CytoSpec - an APPLICATION FOR HYPERSPECTRAL IMAGING



 

File Pulldown Menu

Load
Save
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Import ASCII
Import Binary
Export
Delete
Clear
Plot
Customize
Batch Multiple Files
Exit

Spectral Preprocessing

Calculation of Derivative Spectra
Normalization (Vector, Offset)
Cut
Interpolate
Smooth
ABS <--> TR Conversion
Subtraction
Dispersion Correction
Quality Test
Baseline Correction
Water Vapor Compensation
Noise Correction
Cosmic Spike Removal
Batch Preprocessing

Spatial Preprocessing

Crop
Interpolate/Binning
Filter Images
3D-FSD

Univariate Imaging

Chemical Imaging
Chemical Movie
Frequency Imaging

Multivariate Imaging

HCA Imaging
KMC Imaging
FCM Cluster Imaging
PCA Imaging
VCA Imaging
n-findr Imaging
ANN Imaging
Synthon Imaging
Imaging with Distance Values

Tools

Display Spectra
Set Display Limits
Grid On/Off
Set Colors
Capture
Export Maps
Map Statistics
Display Large Maps
Define ROI
Display Colorbar
Swap Data Blocks
Rotate
Flip

File Information

Show History
Show Instrument Parameters
Show measurement Parameters
Show Additional Parameters
Edit Parameters
About
Using the Help Function

Preface


     
    The CytoSpec program was developed in the year 2000 by Dr. Peter Lasch who worked as a postoc with Prof. Max Diem in the Laboratory for Spectral Diagnosis at Hunter College (CUNY, New York), now Northeastern University (Boston). Starting from the first version in 2000 the software has been continously improved and updated. CytoSpec is now known as a specialized software package for vibrational hyperspectral imaging that supports a large number of different tasks for data import, spectral and spatial preprocessing and uni- and multivariate image segmentation. The program is widely used in a scientific environment and was the workhorse in many scientific studies. The CytoSpec software has been continuously developed over the past years and supports now most of the actual hardware platforms, including 64-bit environments, see 64-bit version of CytoSpec for details.
     
    How to obtain the program:
     
    The full program (commercial version) can be ordered from CytoSpec. Please e-mail your request to CytoSpec (e-mail: order@cytospec.com). You can also obtain a free demo version (limited functionality) of CytoSpec (e-mail:service@cytospec.com).

To obtain the most recent version of the CytoSpec online help go to the CytoSpec web pages.

    Introduction to the program/short description:
     
    The software package available from CytoSpec is a program designed specifically for the analysis of vibrational spectroscopic (IR and Raman) imaging data sets. The major innovation of the CytoSpec software over competing software packages is its structure that manipulates hyperspectral data cubes, rather than individual spectra. Thus, all operations carried out on the data affect every spectrum, with the number of spectra in the data set limited only by the available memory.
     
    The CytoSpec software is a stand-alone, comprehensive package that operates under Windows XP, Windows Vista and Windows 7. Hyperspectral data sets, in the format defined by the manufacturer of the Raman, or infrared, microspectrometer used by the researcher, are imported, converted to and stored in a data matrix format specific to the CytoSpec program.
     
    The software permits the standard spectral manipulations customarily found in single spectra analysis software, such as expansion, smoothing, scaling, normalization, etc. Due to the fact that data sets often contain hundreds or thousands of spectra, a number of statistical approaches to the data are built in the software. These can be classified as uni- and multivariate statistical methods. Univarite methods of analysis, included in the software, consist of various mapping displays of hyperspectral data. In these, the user may select band intensities, integrated intensities, frequencies, intensity ratios, etc., to construct false color maps of the spectral data, which may be considered to be slices through the hyperspectral data cube.
     
    The multivariate methods of data analysis create spectral correlations and maps by including not just one intensity or frequency point of a spectrum, but by utilizing the entire spectral information. These methods include principal component analysis (PCA), unsupervised methods of cluster analysis, and endmember selection methods. The software is configured to permit output of the spectral data in the format used by other software packages, such as the NeuroDeveloper (Synthon), an artificial neural network simulator.
     

System Requirements & Installation


     
    SYSTEM REQUIREMENTS:

Software:

  • Windows XP, Windows Vista or Windows 7. Windows 98 or ME are not recommended!
  • Windows 64-bit versions are supported, but note that the compiled version of CytoSpec is still a 32-bit application. The memory advantage of the 64-bit OS is therefore not fully used (see also CytoSpec 64-bit).
  • software to display these help files, that is a web browser such as Firefox, Internet Explorer or Opera

Hardware:

  • approx. 100 MB of free disk space for example files
  • at least 1024 MB of RAM
  • i386 based CPUs (Intel/AMD)
  • at least an 8 bpp 960x720 graphic display. Supported display modi:
      960 x 720 (minimum)
      1024 x 768
      1152 x 864
      1280 x 1024
      1600 x 1200
      and most wide screen resolutions
  • to run CytoSpec 64-bit a full 64-bit hard- and software environment is required. Please refer to the CytoSpec 64-bit documentation for details.

    INSTALLATION (32-bit):
     
    Commercial version: Once you have received the CytoSpec CD or downloaded the installation archive from CytoSpec's web server, you are ready to install the program. During the install procedure CytoSpec's files will be automatically extracted from the cabinet and all necessary steps for starting CytoSpec will be taken.
     
    • If you have downloaded CytoSpec: unzip the installation archive into a directory of choice. In this directory locate the file 'setup.exe'
    • CD-Version: open the Explorer and locate the CytoSpec setup file on the CD you have received. The file is named 'setup.exe'
    • To start the installation process just double click on setup.exe. The installation program will be started automatically.
    • Determine the place on your hard disk where you want CytoSpec to be installed. Normally the proposed C:\program files\CytoSpec\CytoSpec will do fine.
    • Then simply follow the instructions of the installation program.
    • When setup is complete you can find a new program group and a CytoSpec shortcut on the desktop.
    • Start the program by double clicking on the CytoSpec icon.

     
    Demo version: Download the installation package from CytoSpec's webserver. IMPORTANT: In order to run CytoSpec, an additional license file ('keygen.gen') must be copied into CytoSpec's main directory. This individual license key is NOT included in the installation package and can be obtained only by e-mailing a request to order@cytospec.com.
     
    • Unzip the installation archive into a directory of choice. In this directory locate the file 'setup.exe'
    • To start the installation process just double click on setup.exe. The installation program will be started automatically.
    • Determine the place on your hard disk where you want CytoSpec to be installed. Normally the proposed C:\program files\CytoSpec\CytoSpec will do fine.
    • Then simply follow the instructions of the installation program.
    • When setup is complete you can find a new program group and a CytoSpec shortcut on the desktop.
    • You can copy now the license key file into CytoSpec's main directory (e.g. c:\program files\CytoSpec\CytoSpec)
    • Start the program by double clicking on the CytoSpec icon.
    • Note that the temporary license for CytoSpec demo will expire after 90 days.

    Patching CytoSpec: To manually update (patch) an older CytoSpec version you can simply overwrite the file 'ftir.exe' by a newer one.

    CytoSpec 64-bit: With CytoSpec 1.4.02 a 64-bit version of the program is available (on request, commercial version only). Please refer to the CytoSpec 64-bit documentation for details.

Main Window - Basic Concepts


     
    The main window of the program shows three panels where the spectra and hyperspectral maps are displayed (cf. screenshot of the main window of the CytoSpec program below). The two panels to the right are used to display the spectral maps obtained from original spectral data (upper right panel) or from processed spectra such as preprocessed data, derivatives, and (de)convolution data (see lower right panel). The latter panel is used also for displaying hyperspectral images obtained from multivariate spectral analysis methods: (i) clustering and (ii) endmember selection techniques, (iii) principal component analysis or segmentation maps produced on the basis of analyses by (iv) artificial neural networks (ANN).
     
    The color scheme utilized to display spectral maps can be changed (see button 'set color' of the main window, cf. also the set colors option from the tools pull down menu). In addition, the CytoSpec program offers a lot of useful tools to adjust parameters such as image contrast and color scaling.
     
    The CytoSpec program gives you easy access to the spectra. When clicking into a hyperspectral map (left mouse button) the
     
    • spectrum with the pixel coordinates of the mouse pointer is displayed in the left window,

    • the pixel coordinates of the active spectrum appear in editable text boxes, and

    • some information of how the spectral map was produced is displayed in the field between both panels to the right.
       

    Many options for displaying spectra can be modified (button 'display options', option 'display options' of the tools pull down menu). For more details please refer to the chapter Working with Spectra - Basic Concepts.
     
    One of the basic principles of this software is to ease data import of spectral data into other applications, and also from other programs into CytoSpec. To give an example, selecting individual spectra for ASCII export can be started by a simple click with the right mouse button (for details see chapter export).
     
 
main window
 
    When starting the CytoSpec program the main Window and a command line window (see screen shot below) will appear. While the main window allows to interact with the program, the command line window shows input parameters and displays non-standard errors. These messages are stored in a log-file ('history.log'), which can be found in following directories:
     
      CytoSpec standalone version (32-bit), Windows operating system (W2k/XP/Vista/W7): root directory of the CytoSpec program, usually C:\program files\CytoSpec\CytoSpec\history.log
       
      CytoSpec pcode toolbox under Matlab (64-bit), Windows operating system (W2k/XP/Vista/W7): Matlab user directory, usually C:\users\CytoSpec-user\Documents\Matlab\history.log with 'CytoSpec-user' being the Windows user name
       
    When reporting problems and software failures (bug reports) please send the file 'history.log' to the following e-mail address: service@cytospec.com
 
 
command window
 
Example of the command line window

 
     
     
    This section describes the interactive display mode of the CytoSpec v.2.00.01 program. In this version the following interactive display functions are available:
     
    Mouse mode:
     
    • show: displays the pointer's x- and y-position in the 'spectra' information field

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

    • move 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 spectra can be selected from the main CytoSpec window (the window 'display options' of earlier CytoSpec versions has been removed). The following functions are available:
     
    • display interactively wavenumber and absorbance values (radio button 'show')

    • interactive expand/compress mode (radio button 'zoom')

    • interactive shift mode (radio button 'move')

    • set curves color (list box 'color of spectra')

    • zoom window horizontally/vertically (sliders)

    • display multiple spectra (check box 'add mode')

    • x-y auto scale for active spectrum (check box 'auto scale')

    •  
    In the mouse mode 'show' the pointer's x- and y-position will be indicated in the 'spectra' information field of the main window.
     
    Using the 'zoom' mouse 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. 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' and/or by activating the check box 'processed spec'. If the corresponding map does not exist (e.g. there is no map of original absorbance spectra), an error message will be displayed.


 

Hot Keys & Icons


     
    A number of icons is displayed CytoSpec's main window. The following table will give you a short overview about icons and associated CytoSpec functions.
     
    Additionally CytoSpec provides many Hot Keys to help speed up image re-assembling and exporting data to other applications. You will find that you can save a lot of time by eliminating the need to move the mouse and to choose options from the pull down menus.
     

ICONS
 
clear all load spectral map save all
print customize export ASCII spectra
exit calculate derivatives quality tests
batch mode chemical maps chemical movie
frequency maps HCA maps Synthon maps
autoscale spectra display spectra set display limits manually
grid on/off set color capture spectral plot
capture upper image capture lower image edit
help about

 
HOT KEYS
 
Control-A about Control-C set colors
Control-D display spectra Control-E edit parameters
Control-F frequency maps Control-H help
Control-I chemical maps Control-L load data
Control-M save Matlab Control-O customize
Control-P plot Control-Q clear all
Control-S save data Control-X quit the program
Control-1 capture map from original data (bmp) Control-2 capture map from processed data (bmp
Control-3 capture spectral plot (bmp) Control-4 export mapping data (original data, ASCII)
Control-5 export mapping data (processed data, ASCII)    

Internal Data Organization


 
    Internal data organization: Spectral data are stored in one file consisting of up to four distinct data blocks:
     
    1. Original spectra: the first block is reserved exclusively for the original spectra, usually absorbance or transmission spectra, or Raman intensities in case of Raman maps. This block is usually not modified. Exceptions are the functions 'cut' and 'interpolate' of the preprocessing pull down menu and the functions 'swap data blocks', 'rotate', and 'flip' of the 'tools' pull down menu.
    2.  
    3. Preprocessed spectra: this data block contains the results of data manipulations on original spectra (e.g. preprocessed data).
    4.  
    5. Derivative spectra: the third data block is reserved for derivative spectra.
    6.  
    7. (De)convolution spectra: block number four contains the results of spatial preprocessing: spatial filtering, 3D-Fourier self deconvolution (FSD).
    8.  
     
    Preprocessing, multivariate analysis, and image re-assembling can be performed by choosing one of the existing data blocks (also called source blocks). The results of data manipulation are stored into so-called 'target data blocks'. Please note that existing target data blocks may be overwritten without warning! Target data blocks may vary for distinct functions (see synopses below).

    I. Preprocessing (except functions 'cut', 'crop', 'interpolate', 'binning',
    'derivatives', 'baseline correction', and 'ABS <--> TR'):

      source block   target block
    original spectra
    »»
    preprocessed spectra
    preprocessed spectra
    »»
    preprocessed spectra
    derivative spectra
    »»
    derivative spectra
    (de)convolution spectra
    »»
    (de)convolution spectra

    II. 'Derivative calculation':

    source block target block
    original spectra
    »»
    derivative spectra
    preprocessed spectra
    »»
    derivative spectra
    derivative spectra
    »»
    derivative spectra
    (de)convolution spectra
    »»
    derivative spectra

    III. 'Cut', 'crop', 'interpolate', 'binning', 'rotate' & 'flip':

    source block target block
    original spectra
      
    The functions cut', 'crop', 'interpolate', 'binning', 'rotate' & 'flip' modify the number of points (pixels), or the point (pixel) spacing in the spectral and/or spatial dimensions, respectively. All available data blocks are modified .
    preprocessed spectra
      
    derivative spectra
      
    (de)convolution spectra
      

    IV. Preprocessing: 'baseline correction' & 'PCA
    based noise reduction
    ':

    source block target block
    original spectra
    »»
    preprocessed spectra
    preprocessed spectra
    »»
    preprocessed spectra
    derivative spectra
    »»
    not possible
    (de)convolution spectra
    »»
    not possible

    V. 'Swap data blocks':

    source block target block
    original spectra
    »»
    Data block of original spectra will be overwritten by data block of preprocessed spectra
    preprocessed spectra
    »»
    derivative spectra
    »»
    not possible
    (de)convolution spectra
    »»
    not possible

    VI. 'ABS <--> TR':

    source block target block
    original spectra
      
    Original spectra will be overwritten by converted data. All other data blocks will be deleted!
    preprocessed spectra
      
    derivative spectra
      
    (de)convolution spectra
      

    VII. 'Dispersion correction':

    source block target block
    original spectra
      
    Dispersion correction can be carried out only with transmittance spectra stored in the data block of original spectra. Spectra are dispersion corrected and converted to absorbance spectra. The target data block is the block of preprocessed spectra. Other data blocks are not modified.
    preprocessed spectra
      
    derivative spectra
      
    (de)convolution spectra
      

    VIII. 'Subtraction':

    source block target block
    original spectra
    »»
    preprocessed spectra
    preprocessed spectra
    »»
    preprocessed spectra
    derivative spectra
    »»
    derivative spectra
    (de)convolution spectra
    »»
    not possible

    IX. 'Spatial Filtering':

    source block target block
    original spectra
    »»
    (de)convolution spectra
    preprocessed spectra
    »»
    (de)convolution spectra
    derivative spectra
    »»
    derivative spectra
    (de)convolution spectra
    »»
    (de)convolution spectra

     
     
    Since CytoSpec version 1.1.04 the presence/absence of data blocks is visualized by four LEDs located in the lower left corner of the Main window. A red LED indicates the presence, a black LED the absence of the respective data block.
     
    Memory management modes: CytoSpec offers two distinct memory management modes. This functionality was introduced to allow highly memory consuming operations also with limited RAM resources. With version 2.00.01 and the availability of the 64-bit version of CytoSpec the memory mode 'compress' is obsolete and has been removed.
     
    1. 'speed' the fastest mode, but highly memory consuming. Recommended for HCA with hyperspectral data sets containing up to 128 x 128 pixel spectra (32-bit version, 4 GB of RAM required). In this mode all spectral data are hold in memory with 8 byte precision as float64 values. The option is recommended also for HCA of larger hyperspectral data sets when using the 64-bit version of CytoSpec (Matlab toolbox)

    2.  
    3. 'intermediate' - relatively fast, but less memory consuming. Try this option when datasets are large and/or the amount of installed RAM is reduced. Spectral data are stored with 4 byte precision as float32 values on disk. Only the required data block will be loaded and is held during the calculations in memory. When performing HCA, the distance matrix will be held in RAM.

    4.  
    5. 'compression' - this memory option has been removed with version 2.00.01 (see 64-bit version of CytoSpec for details).

    6.  

Working With Spectra - Basic Concepts

 
 

    With CytoSpec version 2.00.01 (Sep. 2012, built version 338) the window 'display options' has been removed. The functionality is now an integral part of the main window of CytoSpec.
     
    How to select spectra for display? Three methods available to select spectra for display:
     
    Method A: By clicking into one of the hyperspectral maps ('show mode' is activated) the (x,y) pixel coordinates are obtained and used to display the respective spectrum. The (x,y) coordinates are displayed in the edit fields of the main window (see also [x,y] coordinates of active spectrum: Main Window - Basic Concepts ). Depending on the character of the map (map produced from original or pre-processed spectra) the check box ' processed spec' will be checked or unchecked. Note also that CytoSpec cannot display spectra from segmentation maps produced on the basis of multivariate image segmentation methods such as HCA, KMC, FCM, etc..
     
    Method B: Alternatively, spectra can be selected by manually editing the edit fields '(x,y) coordinates of active spectrum' (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.
     
    Method C: Spectra of the pixel coordinates x±1; (y±1) can be displayed by pressing the buttons '+' or '-' (main windows, option 'select spectrum')


 

How to Produce Hyperspectral Maps

 
 
    The basic idea of hyperspectral imaging (or mapping) is to derive for each spectrum of a given set of spatially resolved spectra one single spectral parameter. This parameter is then color scaled and plotted as a function of the spatial (x,y) coordinates. There are a number of techniques, which can be used for hyperspectral mapping. The most frequently used technique is the so called chemical mapping technique. In chemical mapping (also referred to as functional group mapping) the absorbance, transmittance, Raman intensity, half-width, or the frequency of a given vibrational band is color encoded and plotted against the spatial (x,y) coordinates. This univariate imaging techniques permits to visualize the spatial distribution of functional groups, or specific chemical substances and is extensively applied in biomedical science, remote sensing and other research areas.
     
    In the CytoSpec program you can create chemical maps by a number of different methods using the absorbance or transmittance, integral absorptions, Raman intensities, ratios etc. These methods are outlined in the chapter Chemical Maps
     
    Furthermore, the CytoSpec software offers the opportunity to re-assemble so-called Frequency Maps. In this method, the maxima of specific bands are obtained and images are re-assembled by plotting the frequency values as a function of the spatial coordinates.
     
    Other imaging techniques are based on multivariate methods of spectral data analysis. The CytoSpec program (v.2.00.01 , September 2012) offers nine different methods of multivariate spectral imaging:

The Log-File

 
 
    When starting the CytoSpec program the Main Window and a command line window (see screen shot below) will appear. While the main window allows to interact with the program, the command line window shows input parameters and displays non-standard errors. These messages are stored in a log-file ('history.log'), which can be found in following directories:
     
      CytoSpec standalone version (32-bit), Windows operating system (XP/Vista/W7): root directory of the CytoSpec program, usually C:\program files\CytoSpec\CytoSpec\history.log
       
      CytoSpec pcode toolbox under Matlab (64-bit), Windows operating system (Vista/W7): Matlab user directory, usually C:\users\CytoSpec-user\Documents\Matlab\history.log with 'CytoSpec-user' being the Windows user name
       
    When reporting problems and software failures (bug reports) please send the file 'history.log' to the following e-mail address: service@cytospec.com
 
 
command window
 
Example of the command line window

 

Ordering the Program, License & Disclaimer

 
 

    How to obtain CytoSpec?

      Ordering the full version:

    The commercial version (unlimited license, complete functionality) can be ordered from CytoSpec. Please e-mail your request to CytoSpec (e-mail: order@cytospec.com).

      Downloading an evaluation (demo) version:

    You can also download a free demo version (limited functionality) of CytoSpec. In order to run CytoSpec an additional license key file ('keygen.gen') must be copied into CytoSpec's main directory. This individual license key is NOT included in CytoSpec's zip archive and will be send on request. Please e-mail your request together with your name and an institutional address to the following e-mail address: service@cytospec.com.

 
 

    Most actual version 2.00.01 - please report software bugs

Download CytoSpec's standalone demo:
version 2.00.01, built 340, March 2013
size: 7156733 byte
CRC checksum: B33D2C94

    CytoSpec 64-bit version (Matlab pcode, v. 2.00.01)

Download CytoSpec's 64-bit toolbox:
version 2.00.01, built 340, April 2013
size: 800666 byte
CRC checksum: 6680B172

    Test data (binary example files, ASCII data)

Download binary and ASCII test data:

size: 68791131 byte
CRC checksum: 099DC8D0

    Earlier demo version of CytoSpec:

Download CytoSpec's demo:
version 1.4.03, built 331b, April 2011
size: 24956334 byte
CRC checksum: 14826283

    Please note that you accept with downloading the following license conditions:

    You are using the program at your own risk! CytoSpec does not take any responsibility for damages, problems etc. resulting from use of this program. CytoSpec also does not give any warranty for bug-free operation, fitness for a particular purpose or the appropriate behavior of the program.
    The software is provided 'AS IS'. For your personal use you can make copies and run as many instances as required, but it is not allowed to further distribute this software.


 

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