best display results. Using the 3D Luminance Surface dialog box: 1. Adjust the Rotation, Sampling, and Viewpoint text to reflect how you want the luminance values to appear in the display window. 2. Click on Select ROI. Draw a ROI on the image. 3. Click on View. The three-dimensional luminance surface profile appears in the display window. 4. To close 3D Luminance Surface, double-click the Control-menu box in the top left corner of the dialog box. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 40 4.7 2D-FFT The command 2D-FFT on the Analyse menu opens the 2DFFT dialog box. You can use the 2DFFT dialog box to perform forward and inverse discrete Fast Fourier Transforms (FFTs) on images. The forward FFT converts an image from the spatial domain into the frequency domain and displays the resulting spectrum. The spectrum may be edited either by removing unwanted frequencies or by emphasising certain frequencies. The inverse transform then converts the frequency spectrum back into the spatial domain. 2D-FFTs require a great deal of processing power and memory. All FFT computations are performed in single precision floating point math, and are retained for additional analysis after the computation completes. Buttons, options, and information fields: View menu This menu offers you three options for viewing the image. Original Redisplays the original, unmodified image. FFT Redisplays the FFT of the original image. Inverse Redisplays the results of the inverse transform. Retouching Displays the Retouch dialog. Help Click on the Help menu to display on-line help specific to the 2D FFT utility. ROI Specify the dimensions of the ROI you want to use in the Rows and Columns list boxes. You can select from 8, 16, 32, 64, 128, 256, and 512.You can also specify absolute pixel co-ordinates by entering values in the Left and Top edit boxes. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 41 Move This button moves the ROI, using the dimensions and pixel co-ordinates you selected. Forward FFT This button prompts the micron software to perform a forward FFT on the current ROI, then display the frequency spectrum. The DC frequency component is displayed in the center of the ROI. After the forward FFT completes, use the frequency editing tools to remove or emphasise ranges of frequencies prior to performing the Inverse FFT. Inverse FFT This button prompts the micron software to perform an inverse FFT on the current ROI, then display the resulting image. Output LUTs Linear This button sets the Output LUTs to a linear, positive ramp. Gamma This button sets the Output LUTs to a gamma curve, where the slope of the curve is controlled by the value in the associated edit box. This allows frequencies which have low magnitudes to be displayed. DC Value The value in this edit box reflects the DC (or zeroeth) frequency component displayed in the center of the ROI. When you perform a Forward FFT, this value will be the average luminance value for the entire ROI. You have the option of editing the value to change average luminance value for the entire ROI. Frequency Select Symmetric This button allows you to create a box centred about the DC component of the image. Frequencies either inside or outside the box can be subsequently edited. As the cursor moves, the X and Y values display the spatial frequency. Click the left mouse button on the box to complete the selection process. Range This button allows the user to create a box anywhere within the frequency range. Although a single box is displayed, editing will be automatically performed in the symmetric frequency range (selections in the upper right quadrant will also edit the lower left quadrant). Frequencies either inside or outside the box can be subsequently edited. As the cursor moves, the X and Y values display the spatial frequency. Click the left mouse button once to fix one corner of the box. Click the left mouse button a second time to complete the selection process. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 42 Select Last Range Click this button to revert to the last range you used. Frequency Edits Zero outside Zeroes all frequency components within the selected range. Zero inside Zeroes all frequency components outside the selected range. +1 dB inside Increases all frequency components inside the selected range by 1 dB. -1 dB inside Decreases all frequency components inside the selected range by 1 dB. Apply This button applies the Frequency Edit you selected to the area you specified in Frequency Select. Using the 2D FFT dialog box: 1. Use one of the ROI tools to specify the ROI. Click on Move to place the ROI with your mouse. Click on the primary mouse button to set the ROI in place. 2. Click on Forward FFT. The Status of the FFT is displayed at the bottom of the dialog box. 3. To view the frequencies of lower magnitude, click on Gamma ore use the Look-Up Table dialog box. 4. Select the frequency you are interested in editing using either the Symmetric or Range options. 5. Select the type of Frequency Edit you want. Click on Apply. The edited frequencies will be modified. 6. Click on Inverse FFT. The Status of the FFT is displayed at the bottom of the dialog box. 7. To close the 2D FFT dialog box, double-click on the Control-menu box in the top left corner. To minimise 2D FFT, click on the Minimise arrow in the top right corner. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 43 Chapter 5: Image Conversion Tools The image conversion tools operate on files, unlike all the other image operations, that work on loaded image frames. The output then is a new image file, that can be loaded to the viewer to display. 5.1 Merge The merge function combines two 16 bit images with different gain to a high dynamic image with 16 to 32 bit data depth. This can be used to create high dynamic images that have been scanned as two images. Invoke the function by selecting Merge from the Tools menu. Specify the two input files by entering the path or using the Browse button. High reefers to the image with higher gain, Low to the image with lower gain. Specify the output filename. The Input filenames may contain wildcard characters (* and ?). In this case all matching files will be computed. The output file names will be derived from the input filenames. The Gain factor (difference in Gain between the images) can either be entered or calculated internally when autorange is checked. The Data Depth of the output file can be specified. 32 bit is the most common format, 24 bit needs less disk space. The 16 bit format will divide the image by a (variable) factor to fit the dynamic range within the 16 bit range. This possibly looses some dynamic range, but uses the 16 bit format optimally. Start Merge starts the process. To expand the dynamic even beyond this range, it is possible to rescan the plate with lower gain setting (only low channel). Then three Images are created. When merging these images, first merge the two with lowest gain to a 16 bit output. Then merge the resulting to the high gain image to a 32 bit output. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 44 5.2 Binning Binning reduces the resolution of the image by creating macropixel. At a binning of two, four neighbouring pixel are averaged to one pixel, at a binning factor of three there are nine pixel averaged, and so on. The amount of data and noise goes down by binning. Invoke the function by selecting Binning from the Tools menu. Specify the input file by entering the path or using the Browse button. Specify the output filename and the Binning Factor. The Input filenames may contain wildcard characters (* and ?). In this case all matching files will be computed. The output file names will be derived from the input filenames. Start Binning starts the process. 5.3 Convert The convert function allows data export on a file basis. As a Standard there is a TIFF_16 File filter available, other filters can be created on request. Specify the input and output file by entering the path or using the Browse button. Specify the File Format Filter. The Input filenames may contain wildcard characters (* and ?). In this case all matching files will be computed. The output file names will be derived from the input filenames. Convert starts the process. The following example converts all images in the \\images\\ folder into the \\TIFF\\ folder: Input Data File: x:/images/* Output File: x:/TIFF/*.* Note: The convert function can also be invoked automatically. See the Options dialog box.650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 45 Chapter 6: Other Functions 6.1 Emergency Functions: This functions are used to maintain secure operation in fault conditions. 6.1.1 Abort Scan The function Abort Scan in the Extras menu allows the user to abort a running scan, if he decides, that the operation should be interrupted. This can be if after starting the scan you get aware of some wrong set-up, or after the first row of images scanned show some desirable changes in set-up of the scanner. After aborting the scan it is possible to rescan the plates. However be aware, that the scanning process erases the information partly, so already scanned plates will not give the same results as in the first scan. 6.1.2 Forced Unload The mechanical plate loading and unloading is a possible source of errors. Damaged or bent plates may cause errors in the loading mechanism. The instrument will detect the error condition and report it. Possible errors are: • Plate hangs on drum : The Plate sticks to the drum and does not clear off the clams when unloading. • Plate obstructing: The Plate is mispositioned, the clamp Positioner cannot go down because the plate is in the way. • Plate falls into the instrument If a plate jam occurs when loading or unloading plates, Forced Unload unloads the plates in a brute force mode and brings the scanner back to a defined state. Also refer to the troubleshooting chapter below for more information. Do not use, if plates are one-side loose. In this case remove plates manually 6.1.3 Retransmit While scanning, the scan data is stored to a SCSI hard disk within the scanner. When the scan is finished, the data will be transmitted to the computer, converted in image files and stored. If the computer crashes while scanning, the scan of the current row will be continued. => In this case, if the percentage display at the scanner still increments do not reboot the computer, until the percentage is 100% and you hear the drum stopped. => Then switching off computer and scanner and restart the system .The scanner will present a message that there was an interrupted scan. => The scanned data however is on the internal scanner disk and can be retrieved by the function Retransmit in the Extras menu. Start Retransmit before unloading the plates. It 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 46 is a good idea to edit the filenames so not to overwrite files that may already be transmitted. => There may also images be transmitted that have not yet be scanned and show image information from the last scan. => If the images have been retrieved and not all images have been scanned, repeat the scan. (also select other filenames). After completion sort out what images from what scan are ok and rename. 6.2 Transport The scanner has some mechanically sensitive parts, that have to be protected when transported, otherwise damage may occur. Before transporting the scanner execute Transport from the Extras menu and switch off the device after competition. The scanner will automatically protect the sensitive parts. At initialising when the scanner is started next time, the protection automatically will be removed. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 47 6.3 Options The Options dialog box from the Extras menu allows to set general device options. Hardware Available When checked the Software operates the scanner in normal way. If the scanner is not present uncheck this option to operate the software as a stand alone version. Auto Preview after Scan If checked, a preview of every scanned image will be presented to the screen after scanning. Thumb-Nail Zoom Gives the zoom factor of the preview image that is added to the data set. Default is 10. Auto Reduce Bit Depth If Combined Images are created with 32 or 24 Bit Data Depth but only 16 bit or less are used, the images will automatically be reduced to 16 bit and the file extension is changed to .IPL. This function is useful if High dynamic images are mixed with normal images and reduces the amount of data created for the normal images. The conversion time however is somewhat longer (after the scan). This Function is only active in combined mode. (24 or 32 bit) 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 48 Stamp Label from Reflected To identify plates a plate label can be affixed to the surface in the area, where the microscope image information usually is stamped in. When scanned with Signal and Reflected the surface of the plate is scanned in a second image. After the scan Stamp Label from Reflected takes the area from the surface scan and stamps it to the image file, so the plate is easily identified by its number. This function is only active when the reflected light is scanned. If stamping is used, the plate always has to be inserted into the scanner (and the microscope) in the same way, usually the label on top The position of the stamp Area is controlled by 4 parameters in the config.mac file: StampLabel_X controls the distance from the top side of the plate to the label, StampLabel_Y controls the distance from the right side of the plate to the label, viewed when inserting the plate into the scanner. StampLabel_dX and StampLabel_dY controll the size of the label. All values are in mm. Auto Delete Reflected When scanned with Signal and Reflected this option automatically deletes the file with the surface scan after stamping to reduce file space. Comm Channel Serial communication channel the scanner is connected to. Default is 1 Scanner Disk Path Path to the disk drive of the scanner that is connected to the SCSI Bus. Default is P:\\ Undo Steps Number of undo steps that are stored in memory. Setting this value to zero will disable Undo and speed up the system when manipulation big images. Default is 2. Auto Convert Checking this box will convert every scanned image using the selected convert filter. Be aware of the additional disk space and scanning time when using this function. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 49 6.4 Configuration Functions Configuration of the Software and the dialog box settings can be stored in named configuration files using the Save Configuration function in the File menu and can be loaded again with the Open Configuration function. This allows for multiple users or applications and to tailor the system. Configurations are automatically saved on exit to the file MICRON.CFN and loaded again at start-up. 6.5 Help System The interactive help system can be invoked with the F1 key or with Help/Index from the menu. If a dialog box is activated, context sensitive help will be given to that function. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 50 Chapter 7: Troubleshooting 7.1 Clearing Plate Jams The loading and unloading mechanism of the plates may cause plate jams, when the plates are bent or damaged. There are sensors to identify error conditions to warn the operator. Resolving the Jam 1. First make sure that there are no plates in the loading slots. Remove plates. 2. The next step is the software function Forced Unload (Extras Menu). This function provides means to unload the plates in a brute force mode. If needed, the user can interfere and manually lift sticking plates. Do not use, if plates are one-side loose. In this case use next step 3. If this does not work, try to resolve the jam by opening the service flap on the front side of the instrument (See Hardware Manual for details) If it is not possible to resolve the problem by the above, the service technician has to open the instrument. Most of the problems will be obvious while loading or unloading. The following messages may occur: Error conditions while loading: • No Plate loaded No Plate in Slot or plate not correctly positioned. Insert plate or press Abort to abort loading. • Plate not correctly positioned Please correct manually Plate is not correctly positioned in the Loading slot. Make sure to draw the plate to touch the back side. Press continue. • Plate obstructing, clear manually The Plate Positioner cannot go down, because there is something in the way, most probably a ill-placed plate. Check the plate positions and press continue. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 51 • Critical Error, Cannot Recover The loading mechanism is unable to resolve the problems. If this error occurs, while the plates still are in the loading slot, remove all plates from the loading slots if possible. If there are plates already loaded into the scanner, start the scan with the already loaded plates. If this error occurs, when the plates already were drawn into the scanner, it is necessary to open the scanner and clear the drum manually. To do this, see chapter “Manually clearing Plate Jams” below. • Plate not Gripped The loading mechanism is not able to grip the plate to the drum. Press continue and the mechanism will try again. (up to 3 times). • Cannot grip plate - Remove Plate manually After several tries it was not possible to grip the plate. Remove the plate manually. If Plates have already been loaded to the drum, start the scan for this plates. • Plate not gripped Plate will be rejected The loading mechanism was not able to grip the plate (second clamp) to the drum. Press continue and the plate will be ejected to the loading slot. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 52 Error conditions while unloading: • Plate not removed The Plates were not removed from the loading slot. Remove the plates and press continue. • Plate hangs on drum – Clear manually The plate hangs on the drum when the clamp opens. Try to remove the hanging plates by inserting a screwdriver into the loading slot and lifting the plates to clear off the clamps. Be careful not to damage plates or clamps. Press Continue to try again. • Plate hangs on drum, Cannot recover The loading mechanism was not able to unload the plates. If this error occurs it is necessary to open the scanner and clear the drum manually. To do this, see chapter “Manually clearing Plate Jams” below. • Plate obstructing, clear manually " The Plate Positioner cannot go down, because there is something in the way, most probably a ill-placed plate. Check the plate positions and press continue. • Critical Error, Cannot Recover The loading mechanism was not able to unload the plates. If this error occurs it is necessary to open the scanner and clear the drum manually. To do this, see chapter “Manually clearing Plate Jams” below. Manually clearing Plate Jams If the loading mechanism is not able to resolve the problems, a plate jam has to be cleared manually. 1. First of all remove all plates from the loading slots if possible. 2. Switch off Scanner and Computer and disconnect the scanner from mains. 3. Open the service flap at the front side. To do this, remove the casing in front (snap in), open 4 thumbscrews and remove cover. A flashlight may be useful for this work. 4. When accessing the scanner drum from the front service opening, be careful not to bent the clamps of the drum loading mechanism. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 53 5. Manually turn the drum (move front side down for correct direction) to possibly eject loose plates. If plates are unloaded to the loading slots, carefully remove them from the drum. Make sure to turn the drum one full turn to see that there are no plates on the drum, that are not gripped on both sides. If there are such plates, carefully remove them from the drum. 6. When the drum only contains plates that are correctly positioned close the service opening for your security and restart the system. 7. When the micron system and software is running, select Forced Unload from the Extras menu. A forced unloading mechanism is invoked, that removes all plates from the drum. As the software in this state does not know about the loaded plates, (depending on error condition, some may have been removed), the routine goes trough all 5 drum positions to unload. 8. In every step, the user is prompted to make sure, that all plates are clear off the open clamps. To do this, look to the loading slot (a flashlight may be useful) and see that the plate is above the clamps. If not, carefully insert a screwdriver to the loading slot and lift the plates above the clamps. 9. The user will be prompted to remove the plate. If the plate hangs, press the No-Button and the clamp will open again. Now the plate can be removed. If the plate hangs in the clamps and will not clear, put it for a centimetre into the slot and press ok. Remove it as soon as the clamps closed. 10. When the Process is completed, normal operation may continue. More hints and Problem Solving • If the drum cannot be moved, there may be a clamp open and the clamp mechanism may obstructs the drum. In this case start the Extras/ Forced Unload function as described above. This shall solve the problem. • When the Software is started after an error condition, it automatically tries to complete the interrupted load or unload cycle. Simply press continue until the problem is solved or an unrecoverable error is detected. If you switched off the system to avoid a fatal error condition, this may be unwanted, because the user directly wants to manually unload the plates as described above. In this case rename the file MICRON.CFN to an other name. When starting up the Software then it will not go into this completion routine. Rename the file back later after the program exited. 650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 54 7.2 Configuration Problems Understanding the computer configuration The Scanner is connected to the computer by two links: A serial RS232 link (COM1) and a SCSI connection. The serial link is for scanner control, the SCSI for data transfer. At start-up the serial link is opened. If this is not possible, a message is displayed: No Communication with Scanner. Also at start-up, files on the scanner disk are accessed. If this is not possible, a message: “Scanner Disk not found - Check Path and Restart Computer" is issued. The SCSI link is a special configuration. While normal operation, the scanner host adapter controls the disk and the scanner controller. Therefore both devices appear in the Windows Device Manager. When scanning, the scanner controller takes over SCSI control and writes the scanner data directly to disk. In this time the computer is prohibited to access the scanner disk (will result in disrupting the scan) After the scan the computer again accesses the disk and gathers the scan data. The scanner disk also contains files that contain calibration and instrument status information. To avoid data loss, these files are mirrored to the micron/config directory whenever the program terminates normally, where they can be taken to restore corrupted files. As the scanner writes data to the disk in the background, no disk caching is allowed. By configuring the disk as a removable media, caching will be disabled. Resolving serial Problems If the serial connection is not found, first check cables. If the computer has multiple serial devices, make sure to use the right one. The used channel (default is 1) can be entered in the options box in the micron software. In some cases, a conflict with installed devices will prevent correct operation and the software crashes before you get the change to set the correct channel. The first thing is to install the scanner on COM1. If this is not possible, change the default serial cannel in the config.mac file (text editor). Then remove the file micron.cfn, then start the software. An other reason may be that an other running program blocks the channel. Make sure that no other software is running. You also may try to remove the serial channels from the Windows Device Manager and let them detect again. Resolving SCSI Problems In case of computer crashes it can happen that the configuration of the computer gets mixed up, so the scanner disk is not detected any more. (there is the message at start-up: Scanner hard disk not found). In most cases a complete shutdown and switch off scanner and computer helps, so the problem is gone after restart. It is also a good idea to check cables and connectors. If this does not help, try with the Windows Explorer if you can access disk P (or the drive that represents the scanner disk in your system). If this works, close the Explorer and test if in Extras/Options the correct path to the scanner disk is specified. 650-68-08-01.Operating_Manual_MICRON-SW.pdfSoftware Manual Imaging Plate Scanner micron Page 55 If there is no access to the scanner disk check the device manager for correct operation of the SCSI host adapter. In case remove the adapter and reboot (see also the Hardware-Manual chapter C.5. for Computer configuration). It may also be a good idea to use some SCSI diagnostic tool to view the computer configuration (for example the Adaptec SCSI-Explorer) 7.3 Retrieving data after computer crash While scanning, the scan data is stored to a SCSI hard disk within the scanner. When the scan is finished, the data will be transmitted to the computer, converted in image files an stored. If the computer crashes while scanning, the scan of the current row will be continued. => In this case, if the percentage display at the scanner still increments do not reboot the computer, until the percentage is 100% and you hear the drum stopped. => Then switching off computer and scanner and restart the system .The scanner will present a message that there was an interrupted scan. => The scanned data however is on the internal scanner disk and can be retrieved by the function Retransmit in the Extras menu. Start Retransmit before unloading the plates. It is a good idea to edit the filenames so not to overwrite files that may already be transmitted. => There may also images be transmitted that have not yet be scanned and show image information from the last scan. => If the images have been retrieved and not all images have been scanned, repeat the scan. (also select other filenames). => After competition sort out what images from what scan are ok and rename.650-68-08-01.Operating_Manual_MICRON-SW.pdfPage 56 Appendix A: Image Data Format The data created by micron software have the extensions: .IPL Image data first channel, 16 bit per pixel .IPH Image data second channel, 16 bit per pixel .IPR Reflected light data, 16 bit per pixel .IPC Image data, both channels combined, 16,24 or 36 Bit pro Pixel The micron data format consists of three parts: 1. A file header with information about the data. 2. The data ordered line by line in a continuous array of data. The organisation of the data is described in the header. The last pixel repeats the maximum value of the image. 3. A zoomed copy of the image, used for preview. The header is a minimum of 2048 bytes long and consists of ASCII tags and data, separated by CR/LF characters. Every tag entry consists of a key word followed by a blank and an equal sign, followed by the value in ASCII characters. The Tags come in any order. There are some tags that have to be there and some additional ones that are voluntary. Tags that must be present: CREATED = Time and Date String HEADER = Length of header, typically 2048 YPIXEL = Number of pixel in Y typ. 3000..3500 pixel XPIXEL = Number of pixel in X typ. 3000..3200 pixel BYTE PER PIXEL = Data depth in byte, 2,3 or 4 XRESOLUTION = Resolution X in µm YRESOLUTION = Resolution Y in µm, exact value in parentheses THUMB-NAIL-ZOOM = Zoom factor for preview, typ. 10 Additional tags: MAGNIFICATION: gives the magnification in the microscope. OFFSET: gives the value that represents zero dose OFFSET CORRECTION: Indicator whether Offset Correction was used GAIN: Gain setting of the instrument LASER: Laser setting of the instrument PARAMS: Settings file used for this scan FORMAT: Format file used for this scan CHANNEL: Data channel used for this file MICRON-MARK Position, length and text of micron mark overlay COMMENT: is always the last tag and has no equal sign after. It is followed by the comment string that may contain CR/LF characters and is terminated by a null character. 650-68-08-01.Operating_Manual_MICRON-SW.pdf Chapter 1: Scanning Plates3 1.1Loading Plates to the scanner 1.2The scanning Dialog box The Filename specifies a wildcard Filename, that will be used for all loaded plates, and added a running number (_1, _2 etc). The filenames can be edited later. Details on Scanning Parameters Save As: The Settings can be stored to a file to recall it later. Checking read-only will set the file to read-only, so the setting cannot be changed afterwards. 1.4 Details on Scanning Formats 1.5 Import Microscope Data (Annotate) Data Format of Text File 1.6Importing Data from Data Files 1.7Unloading Plates Chapter 2: Viewing Images 2.1Opening Images and Previews Use the Open Preview and Open Image Button to open a preview or full image of the selected file 2.2Look Up Tables 2.3Zooming Images 2.4Saving and Exporting Images 2.5Printing Toolbar Chapter 3: Images Manipulation 3.1Image Filters Edit menu 3.2Rotating Images Center of rotation Aligning Images Image Arithmetic How to combine images Using the arithmetic operations 3.5Region of Interest (ROI) 3.6Undo Chapter 4: Image Analysis 4.1Calibrating Images 4.2 Point Functions Buttons, options, and information fields: File Menu Setting up a data file 4.3 Line Plot Buttons, options, and information fields: Data Information Fields 4.4Area Functions Data Information Fields File Menu 4.5Peak Find 4.63-Dimensional Plot 4.72D-FFT Chapter 5: Image Conversion Tools Merge Binning Convert Emergency Functions: 6.1.1