Help is available for the following functions of the 'tools' pull down menu:
 

display options
display spectra
set display limits
set color (scale colors)
grid on/off
capture
export maps
map statistics
display large maps
define roi
display colorbar
swap data blocks
rotate
flip

 
 

This section describes the interactive display mode of the CytoSpec program. In the CytoSpec v.1.05 program the following interactive display functions are available:
 

• show mode: displays the pointer's x- and y-position in the 'spectra' information field


• zoom mode: can be used to interactively enlarge/reduce a part of the spectrum


• move mode or 'roll' mode':, rolls or moves the spectrum in any direction


Spectra of the mapping measurements are displayed in the left panel of the main window. Options for displaying the IR spectra can be selected from the 'display options' window. This window can be activated by pressing the button 'display options' (see Main Window - Basic Concepts ), or by selecting the option 'display options' from the Tools Menu. To display spectra, the following functions are available:

display interactively wavenumber and absorbance values (radio button 'show mode'   interactive expand/compress mode (radio button 'zoom mode')   interactive shift mode (radio button 'move mode')   set curves color (list box 'set color')   zoom window horizontally/vertically (sliders 'zoom spectra')   display multiple spectra (check box 'add mode')   x-y auto scale for active spectrum (check box 'auto scale')   clear window (button 'clear')

In the 'show mode' the pointer's x- and y-position will be indicated in the 'spectra' information field of the main window.
 
Using the 'zoom mode': to zoom in (out) spectra in the display window you have first to activate the appropriate radio button. Then, if you wish to enlarge parts of a spectrum you have to set the first position for the spectrum window by a left mouse click. Hold the button and drag a frame by moving the mouse to the second point. If you release the mouse button, the contents of the frame will be expanded to the full display window. To reduce the size of the spectrum, you can use the right mouse button in the same way.
A left mouse click, that is when no frame is drawn, expands the spectrum in the display window by 200%. A right mouse click will reduce the size to 50%.
 
The 'move mode' is used to roll the spectrum in any direction. The display limits are shifted so that all spectra displayed are shifted by the same amount. Again, you have to activate the appropriate radio button before you can start. Spectra can then be moved if you click (left button) into the display window and move the pointer to the desired end point (the mouse pointer will change to a closed hand symbol). Release the left mouse button to finally roll the spectra into the desired direction.
 
How to select spectra for display? There are two methods available to display spectra of active hyperspectral maps:
 
Method A: If you click into one of the hyperspectral maps ('show mode' activated) to the right the x/y- pixel coordinates within the map are obtained and the respective spectrum is printed. Furthermore, pixel coordinates of the active spectrum are displayed in the text boxes of the main window (cf. x/y coordinates of active spectrum: Main Window - Basic Concepts ). Depending on the character of the map (map produced from original or processed data) the check box 'show work spectrum' is activated (or deactivated). If images re-assembled by multivariate imaging (PCA, HCA etc.) were activated CytoSpec displays an error message.
 
Method B: Alternatively, spectra can be selected by manually editing the edit fields 'coordinates, x or y' (see Main Window - Basic Concepts ) and/or by activating the check box 'show work spectrum'. If the corresponding map does not exist (e.g. there is no map of original absorbance spectra), an error message will be displayed.

 

This option can be used to manage the appearance of individual spectra, or groups of spectra. You can activate the 'display spectra' window by selecting the option 'display spectra' from the 'tools' pull down menu.
 
Please note: In order to display more than only one spectrum, check the option 'add spectra' in the Display option window. Then, you can display spectra of your choice by clicking in the IR maps generated from original or processed spectra. The 'display spectra' window will show you all active spectra with the x- and y- pixel coordinates. To select/unselect spectra in the 'display spectra' window use the mouse and hold either key 'Shift' or 'Ctrl' pressed (standard windows behavior).

Activate the context menu by selecting one or more spectra and a right mouse click.   set color: allows to set the color of a selected spectrum/selected spectra.   hide from display: hides spectra from the spectral panel. Spectra can be, however, reactivated afterwards.   remove from display: removes spectra permanently from the spectral panel.   cancel: check out what will happen ;-).

 
 

A function that permits to enter display limits of the spectral panel. This window can be activated by choosing option 'set display limits' from the 'tools' pull down menu.

Please indicated the new settings for the x- (in wavenumbers) and/or y- (usually in absorbance units) axis. Wrong settings will produce an error message.

 

 
 

Select and adapt color maps to a given hyperspectral map.

select colormap: allows selection of one of the following color maps: jet, hsv, hot, cool, bone, winter, summer, autumn, copper, prism, flag, lines, colorcube (multicolor mode), ann , blue, red, green, yellow, black, cyan, and magenta (the latter are unicolor modes)   target map: please select whether you want to apply changes to the surface map reassembled from original or processed data.   interpolation: when this check box is activated, hyperspectral maps are interpolated. Otherwise, the maps are textured (see illustration below).   color range compression: permits to increase/decrease the offset and the contrast of the color map. The effects of the respective sliders are illustrated in the image below.   scale manually: permits to modify manually the color map by entering the minimal and maximal z-values into the appropriate edit boxes. When this check box is checked the functions 'set standard', 'invert', and the sliders 'offset' and 'contrast' (color range compression) are deactivated.   set standard: sets the offset and the contrast of the colormaps back to default values.   invert: this immediately inverts the colormaps.   draw: applies the changes to the hyperspectral map.   cancel: closes the application

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Illustration of the effect of the check box 'interpolation':
 
 
     
 


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Illustration of the effects of changing the sliders 'offset' and 'contrast':
(example of the colormap 'jet')
 
 

 


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The colormap ann (artificial neural network): This colormap is particularly useful to generate maps on the basis of classification methods producing crisp class membership values, such as:
 

HCA imaging - images re-assembled on the basis of hierarchical clustering
KMC imaging - images re-assembled on the basis of k-means clustering
Synthon imaging - images re-assembling on the basis of Synthon's NeuroDeveloper(TM) neural network software
ANN imaging - images re-assembled on the basis of the Stuttgart Neural Network Simulator (SNNS)
 

The colormap ann can be individually modified by editing a simple text file 'color.txt'. This file is located in CytoSpec's root directory, usually in C:\program files\CytoSpec\CytoSpec and is read by CytoSpec upon its initialization. When editing this file, please use only color names given at http://www.w3.org/TR/2003/WD-css3-color-20030214/ chapter 4.3. SVG color keywords. Please check for case sensitivity and spaces!
 
An example of the content of the file color.txt is given here: color.txt
 
The settings of the colormap ann define also the following color sequences:
 

1. color options of the 'set color' function available from the context menu of the Display Spectra function.
2. selectable colors available from the listbox 'color of spectra' of the Display Options window.
3. the color sequence is furthermore used to display cluster average spectra of the functions KMC , FCM and HCA imaging (button 'spectra' --> button 'plot averages').

 
 

This option is available from the tools pull down menu and has effect on the spectral plot window, only. The grid ON/OFF function may be useful when the spectral window is exported via the Capture --> Spectral Plot function.
 

 

Capturing maps can be started from the 'capture' option of the 'tools' menu. You can either save the image maps to your hard drive (as bitmaps, map produced on the basis of original, or processed data, spectral window), or alternatively capture the entire program window to the clipboard.
 

Saving images as bitmaps: Images reassembled on the basis of original spectral data (tools --> capture --> upper image ) and of processed spectra (tools --> capture --> lower image ) can be captured. When the corresponding option was selected, it is asked to type in a path and a file name. In order to store the spectral plot you have to select the 'capture --> spectral plot' option of the 'tools' pull down menu. The option Grid ON/OFF activates or deactivates a grid of the spectral plot (tools menu) The standard resolution used to capture the bitmaps is 300 dots per inch (dpi). Files are stored in a standard windows bitmap (.bmp) data format.
 
Capturing program window: Along with storing images or spectra you may also want to take a screenshot of the program window. By choosing the 'capture' --> 'window' option of the 'tools' pull down menu and selecting the resolution (72, 150, 200, or 300 dpi) the entire CytoSpec program window is copied to the clipboard.

 
 
 
 

This function can be used to export the z-data of hyperspectral maps. The z-data are stored in standard ASCII-text files. To export the data you have to select first the option 'export maps --> upper plot (or lower plot)' from the 'tools' pull down menu. Then, in a standard windows dialog box you will be asked for path and file name of the data file to be stored.
 
Data are stored in files of the following structure (example of a map of M x N pixel spectra, [xdim x ydim]):
 

The ASCII-files are now available for import into other programs such as Excel, Origin or Matlab. Please note that the standard extension of the ASCII data files will be '*.dat'.
 

 
 

This function can be used to visualize the distribution of the actual z-data (e.g. intensities) as a histogram. The option 'map statistics' is available from 'tools' pull down menu. You can do map statistics from maps assembled from original data (upper hyperspectral image), or from maps from any type of processed data (lower map).  
 

# of pixels: displays the total number of pixels of the hyperspectral map.
 
# of bad pixels: displays the number of pixel spectra that had failed one of the Quality Tests.
 
mean: displays the mean value of the data used for imaging.
 
median: shows the median of the image data.
 
std: the standard deviation.
 

 
 
 
 

 

The function 'display large maps' is available from the 'tools' pull down menu but also from the context menus of hyperspectral maps (click by the right mouse button). When this option is chosen, a new window will appear showing the maps in the true spatial aspect ratio.
 

The following information can be obtained from the 'large maps':
 

1. the pixel position of the cursor,
2. (x,y) spatial position of the cursor (obtained from the parameters SZX and SZY)
3. the z-value (absorbance, transmittance, Raman intensity etc.) found at the current cursor location is given.
 

Further options:
 

1. the map can be stored as a bitmap by selecting 'capture to bitmap' ('tools' pull down menu).
2. a colormap is available by selecting 'display colorbar' ('tools' pull down menu).
3. the window can be deleted (chose 'exit' from the 'file' pull down menu).
4. one can define Regions of Interest (ROI)

 

 

 

The 'define region of interest (ROI)' function is available from the 'tools' pull down menu but also from context menus of hyperspectral maps (click right mouse button). When this option is chosen, a new window Large Map will appear that shows the map in the real spatial aspect ratio. Furthermore, an additional window 'define ROI' pops up which permits to define up to three different regions of interest in one hyperspectral map.
 


listbox ROI #: Up to three different regions of interest can be defined. Switching between active ROIs can be achieved by selecting the appropriate option from this listbox.   edit fields 1: - 15: Please enter the (x,y) coordinates of ROI boundary points (in pixel coordinates) in the respective fields. Alternatively, you can click into the 'large map' - the pointer coordinates will be transferred automatically.   calculate average spectrum: a tool that permits to calculate/display and store average spectra from all active regions of interest. Works in exactly the same way as the option 'spectra' described in the HCA imaging and KMC imaging functions.   Mann-Whitney test: not yet implemented   t-test: not yet implemented   checkbox outside modus: this option permits to switch between the in- and outside modus of the ROI definition procedure.   checkbox add ROI point: fields that permit to define additional ROI boundary points are added.   checkbox delete ROI point: ROI points are deleted.   checkbox delete ROI: all ROI point definitions made so far are deleted.   display ROI causes to draw the region of interest in the window 'large map'   cancel the routine is aborted


 

When a region of interest has been marked within the hyperspectral map, the option 'apply ROI --> preproc data' from the pull down menu of the large window becomes activated (see screenshot below). The function 'apply ROI --> preproc data' creates a new set of preprocessed spectra from the data block of original spectra by transferring only spectra located within the actual ROI area. Spectra from areas outside are omitted and filled by NaN-values (Not a Number). Consequently, spectra originating from non-ROI areas are excluded from all further image processings and appear in any hyperspectral map generated on the basis of preprocessed data as black pixels. Thus, the 'apply ROI --> preproc data' function works similar to a manual Quality Test .
 

 

 

This function is available from the 'tools' pull down menu and displays a colorbar of the active map. It use the active colormap which is available from the Set Color function of the 'tools' pull down menu. Colorbars can be obtained for all types of maps, including Large Maps) and maps produced on the basis of crisp class membership values such as
 

HCA maps - images re-assembled on the basis of hierarchical clustering
KMC maps - images re-assembled on the basis of k-means clustering
Synthon maps - images re-assembling on the basis of Synthon's NeuroDeveloper(TM) neural network software
ANN maps - images re-assembled on the basis of the Stuttgart Neural Network Simulator (SNNS)

 

Example of a colorbar for a KMC map. Note that in the example below the active colormap is of type ann. Although only five clusters are displayed, the colorbar will always show the complete colormap which consists here of 27 different colors. Note furthermore, that the sequence of colors is defined by the numbering of colors in the text file 'color.txt' (can be found in the root directory of CytoSpec, usually C:\program files\CytoSpec\CytoSpec). For details, see ann Colormap

Four examples of the colorbars of the types jet (top left), hsv, colorcube and hot (clockwise direction).

 

 

Overwrites the original data block by preprocessed data. This function is rarely required; however, the test for thickness of the Quality Test (which can be performed exclusively on original data) may require offset corrected spectra. As original data are lost it is highly recommended to store the data before performing the 'swap data block' function. If the block of preprocessed data is empty CytoSpec returns an error message. To avoid overwriting of the original data as a result of typing error, CytoSpec asks for confirmation (see window below).
 
 

 
 

Data blocks AND images are manipulated such that the spatial dimensions are changed (i.e. swapping the x- and y-dimensions) while the spectral dimension remains unaffected.
 
Rotate function can be used to rotate the data arrays by 90 degrees clockwise/counterclockwise, or by 180 degrees, respectively.
 
The Flip function permits the flip the spectral hypercube along the x-axis ('flip horizontally'), or alternatively along the y-axis ('flip vertically')