There are several classes of image data structure.
On-line plotting of light intensity changes over time is an important function during physiological experiments. There are two ways for time course plotting of image data. Both display mean values within Region Of Interest (ROI) defined by the user. "Segment plot" plots throughout one segment of images. "File Plot" plots throughout one segments (stack of segments a file). There are advantages and disadvantages for both methods.
When doing file plot, caution should be taken about some options in the "image-option" menu. As described above, "keep prev ROI's", "keep previous background, ratio & [Ca] param" affects time course plot, and if "save back parameters" is checked, all headers of segments in a file are overwritten with "previous" parameters.
For detailed description about time course plotting, refer to the "ROI Manipulation and Time Course Plot Windows" section.
When image is analyzed quantitatively, signal must be separated from the background. This is very important issue especially when ratioing method is used. There are several methods in this program for this purpose. Refer to "background subtraction..." section for detail.
This method can be combined with other methods. This method uses other image stack(s) which has the same format, but without stimulus, as a bleaching reference.
This method is useful when a background image can be taken before fluorescent dye is loaded into a cell. Use take background button in the CCD-online dialog. A taken background image is copied to the first frame of a sequence of images. Refer to the "take background" section as well as "background subtraction..." section. (This method is obsolete, and is not recommended to use.)
Average value of the last ROI is used as a background value.
It is useful for brain slice imaging. After taking images, autofluorescence image stack can be taken from view field without target cells by moving a microscope stage.
Another image can be overlaid on the current image. This overlay
is temporary (overlay information is not stored in the header
in the file) and can be set/reset through "overlay
and linking..." option in the "local image option
menu" or drag'n dropping an Image
window's Proxy Icon onto a destination Image window. Although image
with another overlaying image can be
copied to the clipboard and pasted to other applications as it
is, some application can not print the overlaying image (Photoshop
is ok, MacDraw Pro is n.g.).
A set of A/D (electrophysiological) data can be linked to an image segment. When an A/D data is linked to an image segment and files including those data are opened, A/D data is automatically selected when image segment is advanced in a file. A linked A/D data can be sent to Igor with a coupled segment image data. This linking can be made by "overlay and linking..." option of the "image-options" menu, or by enabling link with oscillo check box in the CCD on-line dialog.
The "display linked AD sweep" item in the image option menu (Menu Bar) switches whether linked A/D sweep is overlaid on time course plot or not.
Synchronicity of time course plots of simultaneously recorded
image data and A/D data is guaranteed. But this does not mean neither
that image recording and A/D recording are started simultaneously nor
that the time difference of the starting of the two recordings are
constant from run to run. Usually A/D record precedes image recording.
TIWB merely display the plots with this time difference as offset of
one plot. This offset is also transferred to Igor Pro when time course
plot is sent to Igor Pro.
Image time course is plotted at centers of exposure durations.
In certain hardware configurations, it is possible to keep time
difference of the starting of the two recording constant from run to
run (in fast read mode).
use Trigger A/D start by PCI-1200 OUTB1 port on fast read mode option.
In data acquisition mode, four windows are displayed. "CCD on-line" dialog box is the main panel for image data acquisition. "full" and "seq" windows display full pixel image and sequential, binned image, respectively. "Image browser" dialog box is used to adjust contrast, move frames in a sequential image data, select raw or ratio display, etc. In this section, functions of the "CCD on-line" dialog box are described. The use of the remaining windows is described in the "Browsing data (off-line)" section.
This window appears by selecting "CCD On-line" in the Window menu. If no camera is connected or camera initialization failed, this menu item does not appear.
This box sets conditions for "full pixel image" acquisition. Full pixel images can be acquired anytime.
It saves the current displayed image. It displays a save dialog box with a default file name which can be changed by the user. The same name as the current seq data id is assigned to the file name of the full image. A file name extension ".dts" is added to the end of the file name which can be deleted.
It starts continuous acquisition for focusing. During focusing, this button changes to "stop". If exposure duration is set too short, the actual duration is displayed in red at the bottom of this box.
When a frame-transfer or interline camera is used, focusing is much faster and is terminated by just clicking a mouse button whenever mouse cursor is placed (Shape of mouse cursor is changed to show this state).
- OS-X version: During this "fast focusing", user can change automatic gain adjust factors by up, down, left and right arrow keys.
- OS 9 version: During this "fast focusing", user can change subregion size (3 steps; full frame, 2/3 size and 1/3 size) through the keyboard without terminating focusing. User can also change binning factor and automatic gain adjust factors by keyboard. (The key operation in focus mode is different for Scion LG-3 frame grabber.)
| key | action |
| space key | toggle subregion size |
| '8' and '2' keys | increase or decrease subregion size |
| '4' and '6' keys | increase or decrease binning size |
| up, down, left and right arrow keys |
change automatic gain adjust factors |
Duration (exposure time) for focus or single frame ("take").
It defines which filter (wavelength) should be used for focusing, which is enabled only when an appropriate filter exchanger is equipped.
It takes a single frame. Filters for this single frame taking are chosen by the check boxes ("f1", "f2", "ex1",.. check boxes) below this "take" button. With multiple filters are chosen, "take" takes multiple images of multiple filters at once. This function is enabled when a filter exchanger is available.
Parameters (binning, duration, auto/fixed gain, filter type) are stored and can be quickly retrieved by this menu. This is useful when longer exposures for fluorescence and shorter exposures for transmitted images are switched.
With this check box on, a child dialog appears which attaches to the left of the "ccd on-line" dialog. This function provides another way to take tame-lapse images. With the "autoRun" check box on, single frame mode of "full frame" images are taken at the interval set in this dialog. The number of frames is either not-limited ("free" mode) or limited ("train" mode). With "autoSave" checkbox on, image data is added to a file selected through "file" button in this dialog (it has a default file name). This "easy time lapse" function enbles two independent time-lapse recordings with "seq" image functions. For example, when "seq" is taking Fura-2 ratio pairs every 2 seconds with binning 5x5, you can take RFP signal and GFP signal from the same cells every 30 seconds with binning 1x1 with the "easy time lapse" function. If the "seq" time lapse and "full" time lapse should be synchronized, "auto run" and "link full frame" functions in the "segments" box in this dialog should used instead.
It determines the binning factor of the full frame (serial and parallel binning factors can not be different). Larger binning factors increase focusing frequency, but reduce the spatial resolution of the full frame image. Binning factor can be changed by keyboard without termination of focusing in "fast focusing" mode.
When it is off, only areas corresponding to sub-regions in the "seq" image are exposed (this is not a "full pixel image" any more). This mode increases focusing frequency. When subregion size is changed in "fast focusing", this "seq image-dependent subregioning" of the full frame image is canceled.
It enables automatic gain, i.e., minimum and maximum values are set in min/max of display image browser window. In other words, images are displayed with the appropriate contrast. Cut-off threshold for upper and lower limit can be set through the "cut-off" button right to this check box, which controls contrast of the image.
Cut-off limit in auto gain focusing can be set through this button. This changes contrast of the image. Cut-off limits can also changed by keyboard in focusing (in "fast focusing"). Lower and upper cut-off values are set by %.
Focusing is averaged by number of frames set in the text box. This is live averaging. Taking a single frame by pressing the "take" button is also averaged.
Background is subtracted in focusing image (and also taking a single frame by "take" button). Raw image data is processed before display, and the data saved to the disk is also after processed. Background image is taken by pressing the "taking background" button. Binning factor must match between background image and focus image, but exposure time does not have to match between them because background image value is normalized by its exposure time before subtraction (actually "division" is done).
Digital CCD cameras allow "binning" pixels and acquisition of "sub-region".
Digital CCD cameras have fixed pixel arrays on their chip surfaces. Incident photons are "transformed" into electric charge (photoelectrons) in each CCD "electric" well, corresponding to each pixel. This array of electric charges is sequentially read by an A/D converter in the X-Y dimension, which makes an image. Some CCD's are capable of reading (A/D converting) more than one electric well at a time, which is called "binning". Binning is done in the Y dimension as well as in the X dimension. The merits of binning are acquisition speed (A/D conversion is one of the rate limit factor for frame rate) and sensitivity. Binning combines multiple electric charges, resulting in a larger digital value, yielding images with higher signal-to-noise ratios (S/N). The disadvantage of binning is lower spatial resolution.
A full frame image uses all the pixels on the CCD exposed surface. Sometimes a sub-region of the CCD is enough for acquisition (e.g. most of the image field is just background). By selecting sub-regions of pixels, the amount of data to be digitized is reduced and therefore higher acquisition rates can be achieved.
You can define sub-regions by dragging rectangles on the "full frame image" window using the mouse when the radio button is on (or by pressing the shift key when the radio button is off).Note: Although this software supports multiple sub-region acquisition, Photometrics's PXL series and Sensys cameras do not accept multiple sub-region due to a bug in the firmware of the CCDs. Sensicam and Princeton cameras do not allow this feature, neither, due to a limitation of the firmware. Photometrics's Quantix accepts multiple sub-regions.
It clears sub-region settings, and a full frame image is acquired.
Binning factors can be set through these.
Radio buttons in this panel indicates whether mouse
selection selects and modifies sub-regions or multi ROI's on the "full
image" window. This selection can be reversed by pressing the shift
key while using mouse on the "full image" window.
The time course of intensity changes can be monitored on-line and off-line by defining ROI's. ROI's can be defined by dragging rectangles directly on the "seq" image window or on the "full pixel image" window. When adding a ROI by dragging a rectangle in the "full pixel image" window, the radio button must be on (or off and pressing the shift key). When "seq" image window is active, the active (blinking) ROI can be deleted using the "delete" key, or moved and altered in shape using the mouse.
It clears all the ROI's. ROI's are also cleared when sub region setting is changed.
It indicates whether mouse selection selects or modifies sub-regions or multi ROI's on the "full image" window. This selection can be reversed by pressing the shift key while using mouse on the "full image" window.
For further description for handling ROI, refer to "ROI manipulation" in the Browsing data (off-line) section.
This box sets conditions for each frame of sequential images. There are three modes of sequential modes.
The fastest mode. The computer does not respond to the user
during the recording and no real-time time course plotting is shown.
This mode is only available for frame-transfer type cameras (PXL-37,
etc.).
With Photometrix PXL series cameras (at least for SCSI-connected types), number of frames seems to be limited to 250 by the camera controller firmware.
In case for cameras with PVCAM driver without external trigger-driven exposure settings, the frame interval is a bit longer than the set "exposure duration", since the interval is a sum of exposure duration and readout time. Unfortunately the readout time can not be obtained from the PVCAM library, the exact interval should be read from the interval recorded (recorded lapse time for each image frame is correct (after tiwb version 5/20/2007)). Or an external trigger device to give an interval clock to the camera will provide exact interval as user set. Ask the author for use of an external trigger device which needs some additional coding.
Real-time images and time course graphs are displayed. Whether this mode is available depends on the hardware configulation, and when available, "continuous" radio button appears.
For slow recordings. During exposures, this mode allows filter changes, etc.
In fast read and continuous modes, a duration text box appears. In discontinuous mode, an interval text box is added.
These values are calculated from the min/max values of full pixel frame image which are multiplied by duration ratio and binning factors.
When it is checked, excitation light is turned off, if appropriate filter exchanger / shutter is implemented.
It measures minimum duration necessary for one frame in the current condition. One frame duration must be longer or equal than this value.
In cases for Photometrics Quantix cameras with "Check exposure time validity by camera exposing out TTL" option and Princeton cameras, displayed "minimum" frame time is very accurate. For other cases, displayed values are not so reliable.
It enables automatic gain, i.e. minimum and maximum data values are set in the image browser display min/max. In other words, images are displayed with appropriate contrast.
Caution: There is no warning in the current version of the program when set duration is shorter than minimum duration. The acquisition will run at the minimum duration, and each frame will hold accurate lapse time information (which will differ from preset duration).
A segment is a sequence of images stored on RAM, or on disk in the "segment on disk" situation (refer to the "Data Structure" section). Frame number or duration of the sequence can be set. The frame number in the dialog box is the number of frames of F1 wavelength. The total frame number (F1 frames, F2 frame(s) and dark frame) is indicated below the F1 frame number box. If a sequence duration is set through the text box, frame number is calculated. The amount of memory required for a frame/segment is indicated. Free disk space is also indicated.
It switches automatic time course plot on / off.
It starts segment recording.
It saves the current segment recorded without asking. File name is determined in the file button in the "segments" panel.
Inserts time mark in plots. Since this time mark information is recorded in the comment field as readable format, to whch user can modify, delete or add notes freely in the comment field in "Image Browser" dialog. When the following rules are kept, part of comment is taken as a time mark.
This panel sets the filter mode in a segment.
It decides whether filter exchanger is used or not.
The hardware configuration dialog box for the filter exchanger appears, when available.
Each light wavelength is set as voltage. Delay can also be set.
Negative values can be set to the "delay" parameter for TIL-monochrometer configuration. When negative values are set to this parameter, light source change is delayed with this period unless light is turned off (dark position). This further decreases unnecessary illumination time before "actural" exposure. To enable this function, make sure that the wiring is done according to a section of the manual, "Both Photometrics PXL/Quantix and TILL Polychrome monochromator controlled with National Instruments PCI-1200".
Choose Modem or Printer port.
When it is on, the first frame of a segment takes a dark image (i.e. no filter), which can be used for background subtraction of "dark current" and background light.
These are extra light path options. One extra frame is added to the segment by each extra option. These are used in cases when epi-fluorescent images (usual B or G excitation) or a bright field frame are needed in addition to the F1 and F2 image set (usually for Fura-2 ratio imaging). Since this mode requires a special light path changing device, these options are available in case this program is customized for a special hardware configuration.
By checking these check boxes, the first and/or last frame of a segment is taken with F2 filter.
By checking it, F1 and F2 filters are selected alternately. In this mode, a set of F1 and F2 images are counted as "one frame". As the duration set in the "each frame" panel is for one exposure (one frame), interval in the "each frame" panel must be double that duration.
By checking it, three colors, i.e., F1, F2 and Extra 1 filters, are used alternately.
When it is on, previously taken background image is copied to the first frame image, and used for background subtraction (refer to "background button" section). If there is no "matched" (same binning factors) background image previously taken, this option is ignored.
A sequence of images is taken in the following order: dark image, extra 1 filter image, extra 2 filter image, extra 3 filter image, body of images. The "body of images" consists of (F2 1st image, F1 images, F2 last image) or (F1-F2 alternate images).
"Segments" is a stack of "segment". Multiple of segment are stored in disk as "segments" consisting of a file. Each segment is stored and loaded to/from a segments file. File name is set by "file" button. Segment is appended to the current file (segments) unless file name is altered.
auto name button determines the automatic naming of a new file. The first digit of the automatic name indicates year (last number of the year), second digit stands for the month (1..9 and o, n, d), third for day of month (1..9, a..). The fourth digit distinguishes segments (1..9, a..z). When "auto name" button is pushed, the selected folder is checked and the earliest segments number is used for the new file name ("6511.dts" for the first file of May 1st, 1996 "6513.dts" in case "6111.dts" and "6512.dts" have already existed). Longer file names can be used instead of this automatic names. File name extension ".dts" is added automatically, which can be deleted.
If this option is on, a segment is automatically acquired with an interval set by the user. Soon after this check box is enabled, acquisition starts, which can be terminated by pressing cmd + '.' keys or clicking stop button in this panel.
With this option activated, experimental conditions and time executed are added to the "Log" window every time a segment is recorded.
Segments of images are periodically taken with this option active, otherwise only full frame is periodically taken if "link full frame" option is on. When both of this "seq" option and "link full frame" option are turned off, "auto run" option is dimmed.
Segment acquired by auto read is automatically saved (appended) to the specified file.
Automatic save is done with interleaving preset number of exposures. Zero means every segment is saved. Refer to the "interleave" section for the definition of the word.
This check box switches filePlot feature. This item is dimmed when "auto save" is disabled. "a. g." check box (standing for "auto gain") switches automatic plot gain of the filePlot in recording.
With this option selected, one full frame image is acquired just before the acquisition of a segment of "seq" images. The camera conditions set in the "full image" panel are used for this full frame image exposure. If "fullAutoSave" option is selected, the full frame images linked with seq images are automatically saved. This option provides the capability of monitoring non-binned, high spatial resolution images prior to the acquisition of faster sequences of binned images (lower spatial resolution).
When this option is selected together with "link with full",
full frame images which are acquired before "seq" images are
automatically saved. The name of the saved file is the same as the
"seq" image file but the extension is changed to ".dtf".
Clicking on this button displays "Take Background Dialog". A background image is subtracted from images of a segment when an appropriate background image has been set to the first frame. To achieve this, background images must have been taken using this dialog prior to acquisition of images, and "1st frame dark" and "use back image as 1st frame" check boxes in filter configuration dialog must be selected.
Taken background images are also used in the "background subtraction in focusing" mode. In this case, duration (exposure time) does not have to be matched.
Background images are always acquired in full frame. The binning factor of the background images must match to those of the target images (duration can differ, though the same duration is preferred). When several binning factors might be used during an experiment, background images with the expected binning factors should be taken before starting sequence acquisition. The different expected sets of conditions, such as binning factors, expose durations (and camera gain for series 200 cameras) must be registered prior to image acquisition. Corresponding (same location) pixels are subtracted from F1 and F2 images during background subtraction. For example, in panel A, the sub-array is a rectangle with address (1,1)-(4,3) on binned map. This map is displayed as "binned" location, that means if binning factor is set as 3x3, the actual CCD pixel address for that sub-array is (3,3)-(12,9). When subtracting the background image, each corresponding pixel (binned) is individually subtracted, allowing the change of sub-array shape during the course of an experiment (panel B). In addition, since the background image is a full frame (but binned) image of the CCD array, there is no need to re-acquire a background image after modifying the sub-array shape. When staining a cell by injecting dye through a patch pipette or microelectrode, a background image can only be taken before dye loading, and therefore the sub-array shape can not be decided at that time. |
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Photometrics and Princeton CCDs allow to set the sub-arrays starting from any pixel. However, if you set a sub-array starting at CCD address (2,2), and set binning 3 x 3, the sub-array can not be aligned with the background image, because background images are always acquired starting at CCD address (0,0) (see figure). To avoid this problem, the sub-array starting points (left top position) are always adjusted to the background matrix in TIWB.
take button takes background images of the selected
conditions.
When the button indicating camera gain/speed in the "CCD online" dialog is pressed, the camera configuration dialog box is displayed.
Select fast or slow AD converter when available. Though this option seems not to work for MicroMax with EEV-57 chip.
With this option on, the start of electrophysiological recording is precisely matched with the starting of camera acquisition (in fast read mode). To enable this, refer to the wiring description section for further connection.
frequency of the A/D converter
gain of the A/D converter
gain of the analogue multiplier (for Cascade and QuanTEM
cameras)
For frame transfer type cameras. Usually frame transfer CCD cameras do not require a mechanical shutter in front of the CCD chip. In cases when the shutter open/close TTL signal is used for other purposes, this signal is available when this check box on.
In fast read mode, exposure timing is controled by external clock. For PXL series cameras, this option is necessary. For Quantix cameras, this is not necessary because of its acurate internal clock. To enable this function, refer to the wiring description section for further connection.
With this option on, the start of electrophysiological recording is precisely matched with the starting of camera acquisition (in fast read mode). Either "exposing output" of camera controller or OUTB1 output of PCI-1200 is used for this trigger. The latter is only available when "use National Instrument's card for external clock in fast read mode" (above) is on. Refer to the wiring description section for further connection.
With this option on, frame rate is checked whether it is shorter than the minimum time. Since this function uses National Instrument's PCI-1200 card, appropreate wire connections must be made.
Camera gain can be selected.
It enables simultaneous electrical (A/D) and optical
recording and stimulation (D/A). "Record" button starts electrical
recording and optical recording of one segment. Electrical recording
starts simultaneously with a segment acquisition, so that the lapse
time recorded for each image frame is precisely matched with the time
in the electrical record. "Link CCD and Oscillo" dialog appears when
this option is active.
When image sequence is automatically run repeatedly through the "auto run" function and electropysiology record is linked to it, the "c" variable (stimulator counter, refer to this)
is incremented every time image sequence is executed. Duration of
electrophysiology sweep should be shorter than the duration of image
sequence to be linked every time.
When this box is checked with the "link with oscillo" check box on, A/D data taken simultaneously with a segment of images is saved automatically when the image segment is saved. It does not mean that the A/D data is saved automatically at the end of A/D recording. But, A/D data is saved when "save" button in the "segment" panel is pressed or when a "segment" recording of images finishes with automatic saving of segment recording enabled. If an A/D data is saved in this fashion, the data header of the image segment holds linking information to the A/D data.
With this option on, an independent A/D segment(s) is executed before a pair of an image segment and A/D segment. For this "preceding A/D segment", a separate parameter for stimulator and oscillo panel can be used through the "exp.set" menu. With "enable train" option on, this preceeding A/D segment is executed with train option in the "oscillo panel" on. With "auto save" check box on, this preceeding executed A/D run is automaticly saved to the disk,. This function is used for cases such as pre-pulse stimulatio before imaging, etc.
With this option active, image acquisition is withheld
according to the preset pattern. An image acquistion (one
segment) and an A/D session are paired for "on" times, and then A/D
sessions are solely performed for "off" times. The number of
train preset in the CCD panel applies for the total A/D sessions.
Refer to the "Linking A/D data" section in the "Concepts" section of "Imaging".
Through this menu users can use store/recall protocols for seq image parameters. Inside the program, all the parameters are stored when a parameter set is made/updated. And limited number of parameters are recalled when a named parameter is selected in this "exp.set" menu. The kind of parameters to be recalled can be chosen in a dialog which is specific to each parameter set.
Data files can be opened by "Open" in the "File" menu (shortcut: cmd+O), by double clicking the data file icons, or by drag-and-drop the data file icons over the icon of TIWB. Available data formats are listed below.
Images can be closed by clicking the close box at the left top corner of the window or by "Close" in File menu (shortcut: cmd+W) when the image window is active.
Since 2012.10 "Save as..." functions were moved to "Export..." menu in the "File" menu.
Images can be copied to the Clipboard by "Copy" in Edit menu (shortcut: cmd + C). The color is the same as the displayed LUT, but the contrast, i.e. 12 bit or 16 bit data precision, is not preserved in the data exported to the Clipboard. PICT option setting affects the style of images.
To copy the LUT color belt in the Image browser, use "Copy Special" in the Edit menu.
If Copy command is evoked when the Image Browser dialog is at the front (active), a corresponding Image is copied to the Clipboard.
At "Copying", image parameters (ROI locations, contrast conditions, ratio options, etc.) are also copied into a special memory area, which can be "Pasted" onto other image data through the "Paste Special.." and "Paste Special" in the "Edit" menu. By this way, parameters of an image data can be easily transfered to other image data sets.
Images can be exported to Igor Pro software by selecting "Export to Igor" in "Export..." submenu in "File" menu (shortcut: cmd+E) when an image window is active. Time course plots are also exported to Igor Pro. A linked A/D (electrophysiological) sweep can be sent to Igor if the image has a linked A/D data (refer to the "linking A/D data" section in "Concepts").
This function enables easy creation of experiment summary by printing resultant Igor Layout.
Some parts of time-course plot (peak detection range, etc.) are put in a different draw layer in the Igor Graph window. Draw Layer can be selected in Igor to crick the "Environment icon" (a tree is drawn) shown at the left part of Graph windows with pressing an option key.
Sends current image to Igor. Image properties are determined by PICT options which is equivalent to "PICT options" (in image-option Menu).
When "as PICT" is chosen, image is exported to Igor as a PICT image data.
When "as 2D wave" is chosen, image data is exported to Igor as a 2D wave data, which can be further analysed in Igor.
Sends reference image(full frame image) to Igor.
Sends each plot to Igor.
Line thickness (in Points) for time course plots of images in Igor graph. (ranging 0.0 - 10.0).
Graphs created in Igor by exporting plots in tiwb uses multiple graphics layers defiend in Igor. Most of the elements of graphs are put to the "UserFront" layer which is the default layer. Color boxes indicating time range for Fbase calculation, fitted range, etc. are put to "UserBack" layer. The dotted vertical line indicating the time point of the image frame is put to the "ProgFront" layer (from the version of tiwb 2005.10.25).
How to access layers in Igor graphs: Activate a Graph window in Igor. Select "Graph"->"Show Tool" (cmd+T). Two buttons appear. Press the second button (drawing) to enter Drawing mode. Hold down the "Draw environment popup" icon (treen and grass) with the option key pressed, layer menu appears. For detail, refer to the Igor manual ("Drawing" section).
If it is on, sent linked A/D plot is merged with ratio plot.
An A/D plot exists as a wave in Igor. By setting number of graphs in this text edit box, more than one graph of A/D sweep are displayed in Igor, which share the same wave. This multiple graphs are convenient when a number of "expanded" sweeps are needed to be displayed from one long A/D record.
Line thickness (in Points) for A/D plots in Igor graph (ranging 0.0 - 10.0).
Since interval of time-lapse image frames are not always regular, time-course plots are sent to Igor with "lapse" wave which is used as the x-axis data. When this option is checked, time-course plots in Igor does not accompany corresponding "lapse" wave. This option is useful for Igor functions which receive constant interval data (e.g., fit functions).
Raises plots onto a Layout in Igor.
If it is on, sent plots are added to an existing Layout in Igor. If there is no Layout in Igor, a new Layout is produced. To create a Igor Layout of multiple data sets, check this option.
Sends information text (the contents of which is determined by "log format..." which is equivalent to "Log Format..." in Edit Menu) to a Layout in Igor. Size of font for this memo text in Igor layout is set by font size text box.
When composing a Layout sheet of Igor with multiple data sets, you can choose an area of divided Layout sheet to send components of data (graphs and images). Layout sheet can be divided into n x n parts by setting numbers in the "division of layout" text boxes. Target layout part can be chosen by single clicking the part in the divided boxes in this dialog. Locations of each element can be modified by dragging elements displayed in this dialog.
Images and plots can be exported to Microsoft Excel by selecting "Export to excel" in the "export" submenu of File menu. Since plots with data values are exported, format of charts in Excel can be modified. However the look of charts are not good enough for publication without manually modifications on Excel. If one need a publication-quality charts easily, "export to Igor" option is recommended.
Data from the active image window can be saved as different format files using the "Export..." option in the "File" menu.
Refer here.
The standard TIFF format supports the feature of image stacks within a file. When frame option is chosen, number of frames from the current frame of the current segment are saved. When segments option is selected, all frames of segments starting from the current segment are saved. If pack into a stack option is checked, all frames are saved as one stack in a TIFF file. In this case, all saved frames of all segments must be of the same size. If this option is not checked, each segment is saved into separate TIFF files named as "*.n" (n represents image frame). Larger numbers than actual ones are accepted for frame numbers and segment numbers (e.g. 999). When image display mode is set to "ratio", ratio images are saved instead of raw images.
If bit depth of image data is larger than 8 bit, data is converted to 8 bit pixels according to the current setting of display range. Color look up table (CLUT) is not included in the saved TIFF file.
If bit depth of image data is larger than 8 bit, data is converted to 8 bit pixels according to the current setting of display range. Color look up table (CLUT) is included in the saved TIFF file.
When data is 12, 14 or 16 bit, this option is enabled. This format is not standard TIFF format, although data is not deformed.
Full color TIFF is made. In this format, ROIs are also included if PICT option setting is properly set.
Image frames can be packed into a QuickTime movie file. Video playing frame rate can be set through "movie speed" text edit box. 1x means that the movie will be replayed at real time (for playback at 10 frames / sec of a sequence of images acquired at 1 frame / 10 sec, set value 100 in this text box). The range of frames can be selected by segments or frame basis: When frame option is chosen, number of frames from the current frame of the current segment are saved. When segments option is selected, all frames of segments starting from the current segment are saved. PICT option settings affect the style of the images.
Some movie formats can be chosen. "animation" does not compress image data, which makes huge movie files. "jpeg" option reduces movie file size to 1/10 or so, and the quality of the image seems not to be bad when the "normal" in "quality" is chosen. Thus the "jpeg" movie formst is recommended.
Movie files created by versions of tiwb before 12/18/2003 held movie data in their resource fork. This format can not be read in Windows environment (e.g., QuickTime Player for Windows, and Power Point for Windows). Tiwb after 12/18/2003 stores movie data in the data fork, which can be read in Windows environment. To make movie files created by the old tiwb versions readable in Windows environment, movie files are needed to be read by QuickTime Pro player in MacOS and saved as another file to convert the data format.
Sequential 3D images are saved as a QuickTime Movie format file. Among choosable movie formats, "jpeg" format seems to produce reasonablly small size of movie files. The "animation" format does not compress the image data, resulting movie files of huge file size. Tune the size and quality with the quality option.
Note: Images can be directly ported to Igor Pro, ImageJ or Microsoft Excel software by export functions.
The "Image Browser" window is the main control panel for browsing image data on-line and off-line. In this section, the functions of this dialog box are described.
This dialog box appears when an image window is activated, and controls contrast, Look Up Table (LUT), frame transition, etc.
The side drawer part (right part in this figure) is available only in OS-X versions. This drawer is shown/hidden by pressing the "Toolbar control" button indicated in the figure. Checkboxes in this drawer part are equivalent to those in the "Image Option (global)" menu in the menu bar.
Images can be displayed at any desired magnification. Magnification can be set using the slide text edit box located at left top of the Image Browser Dialog.
When F1 and F2 images are displayed together, the LUT range of them can be manipulated separately. This button toggles the manipulated LUT.
This menu enables digital filtering of the image. "smooth",
"gaussian", "more smooth", "sharpen" and "more sharpen" can be chosen. The same
algorithms are used described in "Filter
functions (Digital)". The "Filter Functions" in the local
image
option menu creates a new image window, and this "filt" menu just
change the image display. Image data is not altered. Usually
"smooth" or "gauusian" are most used than other filters. Though this
filter function does not
modify original image data, it affects image calculations. Time
course
plot and ratio image calculations are performed after filtering.
Filtering before ratioing is very powerful method to reduce noise.
The "remove outlier" filter replaces a pixel by the median of the
pixels in the surrounding if it deviates from the median by more than a
certain value (the threshold). When this filter is selected together
with other on-line filters, "remove outlier" is performed, the another filter is applied.
Information attached to the image is displayed in the information box at the bottom. This information text can be copied to the "Log" window by selecting "Log" in Edit menu (shortcut: cmd+M).
A comment text can be attached to a segment of image. By
clicking "info comment" button, information box turns to be comment
box. The length of the comment is limited to 127 characters.
"mark" tag: time mark is recorded in the comment text starting with "mark" keyword (e.g., "mark1.1sec"). For detail, refer here.
"pix" tag: pixel mark is recorded in the comment text as pix(XX,YY) format (e.g., "pix(50, 50)").
In this software, images are displayed in a 256 scale. LUT is a table of 256 colors corresponding to pixel values. Several LUT's can be selected from the LUT menu in the Image Browser Dialog. If a user wish to use a special LUT, LUT's can be modified or appended by editing the 'clut' resource of the software, although the modified resources will not be preserved in newer versions of the software (if there is a strong need to use custom LUT's, make a claim to the author to improve custom LUT usage).
The "original LUT" item in the LUT menu is available when an image data has an original LUT. TIFF and Olympus Fluoview format files can have original LUT.
Use of Magenta instead of Red in presentations is encouraged
for colorblind people. 
Image contrast can be changed by dragging the small triangle tags to the right of the LUT. LUT can be inverted by dragging one triangle tag over the other. Top/bottom limit indicating buttons display a dialog box allowing to set display contrast by set values. The left value in up/low limit is the maximum (or minimum) value of LUT which determines the image contrast. The right value is the maximum (or minimum) value of the range of the displayed LUT. By pressing the "optimize" button, LUT display contrast (max/min) can be expanded (optimized) within these range values. This "optimize" button is equivalent to the "LUT range optimize button" in the right upper coner of the image windows.
When an image file holds multiple segments of images, segments can be selected by this panel.
Replots "filePlot" with the current ROI's (refer to the "Time Course Plotting" section).
Segment within a file are automatically advanced. If both animate check boxes in segment and frame panels are selected, all frames in each segment are animated, then the segment is advanced. The animation runs when the image window is active (selected) and mouse cursor is outside the window. The animation rate can be changed by "animation rate" in the "image-option" menu.
This option sends the number of segments set in the edit box to the Layout Window, starting with the current segment. When negative values are used, the order of the printed segment is reversed (zero is not allowed). All frames in each segment are exported. Settings in PICT options affect the style of the images to be sent/printed.
When an image segment holds multiple frames of images, a particular frame can be selected using this panel.
frame can be changed by clicking left and right arrow button. Left and right key on the keyboard can also be used.
"replot" button appears in the "segment on disk" situation. The time course plots of the segment are not automatically updated press this button to update segment plots.
If this number is more than 1, number of frames after the current image frame (including the current frame) are averaged and displayed. Time course plots are not affected by this averaging.
This menu allows the selection of raw image, ratio image or mask to be displayed in the image window.
ROI's can be added and modified by directly dragging them in the image window.
When the mouse button is pressed in the image area with shift key pressed, positions of mouse click are recorded in the "Log Window". This text information can be copied and pasted to other applications (e.g., Excel) to calculate distances of points in the image, etc. After three points are recorded in the memo window, an additional figure can be also printed in the memo window, which indicates the angle of the two lines drawn. The format of the figures printed in the memor window can be chosen in "position length measure options..." menu item in the "image-option" menu.
There are two methods for tracking particles through a stack of images manually.
Image contrast is optimized, i.e., display range is expanded to the min/max range of the pixels. This button is equivalent to the "optimize" button in the Image contrast setting dialog.
These two triangle buttons are equivalent to the small triangle tags right to the LUT in the image browser window, which enable changing image display range.
When Option key is held down with these operations, all
the image frames following the current frame are also shifted or rotated. When option key + shift key is held down, all the frames are shifted/rotated.
When Control key is held down with these operations, all
the z slices following the current zslice are also shifted or rotated. When control key + shift key is held down, all the zslices are shifted/rotated.
This shift/rotate
feature us useful when a target cell changed its location or angle, and
F/F0 calculation must be done. Or two images taken at different
times (and cell moved during that interval) needed to be
overlaid.
It shows much faster animation than the "animation" function (of frame) in the image browser dialog. Short cut key: cmd + right arrow key (start and stop).
Frame can be moved back and force by these buttons.
Shows this page.
ROI's can be added and modified by directly dragging them in the image window.
For basic concepts of time course plot, refer to the "Concepts" chapter.
Time Course Plot WindowBy default, all frames of a segment share one set of ROIs. But each frame of a segment can hold separate sets of ROIs, which is useful for tracking moving targets, which is enabled by changing a ROI by dragging with the option key pushed. Once this action has been made, a list of ROI shape information for each frame is prepared in the memory, and each frame holds each ROI shape. To know whether a segment of image holds a single set of ROI or each frame holds separate ROI set, see the "frame" panel of the "Image Browser dialog". In the "multiple ROIs" mode, "clrFrRoi" button appears. To remove "separate sets of ROIs", push this "clrFrRoi" button. To change the shape of a ROI of a particular frame, option key must be held down. Without holding down the option key, the shape of the corresponding ROI of all the frames are changed (other ROIs' shape are not altered by this action). By pressing down the option key and ctrl key, shape of the corresponding ROI of the succeeding frames (including the current ROI) are changed. In the file, this "multiple ROI" information is stored not in the header of the data packet but appended to the image data, so that if an image segment does not locate at the end of a file, and if it was not saved with the "multiple ROI" information, newly added "multiple ROI" information can not be added into the file. If a segment of image was saved with "multiple ROI" information, or if it is located at the end of a file, newly added "multiple ROI" information is stored in the file, if "save back header" option in the "image-option" menu is checked.
When an image segment has multiple frames, the time course of intensity changes in each ROI is plotted in separate windows for raw1, raw2 and ratio values. These plots are automatically updated responding to ROI changes. "raw1" corresponds to the mean values of raw data of F1, "raw2" corresponds to those of F2 in alternate filter mode, and "ratio" corresponds to those of ratio data values. A vertical line in the plot windows indicates the current frame, which can be dragged to change the current frame.
Background values used for subtraction (in ratio display mode) are also plotted as dotted lines in the raw plots.
Plot option menu
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Segments of images can be plotted as "File Plot". By clicking the "FilePlot" button in the segment panel of the image browser window, the time course of the current ROI's is calculated and plotted throughout the current image file. To change the plot range, click inside the plot window and a dialog box with upper/lower limits appears.
File plot can hold a set of ROI's, background parameters, etc (called "PARAMETERS" in this paragraph). Whether using a single set of PARAMETERS for all segments or using each set of PARAMETERS of each segment can be chosen. The latter case is enabled by selecting "keep prev ROI's" and "keep previous background, ratio & [Ca] param." options in the "image-option" menu, which is useful when target is moving so that each segment should have different ROI's.
This menu sets general parameters for image browsing functions (parameters for each image are changed through "image option menu (Local)".
When checked, changes in parameters (e.g. LUT type, display min/max, ROI settings, etc.) are saved back to the data file header. In on-line mode (full & seq images), this function is inactivated. In case of TIFF files including Olympus Fluoview files, information (ROI information, LUT, etc.) is written to /retrieved from an external file of a name with extension ".tiprm". This addisionally made "*.tiprm" file can be discarded which does not affect the original data file, just lose information added by TIWB. TIWB versions before 2002.9.28 stored this type of information in resource fork (resource type 'tihd') of the data file instead of an external file. This way caused data file crash if the data file is located in Windows machine and is accessed through Mac OS-X SMB file sharing mechanism. Saving back is done when a file is closed or a segment is moved to another segment in a file (in case of TIWB format).
ROI's IDs are displayed with the frames. Size of the font can be chosen.
ROI's shape cannot be changed, just moved.
Keeps parameters listed below when segment is changed.
ROI's are kept when segment is advanced in a file.
LUT range is kept when segment is advanced in a file.
Window position of the image is not changed when segment is advanced in a file.
Mask area is preserved when segment is advanced in a file, provided that conditions such as number and shape of sub-regions, and binning factors, are the same.
Background parameters, [Ca] calculation parameters and ratio parameters are kept when segment is advanced in a file.
ROI color combination is preserved when segment is advanced in a file.
Format for image output used for Clipboard Copy, Print, QuickTime Movie, Export to Igor Pro, etc. is defined through this dialog box. Preview is displayed in the dialog.
This menu is equivalent to the "Export Image Format..." in the "Edit" menu in case for image data.
ROI frames are added to the image.
magnification is ignored 100% magnification is used for image.
the full pixel array of the CCD imageis set as the boundary. If this option is off, a rectangle including all sub-regions is used for image boundary. This option is ignored when both F1 and F2 images are used in alternate filter mode.
when active (blinking) ROI exists, only that area is used for image. This option dominates to "boundary is full CCD pixel area". This option is ignored when both F1 and F2 images are used in alternate filter mode.
Image is made up with a shown part of the image, i.e., (a part of ) image displayed in the window,
When alternate filter mode is selected, image type (F1 or F2 or both) for image output can be selected.
Time course plots are also included in the resulting image. The shape(s) and relative position(s) of plot(s) are as they are on the screen.
With the "Use Plot in Igor if exist" check box active, graph windows of time course plots exported in Igor are used instead of the tiwb intrinsic plot windows if exported plots exist in Igor.
Contents of the 3D display window is included, if it is opened.
If this is checked, the main image window is not included.
Linked sequence of images is also included. In making a QuickTime movie with this option active, linked stack of images is also updated for each frame.
selects behavior of layout / print buttons in the Image Browser Dialog.
Switches whether linked A/D data is overlaid onto time course plots or not.
Sets the animation frame rate.
With this option on, mouse cursor shape does not change when it is moved onto image windows.
Format of the length/angle
measurement is set by this menu item.
This menu sets "local parameters for each image", i.e., parameters inherent to each image. General parameters (e.g., save back header switch, PICT option, etc.) are changed through "image option menu (Global)".
The mode of background subtraction prior to ratioing is selected in either of the below dialog boxes. For TIWB native sequential image data, the complex dialog opens, and for other image format files and for on-line mode the simple dialog box opens.
Note: The simple dialog box appears in on-line image windows ("full" and "seq" windows) in acquiring images. Once image data is saved as a file and opened as off-line image data, the "complex dialog" is applied. During acquisition, the recording files can be opened as off-line files at the same time.
Background values are not subtracted.
First frame of the image sequence is used as a background image. This is used when "1st frame dark" and "use back image as 1st frame" options in the on-line dialog are selected.
The mean pixel value of the ROI defined last is used as a background value. When only one ROI is chosen, ratio value is 0.
A pair of user-defined, fixed values is used for background subtraction.
It is effective to mask noisy background in ratio images. Ratio image is calculated by dividing an image by another image. When background subtraction is properly done with a fluorescence image, pixel values in the background area (image area with no object) should be close to "0". When dividing images have been processed by background subtraction, backgourn area in resulting ratio images has non-sense values due to division of very small pixel values. With this option on, pixels in dividing image of which values are less than the threshold value are masked in ratio calculation. This function just "masks" pixels in image display and does not exclude masked pixels in averaging ROI pixels for timecourse plot.
When it is on, pixels of which values are below than the lower limit of the display range are ignored in averaging a ROI's pixel value.
When it is on, pixels in masked area are ignored in averaging a ROI's pixel value.
Through this check box, reference (full frame) image can be set/reset. Same as the "reference (full frame) image..." option in the local image option menu.
Bleaching correction is done by the following method: Fc(t) = F(t) * (fitfunc(t0) / fitfunc(t)) or Fc(t) = F(t) + (fitfunc(t0) - fitfunc(t)).
Fc(t) is compensated F value at time t. F(t) is a F value after autofluorescence correction at given time t. t0 is lapse time of the first frame. fitfunc(t) is a function with fitted parameters. Data used as bleaching baseline are taken by two different ways. One uses part of data points of the same image stack (see "Fit to First and/or Last frames"), and the other uses different image stack(s) (see "Fit to External sweep").
With "fit along each pixel" check box on, bleaching correction is caliculated for each pixel of image frame separately along the time stack, i.e., each pixel of an image has different fitted function. When this option is turned off, time course of sum of all pixels in each frame is fitted and used, in this case only Fc(t) = F(t) * (fitfunc(t0) / fitfunc(t)) method is available. Using Fc(t) = F(t) + (fitfunc(t0) - fitfunc(t)) method with fittine time course of sum of all pixels of each frame will result in much deformed shape (especially for images containing mixture of blight and dimm pixels).
Fit function is chosen from linear, square curve, and exponential curve. Square curve seems to be practically preferred to linear function in accuracy and to exponential function in speed.
Suppose that a time-course plot has an entire
response of a fluorescence chagne, i.e., baseline, a transient, and
returning to the baseline, this mode takes the initial baseline points
and the latter "returned baseline" points as a bleaching correction
base. Ranges of points taken as baselines are set as frame numbers in
edit text boxes appear in the dialog box. Whether to use either the
initial baseline and the last balise, or both can be chosen by
selecting checking boxes. In the plot window, ranges used for baselines
are displayed as green area (see the figure). Of course, the more the
base points, the better fit result, and taking the both initial
baseline and the last baseline procuces better re
sult than using only
the initial baseline, if possible.
When a control image stack is taken for bleach correction, i.e., image sequence was taken in the same condition without stimulus, bleaching correction fit function can be calculated using these "no-stimulus" image stack. Image format must be the same (e.g., binning factor, expose time, subregion shape, etc.).
fit functions for each ROI are also shown in time plot graphs.
When this check box is checked together with "show
fit func" box, not bleach-corrected but raw data plots are displayed
together with fit functions. The figure indicates a raw sweep and fit
result for the first and last points with this "w/ raw data" option on.
In this case, ratio plots are calculated against "not-corrected" data
although "bleach correction" is specified.
refer to the simple dialog
refer to the simple dialog
refer to the simple dialog
ratio parameters (F1/F2 or F2/F1, etc.) and [Ca] calculation parameters can be selected here.
Method of ratioing used in ratio image and ratio time course plot. Ratio is calculated by any of the following methods after subtraction of background value. The background value is determined by several ways selected in the "background subtraction" dialog.
The first frame of an image segment or an image frame in a separate file can be used as Fbase image. In the latter case (F - Fbase), difference between F and Fbase is displayed as "ratio" (though actually it is not ratio). In opening an image segment which uses Fbase image in external file, the location of its Fbase image file is always asked.
calculate Ca concentration basing on ratio calculated as above and display [Ca2+] instead of ratio
applies default parameters.
use current parameters as the default parameter set.
Duplicate the active ROI
locate active (blinking) ROI to the center of the window.
Clears all ROI's.
A window appears through which ROI positions can be edited. Order of ROIs can be organized by drag'n drop an ID tag onto another.
With this option active, ROIs are displayed and calculated as oval.
Change ROI colors.
When this option is checked, the automatic plot recalculation by changing ROIs is truned off.
ROIs are set by a watershed algorithm. Automatic segmentation by a watershed algorithm (Vincent & Soille, IEEE Transactions on Pattern Analysis and Machine Intelligence 13, 583-598 (1991)) is performed. The segmented parts are filtered by intensity (whether the highest pixel intensity of a part exceeds a set threshold value, or not) and size (whether the pixel number of a segment is larger than a set threshold number, or not). ROIs of the same set size are assigned to each filtered part with the centers of ROIs matching to the brightest pixel of the filtered parts.
Refer to the particle tracking section.
Select/deselect overlay image and linked A/D data. Another
image can be overlaid on the current image.
Overlaying an image to another image can also be done by drag'n dropping the Proxy Icon.
An A/D data can be linked to an image segment. Refer to the "Linking A/D data" and "Overlay image" sections in "Concepts".
Usually full frame images are taken for higher spatial information, while lower spatial resolution images (binned and with sub-regions) are taken for sequential images for time course measurements. The latter are usually not enough for precise localization of a specific structure. A reference image can be opened by this menu, containing ROI's from the original image.
A dialog appears which manipulates mask area. Mask can be set by "set area below lower limit" button or manually. This mask is either used merely for display purpose or can be used to neglect pixels for average pixel value in ROIs, which can be selected in background subtraction dialog. The mask information is not stored in the data file.
Area with pixels with values below the current display minimum is set as a mask. Display minimum value of F1 image is used (even in ratio display mode).
It clears mask.
display mode of the image window can be selected.
pen width for manual drawing of mask area on image window.
Analyze Dialog appears, in which statistical analysis of pixel data is done (average, deviation, rms, etc). Results are displayed in the Log Window.
Procuces a copy of the image by calculating from two images.
Digital filter for images. It filters a set of images by temporally and/or spatially. Details are described in "Filter Functions".
A dialog box for the Line Compression function appears.
Calculates a diffusion constant from a FRAP image stack.
Calculates a diffusion constant from a FRAP image stack of a thin structure.
Refer to the particle tracking / QDot tracker section.
Automatic segmentation by a watershed algorithm (Vincent & Soille, IEEE Transactions on Pattern Analysis and Machine Intelligence 13, 583-598 (1991)) is performed. Size and average intensity of each segment is shown by histograms and a scattered plot. Automatic ROI placement by a watershed algorithm can be chosen for automatic ROI selection through the "ROI menu" in the menu bar or contextual menu (right mouse).
Refer to the 3D Display section.
pixcel position indicator can be selected.
Scale length and pixel resolution are set. Scale font size 0 means that only a scale bar is displayed.
Rotate the image (just for display; data is not altered).
Procudes a copy of the image of which data consists of floating point (32 bit) pixels.
Produces a copy of the image with pixel value inverted.
Shows a histogram of pixel density. If ROIs are set for an image, histogram is generated for each ROI. Without selecting a ROI, histogram of pixels of the whole frame is generated.
By selecting the "Analyze..." item of the Local Image Option Menu, Analyze Dialog appears, in which statistical analysis of pixel data is done (average, deviation, rms, etc). Results are displayed in the Log Window.
When checked, data id is displayed in the memo window.
When checked, frame number is displayed in the memo window.
For this operation, pixel masking affects the result ("ignore pixels under display range" and "ignore masked pixels" options in the "background subtraction" dialog.
Select which image to be analyzed.
Calculate a regressed line from pixel location information; pixel values are not considered.
Searches a boundary line... (more explanation needed...).
Particle Analysis is ...
Through the Analysis submenu in the Local Image Option Menu, filter functions are available. These filter functions create another Image data which can be saved as a separate data file. Original image data is not altered. "Online filter function" also provides digital filtering of image data, which also does not alter the original image data but just change the image display.
These filters are implemented using 3 x 3 spatial
convolutions, where the value of each pixel in the selection is
replaced with the weighted average of its 3 x 3 neighborhood.
The 3 x 3 tables shown below are the coefficients (weighting factors)
for the filters.
Blurs (softens) the selected area. It can be used to reduce noise in an image. Hold the option key down for increased blurring. |
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Increases contrast and accentuates detail in the selection, but may also accentuate noise. To minimize this problem, you can Smooth and/or Reduce Noise before using Sharpen. Hold the option key down for increased sharpening. |
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"Edge preserving smoothing" is one of the "selective local averaging" methods which enables noise reduction with less blurring edges. One area of which intensity variance is smallest is selected from surrounding nine areas shown in the right figure, of which mean intensity is assigned to the center pixel. |
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Produces a shadow effect, with the light appearing to come from a direction specified in a dialog box. Three of the kernels used are shown below. |
West | NorthWest |
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These filters rank (sort) the nine pixels in each 3 x 3 neighborhood and replace the center pixel with the median, minimum (lightest), or maximum (darkest) value. Use the Median filter to reduces noise. The Minimum filter erodes (shrinks) objects in gray scale images. The Maximum filter dilates (expands) objects in gray scale images.
User defined function is applied to each pixel. Use v (as intensity), x and y (as position) as variables.
Consecutive images can be averaged together or multiple images in a stack (as segments) can be processed as a image stack. This feature is used in conjunction with the spatial filter functions.
Use 0 not to average frames.
Multiple frames are processed starting from the current frame.
All frames in the stack (segment) are processed.
By selecting "3D View..." items in the local option menu, dialog boxes for 3D display appear. In 3D image windows, the 3D images can be rotated by just dragging in on the 3D images. Altering zooming factor etc. can also be done through changing values in the "drawer window" attached to the 3D image windows. 3D image can be copied by the "Copy" menu (cmd + C).
Sequence of 3D image motion can be recorded as a movie through the "create movie..." button in the drawer window attached to the 3D image window or just dragging mouse with control key pressed. The former way creates "dense" in time movies. The latter creates any motion of 3D image reflecting the mouse motion, which is easy by results in rather "sparse" in time.
Pixel intensity is converted to z-value to create a 3D image. "Overlaied image" may be displayed together if any.
If an ROI is active (selected), area where converted to a 3D image can be chosen from the active ROI or the whole frame.
In case of large size images, calculation takes huge time. With these x and y factors more than one, (x * y) pixels are averaged to make a rough image, then 3D image is calculated. This makes faster but rougher display.
If this is checked, 3D image is displayed as a wireframe instead of rendered image. This is much faster and requres much less memory.
Gain for Z axis. Z axis is determined by the pixel value * z-gain.
When it is on, pixels of which values are lower than the lowest display value are cut off, displayed as the same Z value as the lowest display value.
When it is on, pixels of which values are higher than the highest display value are cut off, displayed as the same Z value as the highest display value.
Surface of a cluster of pixels in 3D image data are extracted and 3D structures are reconstructed. Stack image files created with Olympus fluoview (XYZ image) is applicable for this mode. Currently, the largest lump of pixels is extracted and other smaller ones are removed. Each extracted particles are colored differently. Each particle can be shown/hidden through the check boxes listed in the drawer window.
pixels of intensity higher than this value are picked up.
refer to the "3D View of a 2D image" section above.
only larger particles (clusters) are picked up.
Particles (clusters) consist of pixels smaller than this number are not picked up.
display axis indicator
Time lapse image frames are stacked to create a 3D image. Clusters composed of pixels above the threshold value are reconstructed in the 3D space.
By selecting this menu item, a cross-sectional image is calculated and displayed. If an image segment holds more than one image frame, cross-section along time axis is shown. Otherwise if it has z-section image segments (currently applicable only for Olympus Fluoview XYZ format files), cross-section along z axis is shown. With Olympus Fluoview XYZ format files, dimensions of XY and Z directions are mached.
Selected images are stacked in a 3D space.
2D image data displayed is exported to Igor and 3D view of it is created in Igor.
Particle movements are tracked and analyzed. Particles are displayed in a 3D view.
For QDot tracker, refer to Bannnai et al, "Synaptic function and neuropathological disease revealed by quantum dot-single-particle tracking" in "Single Moecule Microscopy", Neuromethods vol. 154, doi:10.1007/978-1-0716-0532-5_7,
Compress frames to a stack of lines. This function can be accessed through the Image Local Option menu.
When a Macintosh has QuickTime-compatible video digitizer capability (e.g. AV-type Macintosh Quadra 840AV, PowerMac 7500/7600/8500), a live video image can be displayed as an image window. If this program detects the video digitizer function in the Macintosh, it adds the "live video" item to the window menu. By selecting the "live video" item in the window menu, a live video window appears. Live video is displayed when "animate" check box in the image browser dialog is checked and live video window is activated (selected). To stop monitoring live video, de-activate video window (click other parts of the screen). By clicking inside the live video window, you can change video digitizer properties. To save images taken through this function, select "Save as.." in the file menu with this image window active.
