Thermo ImageQuest Version 1.0.1

Transcription

1 Thermo ImageQuest Version User Guide XCALI Revision B May 2009

2 2009 Thermo Fisher Scientific Inc. All rights reserved. Xcalibur is a registered trademark of Thermo Fisher Scientific Inc. in the United States. Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries. LTQ XL, LTQ-Orbitrap, and ImageQuest are trademarks of Thermo Fisher Scientific. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Samples courtesy of Roswell Park Cancer Institute, Buffalo, NY. Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc. The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser. Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or errorfree and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any use of this document, even if the information in the document is followed properly. This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents. Release history: ImageQuest For Research Use Only. Not for use in diagnostic procedures.

3 C Contents Preface v Chapter 1 Overview of ImageQuest The ImageQuest Main Window Using ImageQuest Views Optical View Ion Chromatogram View Two Dimension View Three Dimension View Spectrum View Averaging Spectra Data Set Tree View Sky View Using the Cursor Chapter 2 Using ImageQuest to Process Images Changing the Spectra View Range Changing an Optical Image View Changing the 2D Setup View D View Impact on Other Views Changing the 3D View Changing a Combo File View Chapter 3 Working with MALDI Files and Data Sets Working with Files Viewing Data from a Raw File Opening a Raw File Working with Data Sets Creating a New Data Set Editing a Data Set Overlaying Data Sets Animating Data Sets Printing Reports Saving Data Opening a Previously Extracted Data File Thermo Scientific ImageQuest User Guide iii

4 Contents Appendix A ImageQuest Reference Buttons Toolbar Buttons File Buttons View Buttons Spectrum View buttons Spectrum Rescaling Buttons Data Set Buttons Menus ImageQuest Tabs Scale Tab D View Tab D View Tab Dialog Boxes Create New Data Set Dialog Box ImageQuest Options Spectrum Display Options Mass Tolerance Information View Data Set View Raw File View Position File View Tune Method View Instrument Method View Scan Header View Spectrum List View Status Log View Error Log View iv ImageQuest User Guide Thermo Scientific

5 P Preface This guide describes how to use ImageQuest to process and analyze tissue imaging data from a MALDI instrument on LTQ XL and LTQ-Orbitrap mass spectrometers. Contents Related Documentation Product Requirements Contacting Us To provide us with comments about this document, please click the link below. Thank you in advance for your help. Thermo Scientific ImageQuest User Guide v

6 Preface Related Documentation Thermo ImageQuest includes Help and these manuals as PDF files: Thermo ImageQuest Help Thermo ImageQuest User Guide To view product manuals Go to Start > All Programs > Thermo ImageQuest > ImageQuest Manual. After installation, the manual can be located at C:\Program Files\Thermo\ImageQuest\Manual\ImageQuest.pdf. To open Help From the Thermo ImageQuest window, choose Help > ImageQuest Help. If available for a specific window or dialog box, click Help or press F1 for information about setting parameters. For more information, including upcoming application notes, visit Product Requirements You can run ImageQuest on a computer connected to a Thermo Scientific instrument or on a separate workstation computer. To run ImageQuest, you must meet these minimum requirements: System PC Instrument (supported or required) Requirements 2 GHz processor with 2GB RAM CD-ROM drive Video card and monitor capable of 1280x1024 resolution (XGA) 75 GB or greater available on the C: drive NTFS format MALDI on LTQ XL MALDI on LTQ-Orbitrap Software Microsoft Windows XP Professional with Service Pack 2 Xcalibur or higher LTQ 2.5 vi ImageQuest User Guide Thermo Scientific

7 Preface Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need. To contact Technical Support Phone Fax Knowledge base Find software updates and utilities to download at mssupport.thermo.com. To contact Customer Service for ordering information Phone Fax Web site To copy manuals from the Internet Go to mssupport.thermo.com and click Customer Manuals in the left margin of the window. To suggest changes to documentation or to Help Fill out a reader survey online at Send an message to the Technical Publications Editor at techpubs-lcms@thermofisher.com. Thermo Scientific ImageQuest User Guide vii

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9 1 Overview of ImageQuest The Thermo ImageQuest software supports MALDI tissue imaging on LTQ XL and Orbitrap mass spectrometers, providing the ability to analyze, manipulate, and process tissue imaging data in a targeted analytical procedure that makes it easier to identify the intensity and location of an analyte. ImageQuest does the following: Displays MALDI images in two- and three-dimensional figures Displays the tissue optical image and supports image manipulation including overlap of images Provides ion chromatogram and MSn spectra visualizations of the 2D-image Saves extracted data Prints files Exports figures to the clipboard This chapter provides information about the ImageQuest main window, its views, functions, and features: Contents The ImageQuest Main Window Using ImageQuest Views Using the Cursor Thermo Scientific ImageQuest User Guide 1

10 1 Overview of ImageQuest The ImageQuest Main Window The ImageQuest Main Window The ImageQuest main window (see Figure 1) consists of the following: A title bar containing the sample name and the application name A menu bar A toolbar The selected view - Tool buttons provide access to specific views or view all options. Figure 1. For example, click ImageQuest main window to display the 2D view. Title bar Menu bar Main toolbar Data set area View pages Histogram Sky view Status bar 2 ImageQuest User Guide Thermo Scientific

11 1 Overview of ImageQuest Using ImageQuest Views Using ImageQuest Views The Main screen also displays the following views: Optical View Ion Chromatogram View Two Dimension View Three Dimension View Spectrum View Data Set Tree View Sky View Figure 2 displays the location of views on the main screen. Figure 2. ImageQuest views Two dimension view Optical view Three dimension view Ion chromatogram view Spectra view Thermo Scientific ImageQuest User Guide 3

12 1 Overview of ImageQuest Using ImageQuest Views Optical View In the optical image view, the software displays the scanned positions as blue dots. The information in this image is contained in the MALDI position file prior to data acquisition. The software can display the scan dots on top of the optical image depending on the density of the array. Figure 3. Optical View Ion Chromatogram View The Ion chromatogram view displays intensity in relation to the scan number. Each scan is a specific location (pixel) on the tissue. Use this view to see where the most intense scans are. You can select a point in the 1D view to display a spectrum at that point (see Figure 4). The software also displays the correspondent points in the 2D and optical images. You can also select a range to average all the spectra at that range. See Averaging Spectra on page 8 for more information. The software also displays the correspondent rectangle over the selected range in 2D and optical images. Figure 4. One Dimensional Image 4 ImageQuest User Guide Thermo Scientific

13 1 Overview of ImageQuest Using ImageQuest Views Two Dimension View Two-dimensional images displays intensity in relation to X and Y axes. In the Rainbow scale, the intensities are color coded to identify peak intensity: blue and green colors represent a lower intensity; red and orange colors represent a higher intensity. To change the 2D view, see Changing the 2D Setup View on page 14. Use the two-dimensional image to do these tasks: Select any position in the 2D graph and display a spectrum at that point Select an area by dragging the cursor over the area to average all the spectra in the area Zoom in a selected region, and pan the zoomed region around by dragging the mouse Measure a distance between two points Get intensity and X and Y coordinates by placing the cursor on the 2D image at a specific location Figure 5. Two-dimensional Image Thermo Scientific ImageQuest User Guide 5

14 1 Overview of ImageQuest Using ImageQuest Views Three Dimension View Three-dimensional images display intensity in relation to the X and Y positions. The intensities are color coded and presented on the Z-axis. Use this view to see where peaks exist in a multi-dimensional field or rotate the view to find peaks for further study. Control display parameters of the three-dimensional object by using the 3D setting tab. See Changing the 3D View on page 18. Use the three-dimensional image to rotate the 3D object by quickly dragging and releasing the mouse. The image rotates in the direction of the mouse movement. Double-click the object to stop the auto-rotation mode. To make it easier to locate relevant peaks when examining a new image, rotate the three dimension view to match the two dimension view. Figure 6. Three-dimensional Image 6 ImageQuest User Guide Thermo Scientific

15 1 Overview of ImageQuest Using ImageQuest Views Spectrum View The spectrum view displays mass spectra, making it easy to see the location of peaks. If you select an area to average spectra, the view displays in red; if you select a single point, the view displays in black. You can zoom into or out of an area using the toolbar buttons (see Changing the Spectra View Range on page 12). Figure 7. Spectrum View Thermo Scientific ImageQuest User Guide 7

16 1 Overview of ImageQuest Using ImageQuest Views Averaging Spectra To find areas of higher general intensity, average spectra within a selected area. To average a spectra 1. Click on the toolbar. 2. In the two dimension view, select the area to average by dragging the cursor diagonally. ImageQuest outlines the area in the two dimension view. The Spectrum view shows the averages in red. The Ion Chromatogram view shows the averages at any one point. 8 ImageQuest User Guide Thermo Scientific

17 1 Overview of ImageQuest Using ImageQuest Views Data Set Tree View The Data Set view always displays information about the current data sets in the upper left corner of the main screen. Data sets are files that store specific information, including the changes you make to raw files. The current data set is highlighted. Use this view to see which views are available and to determine the composition of combo data sets. It displays the following information for all current data sets (TIC, mass range, base peak, mass range / TIC) and displays different ions for different data set types and plot types. Use the toolbar below the view to create a new data set, edit duplicate sets, or delete a data set. You can only edit single data sets. Figure 8. Data Set Tree view Data set toolbar Sky View ImageQuest displays a two-dimensional image of the current data set with the colors inverted in the lower left corner of the application. This view helps you to keep track of the high level view of the image. When you zoom into a specific area, this view always displays the entire image. Inside the Sky View image, the zoom area appears as a square that you can use to orient yourself in the zoomed image. To return the two-dimensional view to a full view, double-click the sky view. Figure 9. Sky view Thermo Scientific ImageQuest User Guide 9

18 1 Overview of ImageQuest Using the Cursor Using the Cursor Within the Ion Chromatogram and Spectrum previews, use the cursor in three ways to scale the display: Click in the cell to select a point. Drag a line parallel to any axis to select a range. Drag a line in any diagonal direction to select an area. Table 1 describes the effects. Table 1. Effect of cursor action in an active cell Cursor Action Drag parallel to X-axis Drag parallel to Y-axis Drag diagonally Double-click Effect Rescale a graph showing selected X range only, retain the same Y range. Rescale a graph showing selected Y range only, retain the same X range. Rescale a graph showing both the selected X and Y ranges. Close cursor action and return to the original view. 10 ImageQuest User Guide Thermo Scientific

19 2 Using ImageQuest to Process Images This chapter describes how to modify specific image views in the ImageQuest main window in order to more carefully target your analysis towards the location and intensity of the analyte. Contents Changing the Spectra View Range Changing an Optical Image View Changing the 2D Setup View Changing the 3D View Changing a Combo File View Thermo Scientific ImageQuest User Guide 11

20 2 Using ImageQuest to Process Images Changing the Spectra View Range Changing the Spectra View Range You can control the range of the X and Y display axes using either menu commands or toolbar button commands. Use the following buttons to control the range of the display for spectra and chromatograms: Zoom in Y from the current baseline to show more detail. Zoom out Y to show more data. Normalize Y intensity scale. Zoom in X at the center to show more detail. Zoom out X at the center to show more data. Display all data on X-axis or all text in report. Reset scaling to full scale for both X- and Y-axes. Display Y-axis data in normalized mode. Display Y-axis in creep mode. Display Y-axis in fixed mode. 12 ImageQuest User Guide Thermo Scientific

21 2 Using ImageQuest to Process Images Changing an Optical Image View Changing an Optical Image View Use this image to compare the optical image with the mass spectrometry data in the two- and three-dimensional views. The comparison helps you to more quickly understand the mass spectrometry data. To show the scan range 1. View the optical image. If no optical image appears, move the margins for the 2D and 3D images apart. 2. Click to show scan dots. Click again to remove the dots. A screen of dots covers the image area. Figure 10. Image view with scan dots Thermo Scientific ImageQuest User Guide 13

22 2 Using ImageQuest to Process Images Changing the 2D Setup View Changing the 2D Setup View Use the 2D tab to change color schemes, the number of 2D contours, and other options in the 2D view as necessary to focus on a region of interest within the tissue. All data sets can access the 2D setup tab except the frame data sets of a series data set. To change the color Click another color from the Color Scheme list. Unless you select the Invert Color Scheme check box, red indicates the highest intensity in the Rainbow option. Select Monochrome using two different colors to enhance image contrast in overlay images. Select Gray Scale to view features that are closer to the look of the features on the optical image. You can also select a color that displays the data better or one that represents a personal visual preference. To change the text color 1. Select a text color by clicking the Text Color browse button. 2. When the color options appear, select a color. To change the style from the Style list Select one of the following options: Image Contour Image and Contour View the original image. View the image with contours outlined. The Number of Contours option is only active when you select Contour or Image and Contour. Increase the value to narrow the contours. View the original image with contours outlined. 14 ImageQuest User Guide Thermo Scientific

23 2 Using ImageQuest to Process Images Changing the 2D Setup View To set the smoothing level from the Smoothing list Select one of the following options: None Linear Spline Apply no smoothing. Apply linear smoothing, creating clearer images. Improve the Image and Contour views. To invert colors on the 2D and the sky view Select the Invert Color Scheme check box. Note The Link Combo Color Schemes option and the opacity sliders are only active in a combo data set. To measure the distance between two points 1. Click on the toolbar. 2. Click on the first point in the 2D view and continue to hold the mouse down. 3. Drag the mouse to the second point and release the mouse. A line links the two points, and ImageQuest displays the coordinates and the distance between the two points in microns on the 2D view, similar to the following: Figure 11. Measured distance between two points Thermo Scientific ImageQuest User Guide 15

24 2 Using ImageQuest to Process Images Changing the 2D Setup View To zoom in on a 2D view 1. Click on the toolbar and move the cursor to the 2D view. 2. Click and drag the mouse over an area that you want to enlarge. 3. Click the mouse in the selected area. The software displays an enlarged image. Figure 12. Zoomed area of an image To review the location of the zoomed area, see the sky view. The area is displayed in reversed colors in a box. Figure 13. Sky view of a zoomed image 16 ImageQuest User Guide Thermo Scientific

25 2 Using ImageQuest to Process Images Changing the 2D Setup View 2D View Impact on Other Views If you click the position cursor in the two-dimensional view, the software updates the view in these ways: In the optical view, the mark also moves to the clicked position. The 1D image displays a red line on the same spectrum. The status bar displays the spectrum intensity at this point. A line in the histogram in the data set view marks the correct intensity in the histogram: If you click an item in the data set tree, it displays the graphs of the data set. Click toolbar buttons to move the cross position to left, right, top, or bottom. You can use the 2D view to average spectra. See Averaging Spectra on page 8. Thermo Scientific ImageQuest User Guide 17

26 2 Using ImageQuest to Process Images Changing the 3D View Changing the 3D View Use the 3D tab to change color schemes, the number of 3D contours and other options in the 3D view. All data sets can access the 3D setup tab except the frame data sets of a series data set. To change the color from the Color Scheme list Select another color from the Color Scheme list. Select Monochrome color scheme using two different colors to enhance image contrast in overlay images. Select Gray Scale to view an image that is closer to the optical image. You can also select a color that displays the data better or one that represents a personal visual preference. To change the style from the Style list Select one of the following options: Solid Surface Wire Frame View the image as a solid object. View the image as a wire frame. Use this view to see spikes from all angles. 18 ImageQuest User Guide Thermo Scientific

27 2 Using ImageQuest to Process Images Changing the 3D View To change the contours from the Contour list Select one of the following options: None XZ Contour YZ Contour XY Contour Apply no contouring to the image. Apply contours only to the XZ-axis.The Number of Contours option is only active when you select a contour option. Increase the value to have finer contours. Apply contours only to the YZ-axis. Apply contours only to the XY-axis. To invert colors on the 3D Select the Invert Color Scheme check box. To rotate the 3D view Click and turn the mouse on the 3D object to rotate it in any direction. To auto-rotate the 3D view 1. Click the cursor on the view. 2. Turn the cursor using the mouse and release the mouse. 3. Click the 3D view to stop the auto rotation. To enlarge or minimize the 3D view Right-click the 3D view and drag the cursor up to enlarge or drag it down to minimize the view. Thermo Scientific ImageQuest User Guide 19

28 2 Using ImageQuest to Process Images Changing the 3D View To show only the areas of maximum intensity 1. Move the color slides to exclude colors. 2. Select the Clip Data Using Color Range check box from the 3D page to highlight areas of maximum intensity. 3. Move the slide to display the red/yellow colors. ImageQuest does not display colors outside the range of the sliders. You can change the color range within the plot range. To show the outline box Select the Show Out Line Box check box from the 3D page to view a three-dimensional box around the object. This outline remains as you rotate the view. 20 ImageQuest User Guide Thermo Scientific

29 2 Using ImageQuest to Process Images Changing a Combo File View Changing a Combo File View Use the Combo File function to compare views. To create a combo file, click the New data set button and select the combo file option. (See Creating a New Data Set on page 29 for more information.) You can compare the optical image and a 2D view. You can also compare 2D images of several analytes to see the geographical distribution or the quantity of the analytes in the different images. To assist in viewing combined file views, ImageQuest provides two blend mode options: Alpha RGB You can combine an alpha channel with other layers in an image to clearly display translucency. This alpha channel adds an additional eight bits to each pixel in a 32-bit graphics system to represent 256 levels of translucency. Black represents an opaque view and white represents a fully transparent view, while various gray levels represent levels of translucency. An RGB image has three channels: one for red, one for green, and one for blue. Averaging the three RGB channels forms RBG blended image. The averaging is weighted by the percent of opacity of each RGB images. Thermo Scientific ImageQuest User Guide 21

30 2 Using ImageQuest to Process Images Changing a Combo File View The 2D page changes to display combo file options. Figure 14. Combo file options Select the Link Combo Color Schemes check box on the 2D page to make the combined data sets have the same color scheme set or to clear the Link Combo Color Schemes check box to use selected different color schemes for the various data sets. When the data set color schemes are different, you can only select single color schemes (red, green, blue). To invert an image stack, use the alpha blend mode to bring the bottom image to the top. To apply a blend mode On the 2D tab, select either Alpha or RGB from the Blend Mode list. To change the level of opacity in a combo file 1. To switch from one overlay view to the other, adjust the Alpha Blend Percent or either of the two RGB opacity slides. 2. Move the slides to view either layer at full strength or to view a blend of layers so that the overlap displays the information you want. When viewing overlapped images, the images are more precise in the middle of the slide. 22 ImageQuest User Guide Thermo Scientific

31 2 Using ImageQuest to Process Images Changing a Combo File View The following figures show changes to the original images after applying specific blend modes. Figure 15. Two original images (2D image on the left, image on the right) Figure 16. Images after applying blend options (Colored image on the left, grayscale image on the right) Thermo Scientific ImageQuest User Guide 23

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33 3 Working with MALDI Files and Data Sets This chapter describes how to manage files and data sets to analyze raw file material to find where the analyte is in the tissue sample and how much of the analyte exists. It has the following sections: Contents Working with Files Working with Data Sets Opening a Previously Extracted Data File Working with Files ImageQuest uses files to retrieve and store information. This section has the following topics: Viewing Data from a Raw File Opening a Raw File The following file types are used in ImageQuest. Raw Files Position Files MALDI Data Files You cannot change or save raw files. ImageQuest uses a raw file to extract data only and saves extracted data to a.maldidata file. A position file contains information about tissue location including the optical image of the tissue. ImageQuest uses the optical image of the file to create the optical data set, which can be overlaid with other images of other data sets. See Overlaying Data Sets on page 32 for more information. ImageQuest saves all data set information into a.maldidata file, including the extracted data from the source raw file so that the data processing for a session is not lost and you can correlate mass spectrometry data to a visual image of the tissue. When you open the file, ImageQuest restores the settings to the last time you saved the data. Thermo Scientific ImageQuest User Guide 25

34 3 Working with MALDI Files and Data Sets Working with Files Viewing Data from a Raw File Opening a Raw File Using a suitable raw file that contains MS data, use the various views to search for the location of the analyte of interest. Click to view more information about the acquisition process of the raw file. See Information View on page 50 for more details. To open a raw file (.raw file extension) 1. Click on the toolbar. 2. Select a raw file in the Open Raw File dialog box and click Open. ImageQuest processes the information from the raw file based on changes you make to the views. You can save this processed information as a.maldi data file. ImageQuest saves this file to the same folder as the.raw file unless you specify a different location. Open the file to restore the settings when saved. Saving this file does not change the raw file. If you have created a color image using a scanner or a black and white image using the camera in the LTQ instrument, you can open it at this time. Create the scanner image as a.bmp file in 24-bit color and 600 ppi (pixels per inch). For best results, create the color image before spraying the matrix over the slide. To create a color image, refer to the getting started book that came with your MALDI hardware. Note You can only open files with data collected using the Xcalibur or TunePlus software that supports your version of ImageQuest. Remember to update ImageQuest on stand-alone computers to match the version on your instrument computer. 26 ImageQuest User GuideThermo Scientific

35 3 Working with MALDI Files and Data Sets Working with Data Sets Working with Data Sets ImageQuest uses and stores data in files that are called data sets. Creating a New Data Set Editing a Data Set Overlaying Data Sets Animating Data Sets There are four main data set types: Single Data Set Optical Data Set Combo Data Set Series Data Set The single data set holds the information that the software extracts directly from the open raw file, such as mass range or total ion current (TIC). The optical data set is derived from the MALDI position file. The combo data set is derived from multiple single or optical data sets. You can display the combined data sets at the same time with the same or different color schemes. The series data set is composed from a series of data sets called frame data sets. The software displays a frame data set as a frame in an animation. The frame data set shares the same information with the series data set, except for the extracted mass range value. In a frame data set, the cursor moves across the 2D image displaying MS spectra at each position. This feature is useful for looking at unknown or unexpected peaks. Use the series data set to display still images that might reveal unknown metabolites or unexpected peaks. Thermo Scientific ImageQuest User Guide 27

36 3 Working with MALDI Files and Data Sets Working with Data Sets ImageQuest offers the following plot types to manage data from the raw file. Mass Range TIC Base Peak Mass range / TIC Specify the mass range for an analyte. ImageQuest displays only that range. Display the total ion current, all values in the scan. Display the most intense base peaks of all types. Display the selected mass range divided by the total ion current. This plot type normalizes the specific mass range over the total ion current. You cannot access the scaling setup tab from frame data sets in a series data set. From all other data sets, you can use the tab to change the plot range, scaling type, normalize data or play animation. Select only one of the items listed in the combo list. You can enter any value for minimum and maximum plot values as long as the minimum plot value is smaller than the maximum plot value. A histogram of the selected 2D distribution displays data as number of pixels vs. MS intensity. Histogram equalization involves selecting of the MS intensity range that contains the data of interest in order to optimize visualizing the data. Intensity is displayed on the X-axis using a linear or logarithmic scale. The number of pixels is displayed on the Y-axis. Use the histogram to see how signal intensity is distributed over a plot range or over the raw range. You can move the slide to hide areas with no data. Moving the slide changes the 2D image. Change the plot range by dragging the arrows under the histogram. You cannot change minimum and maximum raw values on the Scale page. These values are obtained while extracting data from the raw file. 28 ImageQuest User GuideThermo Scientific

37 3 Working with MALDI Files and Data Sets Working with Data Sets Creating a New Data Set To create a new data set 1. To open the New Data Set dialog box, click under the data set area. This dialog box presents options for creating a new data set. When ImageQuest creates a new data set, the software automatically generates a data set name based on the data set type, plot type, and mass range. You can manually change the data set name and ImageQuest accepts the new name. 2. Set the plot type to one of the following: Mass Range: Specify the mass range for an analyte. ImageQuest displays only that range. You can enter a single mass in the Mass Range field. The software extracts the mass range of the entered mass +/- the mass tolerance entered in the Mass Tolerance dialog. For example, you can specify for a mass range and set a mass tolerance of The software extracts the mass range to The software automatically selects the mass tolerance based on the mass analyzer: FT or Ion Trap. TIC: View the total ion current range. Base Peak: View all peaks to find those of significant intensity. This feature helps you find an unidentified substance. Mass Range/TIC: View the specified mass range divided by the total ion current. This plot type reduces the intensity of the peaks. Thermo Scientific ImageQuest User Guide 29

38 3 Working with MALDI Files and Data Sets Working with Data Sets 3. Click the arrow in the None box to select a mathematical operator: none, minus, plus, or divide. 4. Select a second plot type. Not all options are possible. For example you cannot select the mass range and select a TIC. You can use this dialog box to display the relative ratio between two peaks or an analyte in a matrix by using an equation similar to the one in the following example: 30 ImageQuest User GuideThermo Scientific

39 3 Working with MALDI Files and Data Sets Working with Data Sets Editing a Data Set Use this dialog box to extract specific data from the raw file. To narrow the data, set the plot type to the following: Mass Range: Specify the mass range for an analyte. ImageQuest displays only that range. TIC: View the total ion current range. Base Peak: View all peaks to find those of significant intensity. This feature helps you find an unidentified substance. Mass Range/TIC: View the specified mass range divided by the total ion current. This calculation reduces the intensity of the peaks. To edit a data set To open the Edit Data Set dialog box, click under the data set area. Use this dialog box to edit a current data set. The view depends on the type of data set you want to edit. You can edit only one data set at a time. Thermo Scientific ImageQuest User Guide 31

40 3 Working with MALDI Files and Data Sets Working with Data Sets Overlaying Data Sets Create a combo data set containing 2 to 4 data sets to overlay the data sets on top of each other using the same or different color schemes. Use combined data sets to see where the highest relevant signal appears on the plate. To create a combo data set 1. Click under the data set area.the New Data Set dialog box opens. 2. Select the Combo option. The following area appears. 3. To combine available data sets, select the check box next to at least two file names. 4. Click OK. Once you have created a combo data set, you can view the merged picture with RGB or Alpha mode. You can also move from a two-dimensional view of one of the merged data set views to the other with various levels of opacity. For more information on adjusting opacity, see Changing a Combo File View on page ImageQuest User GuideThermo Scientific

41 3 Working with MALDI Files and Data Sets Working with Data Sets Animating Data Sets You can animate a series of 2D and 3D graphs as a function of mass range to display unknown metabolites or unexpected peaks. Create a series data set to play an animation of a series of frame data sets. Use this feature when looking at unknown or unexpected peaks in relation to tissue position. The data sets share the same parameters except for mass range. When you turn on animation, ImageQuest scrolls through the data set in the selected direction. To animate a data set 1. Place the cursor in the chromatogram of the 2D graph area that you want to explore. 2. In the TIC area of the Scale view, select a direction and click to start the animation. In a series data set, click range. Ιn a single data set, click that the data was collected. to animate the 2D and 2D with different mass to display a series of spectrum in the order As the red line moves over the chromatogram, the Ion Chromatogram view changes to reflect the new view. 3. To move across the view more quickly, slide the Refresh Frequency button to the left. To slow down the animation, move the Refresh Frequency button to the right. 4. To stop the animation, click. When animation is not running, use the and buttons to manually go from frame to frame or from one spectrum to the next one. You can also use the left and right keyboard arrows to move through the spectra. Printing Reports You can print the current processed 2D images as a report. To print a report 1. Click to display the Print dialog box. 2. Select printer options and click Print. Thermo Scientific ImageQuest User Guide 33

42 3 Working with MALDI Files and Data Sets Opening a Previously Extracted Data File Saving Data The processed images and parameters of all the extracted data sets are saved in the.maldidata file. After you make changes to a view, save the file. To save data 1. Click on the toolbar to display the Save dialog box. 2. Provide a name for the file. 3. Click Save in the dialog box. Opening a Previously Extracted Data File When you open the saved.maldidata file, ImageQuest loads all the images and data sets exactly as you saved them. This feature reduces data processing and extraction time. To open a data file 1. Click on the toolbar to display the Open dialog box. 2. Browse to select the file and click Open. 34 ImageQuest User GuideThermo Scientific

43 A ImageQuest Reference This appendix provides ImageQuest reference information. Contents Buttons Menus ImageQuest Tabs Dialog Boxes Buttons Buttons on the toolbar can be hidden or viewed. To display or hide the toolbar and status bar Choose View > Toolbar to display or hide the toolbar. Choose View > Status Bar to display or hide the status bar. To maximize the display of a Processing Setup view, hide both of these features. Toolbar Buttons The main toolbar has the following types of buttons: File Buttons View Buttons Spectrum View buttons Spectrum Rescaling Buttons The Data Set tab has buttons to assist in working with data sets. Data Set Buttons Thermo Scientific ImageQuest User Guide 35

44 A ImageQuest Reference Buttons File Buttons Use these toolbar buttons to work with data files. Open an existing file. Save the active data. Print the active document. View Buttons Use these toolbar buttons to change the main window view. View all images (Spectrum view, 1D, 2D, and 3D graphs all appear). View 2D image only with the data set view. View optical image only with the data set view. View 3D image only with the data set view. View spectrum only with the data set view. View file and acquisition information. Spectrum View buttons Use these toolbar buttons to view spectra: 36 ImageQuest User Guide Thermo Scientific

45 A ImageQuest Reference Buttons Point to display a spectrum at this location. Average spectra within a selected area. Turn Zoom feature on or off. Measure the distance between two selected points. Copy the selected image to the clipboard. You can paste the copied image into any documents. Use these toolbar buttons to select a spectrum or a position on a spectrum. Go to the previous spectrum at this location. If the Local Average button is turned off and there is more than one spectra at the current point, the previous and next buttons are turned on and you can move to the previous or next spectrum at the current position. Average all spectra at this location. (Turn Local Average on or off). The button is only available when there are more than one spectrum at the current position. Click the button to average all the spectra in an area. Go to the next spectrum at this location. If the Local Average button is turned off and there is more than one spectrum at the given point, the previous and next buttons are turned on and you can move to the previous or next spectrum at the current position. Go left to the next position within the 2D or optical image. Go right to the next position within the 2D or optical image. Go up to the next position within the 2D or optical image. Go down to the next position within the 2D or optical image. Show scan dots in the optical image. Thermo Scientific ImageQuest User Guide 37

46 A ImageQuest Reference Buttons Spectrum Rescaling Buttons Use these toolbar buttons to re-scale a Spectrum preview. Zoom in Y from the current baseline to show more detail. Zoom out Y to show more data. Normalize Y intensity scale. Zoom in X at the center to show more detail. Zoom out X at the center to show more data. Display all data on X-axis or all text in report. Reset scaling to full scale for both X- and Y-axes. Display Y-axis data in normalized mode. Display Y-axis in creep mode. Display Y-axis in fixed mode. The Zoom menu contains equivalent commands. Display the toolbar as a shortcut menu by right-clicking on the appropriate preview or re-scale the chromatogram using the cursor. 38 ImageQuest User Guide Thermo Scientific

47 A ImageQuest Reference Buttons Data Set Buttons Use the buttons below the data Set view to work with data sets. Create a new data set. Edit an existing data set. Copy the selected data set. Delete the selected data set. Thermo Scientific ImageQuest User Guide 39

48 A ImageQuest Reference Menus Menus ImageQuest main window has the following menu options: Menu Option File New Open Save Save As Close Print Print Preview Print Setup Edit Copy View Toolbar Status bar Help Help on Current Item (F1) ImageQuest Help Xcalibur Help How to use Help About ImageQuest Function Open a new ImageQuest window. Open a file. Save file information from the recent session. Save a file under a different name. Close a file. Print a file. Display what the printed image will look like. Display print options. Copy the selected data set. Display or hide the toolbar. Display or hide the status bar. Provide context-sensitive help about a screen. Access ImageQuest Help. Access Xcalibur Help. Provide instructions about using Help. Provide the ImageQuest version number and release information. 40 ImageQuest User Guide Thermo Scientific

49 A ImageQuest Reference ImageQuest Tabs ImageQuest Tabs Scale Tab Scale Tab 2D View Tab 3D View Tab Use the Scale tab to view an intensity histogram, where the X-axis is Intensity (in Linear or Logarithmic scale) and the Y-axis is the number of pixels. Use the logarithmic scale to space out data points that are clustered in the linear scale view. In histogram equalization, the pixels should be distributed evenly over the whole intensity range. Thermo Scientific ImageQuest User Guide 41

50 A ImageQuest Reference ImageQuest Tabs Parameter Definition Values/Options Scaling Type Adjust the grouping of data in the display. Linear: histogram intensity axis is in linear scale. This scaling type is often used to preview data. Normalize Histogram Min Plot Value Max Plot Value Min Raw Value Max Raw Value Refresh Frequency slide bar Display data relative to the one member of the data set Display points showing the distribution of intensities in an image. Narrow the range of the data display by adjusting the minimum value. Narrow the range of the data display by adjusting the maximum value. Narrow the range of the raw data display by adjusting the minimum value. Narrow the range of the raw data display by adjusting the maximum value. Change the frequency to refresh the screen. Logarithm: histogram intensity axis is in logarithmic scale. Use logarithmic scaling to reduce image scope and maximize use of color in relevant area. No Raw Data Plot Value Raw Data Plot Data 42 ImageQuest User Guide Thermo Scientific

51 A ImageQuest Reference ImageQuest Tabs 2D View Tab Use the 2D tab to set up the 2D graph properties. Parameter Definition Options Color Scheme Specify the color scheme for the 2D view. Gray scale Rainbow Banded Blue to red Red Green Blue Olive Green Text Color Specify the color of text in the Opens a colors option box. Click color to choose. 2D view. Style Specify the style of the 2D view. Image Contour Image and Contour Smooth Specify the smoothing style to be used on the 2D view. None Linear Spline Num Contour Specify the number of contours. Valid values: A higher value indicates more contours. Thermo Scientific ImageQuest User Guide 43

52 A ImageQuest Reference ImageQuest Tabs Parameter Definition Options Blend Mode Adjust combined file views. The Blend mode is only available with combo data set RGB: An RGB image has three channels: one for red, one for green, and one for blue. Averaging the three RGB channels forms RBG blended image. The averaging is weighted by the percent of opacity of each RGB images. Reverse Image Stack Invert Color Scheme Move the lower image to the top. Invert the color scheme in both the 2d and 3d views. Alpha: You can combine an alpha channel with other layers in an image to clearly display translucency. This alpha channel adds an additional eight bits to each pixel in a 32-bit graphics system to represent 256 levels of translucency. Black represents an opaque view and white represents a fully transparent view, while various gray levels represent levels of translucency. Select to display. Select to invert current color scheme. 44 ImageQuest User Guide Thermo Scientific

53 A ImageQuest Reference ImageQuest Tabs 3D View Tab Use the 3D tab to set up the 3D graph properties. Parameter Definition Options Color Scheme Select the color scheme for the 3D view. Gray scale Rainbow Banded Blue to red Red Green Blue Olive green Style Select the style of the 3D view. Solid Surface Wire Frame Contour Specify None XZ Contour YZ Contour XY Contour Num Contour Specify Valid values: Clip Data Using Color Range Select colors to delete from the 3D view. Select to use the color range to define data boundaries. Show Out Line Box Display the 3d image of the box around the view. Select to display. Invert Color Scheme Invert the color scheme in both the 2d and 3d Select to invert current color scheme. views. 3D Graph Opacity Specify the level of opacity in the 3D view. Valid values: 1-100% Thermo Scientific ImageQuest User Guide 45

54 A ImageQuest Reference Dialog Boxes Dialog Boxes See the following for information about ImageQuest dialog boxes. Create New Data Set Dialog Box ImageQuest Options Create New Data Set Dialog Box Click the new data button to open a New Data Set dialog box. Use the settings in this box to extract specific data from the raw file. Parameter Data Set Types Single Data Set Optical Data Set Combo Data Set Series Data Set Data Set Name Scan Filter Plot Type Definition Create a single data set that holds the information that the software extracts directly from the open raw file, such as mass range or TIC. Create an optical data set derived from the MALDI position file and that represents the optical image. Create a combo data set by overlaying 2 or more single or optical data sets (up to 4 images). You can display the combined data sets at the same time with the same or different color schemes. See Changing a Combo File View on page 21 for instructions on creating a combo data set. Create a series data set composed of a series of data sets called frame data sets. The software displays a frame data set as a frame in an animation. The frame data set shares the same information with the series data set, except for the extracted mass range value. Define the data set. Specify the experiment used during acquisition (MS, MS/MS, and so on). Specify a type of plot in the 2D and 3D images (Mass Range, TIC, Base Peak, or Mass Range/TIC). 46 ImageQuest User Guide Thermo Scientific

55 A ImageQuest Reference Dialog Boxes Parameter Mass Range TIC: total ion current Base Peak Mass range / TIC Operator Second Plot Type Definition Specify the mass range you are searching for. ImageQuest displays only that range. You can enter a single mass in the Mass Range field. The software extracts the mass range of the entered mass +/- the mass tolerance entered in the Mass Tolerance dialog. For example, you can specify for a mass range and set a mass tolerance of The software extracts the mass range to The software automatically selects the mass tolerance based on the mass analyzer: FT or Ion Trap. Display the total ion current and all values in the scan. Display the most intense base peaks of all types. Display the selected mass range divided by the total ion current. Specify the type of mathematical function to perform on the Plot Type and Second Plot Type. Choose from None, Minus, Add, Divide. 3. Specify a type of plot in the 2D and 3D images (Mass Range, TIC, Base Peak, or Mass Range/TIC). ImageQuest performs the operation selected in the Operator box on the primary plot type and the secondary plot type. Not all options are possible. For example you cannot select the mass range and select a TIC. You can use this dialog box to display the relative ratio between two peaks, or an analyte in a matrix by using an equation similar to the one in the following example: Thermo Scientific ImageQuest User Guide 47

56 A ImageQuest Reference Dialog Boxes ImageQuest Options Spectrum Display Options Select View > Options to open the Options dialog box. This dialog box displays data about the spectrum display and mass tolerance options of the raw file. Spectrum Display Options Mass Tolerance To view spectrum display options, click the Spectrum Display Options tab. Parameter Ion Trap Spectrum FT Spectrum Definition Specify the number of display decimals for an ion trap spectrum. Specify the number of display decimals for an FT spectrum. 48 ImageQuest User Guide Thermo Scientific

57 A ImageQuest Reference Dialog Boxes Mass Tolerance To view mass tolerance values, click the Mass Tolerance tab. You can enter a single mass for the mass range in the New Data Set dialog box. The software extracts the mass range of the entered mass +/- the mass tolerance. For example, you can specify for a mass range and set a mass tolerance of The software extracts the mass range to The software automatically selects the mass tolerance based on the mass analyzer: FT or Ion Trap. Parameter Ion Trap Scans FT Scans Definition Specify the mass tolerance for ion trap scans. Specify the mass tolerance for FT scans. Thermo Scientific ImageQuest User Guide 49

58 A ImageQuest Reference Dialog Boxes Information View Click to view more information about the acquisition process and data files. Data Set View Raw File View Position File View Tune Method View Instrument Method View Scan Header View Spectrum List View Status Log View Error Log View 50 ImageQuest User Guide Thermo Scientific

59 A ImageQuest Reference Dialog Boxes Data Set View Use the Data Set view to review details about the data set of the open raw file. Parameter Definition Scan Filter The scan filter used for the image X Data Points The number of pixels in horizontal direction of the 2D or 3D image Y Data Points The number of pixels in vertical direction of the 2D or 3D image Number of Pixels The total number of pixels of the 2D or 3D image = X data points * Y data points Number of Scans The number of scans of the selected scan filter in the raw files. X Resolution The minimum distance (in microns) between 2 adjacent pixels in a horizontal (X) direction Y Resolution The minimum distance (in microns) between 2 adjacent pixels in a vertical (Y) direction Image Width Width of the image in microns (XResolution * X data points) Image Height Height of the image in microns (YResolution * Y data points) Image X Offset The horizontal distance between the home position and the top left corner of the image Image Y Offset The vertical distance between the home position and the top left corner of the image Thermo Scientific ImageQuest User Guide 51

60 A ImageQuest Reference Dialog Boxes Raw File View Use the raw file view to see details about the open raw file. Parameter Raw File Name Creator ID Creation Date Acquisition Time Sample Name Comment Software Version Instrument Model Definition Name of the raw file Computer ID of the raw file creator Date the raw file was acquired Acquisition time for the image Title of sample given at acquisition Comment entered at acquisition Version for acquisition software Specific instrument used for acquisition 52 ImageQuest User Guide Thermo Scientific

61 A ImageQuest Reference Dialog Boxes Position File View Use the Position File view to review the MALDI image parameters. Thermo Scientific ImageQuest User Guide 53

62 A ImageQuest Reference Dialog Boxes Tune Method View Use the Tune Method view to review tune methods used during acquisition. Instrument Method View Use the Instrument Method view to review instrument methods used during acquisition. 54 ImageQuest User Guide Thermo Scientific

63 A ImageQuest Reference Dialog Boxes Scan Header View Use the Scan Header view to view scan header details.the view displays the scan header of the current displayed spectrum. Each spectrum has a different scan header. Spectrum List View Use the Spectrum List view to review spectra found during acquisition. Thermo Scientific ImageQuest User Guide 55

64 A ImageQuest Reference Dialog Boxes Status Log View Use the Status Log view to review status during acquisition. The view displays the closest instrument status at the time the current spectrum is acquired. Error Log View Use the Error Log view to review acquisition errors. 56 ImageQuest User Guide Thermo Scientific