Release Note of BV_Analyzer Ver.0904

Major changes from Ver.0812

1. Additions in analysis functions - Available for MiCAM ULTIMA, MiCAM02, MiCAM01 data

(1-1) [Together by step num] "Sum mode" is newly implemented in the [Together by step num] function. Until the previous version, this function compresses total frame number by averaging some serial frames.
(1-2) [MaxMin by step num] New Function
For compressing total frame number by replacing some every frame with one frame, which has maximum or minimum values in some every frame.
(1-3) [Activation Map] The function showing / exporting number of pixels that meet conditions set in the [Activation Map] window.
(1-4) Detailed setting for analog signal wave display The [Apply All] button, which applies same settings as an analog singnal display to other analog signals, is newly added to the analog signal wave display sub window.
(1-5) [APD for single] Histogram showing APD time and number of action potentials is on the [APD for single] function. This is specialized for analyzing one action potential.
(1-6) [APD for multi] New Function
Overlap a series of action potentials automatically, making statistical analyses, etc.
(1-7) [Drift Remove2] New Function
Removing drift curve with exponential function. This may be useful when LED light is used as fluorescence excitation source.
(1-8) [Setup display option] sub window - Show a threshold value with percentage of change in light intensity.
- Display total pixel number over threshold level.
(1-9) [Remove by SD] New Function
Noise reduction function using standard deviation of data.
(1-10) [Stripe] - Can manually specify coordinates of start/end points for a line.
- Can save/load settings.
- Can display percentage of change in light intensity on a selected line (curve) on one frame.
(1-11) [BV_Thumbnail] - Show basic information of data on the bottom area when mouse cursor is over data.
- Can load data to the BV_Ana by double-clicking a thumbnail image.
(1-12) [Apply all for wave point & overlay] New Function
Overlap waveforms which have same coordinates on all data of the canvas.
(1-13) [Image export by specifying frame No.] New Function
The function for exporting selected frames in BMP/JPG files by specifying frame number.
(1-14) [Effective pixel num csv export] New Function
Export number of pixels that are over threshold as CSV format.
(1-15) Others The [Change Wave size]/[Change Wave color]/[Bold Wave] on the pop-menu were moved to the new menu [Wave property].

2. Additions in Acquisition Function (Available only for MiCAM02)

(2-1) [NoStim-Stim] mode One cycle in this mode is to acquire data without stimulation first, then acquire data with stimulation. One data is created from (data with no stimulation) - (data with stimulation).
(2-2) [Option] button on the [Acquisition] page New Function
Can change signal pattern outputted from the [EXD2] pin in the [Digital IO] connector on the rear panel of the MiCMA02 processor.
(2-3) Display light saturation percentage in a selected region on a monitor image. New Function
Display light saturation percentage (maximum/minimum/average value) in a selected region (rectangle/circle) during monitoring.
(2-4) Others/ Bug Fixed - The bug that the software aborts when unchecking the [preview2] during acquisition.
- The problem during monitoring with over 150msec sampling time.
- When dual camera are used with the [dual calculation process] selected, original data from camera1, camera2 and created data are appeared on the canvas. For example, when the [sub] on the [dual calculation process] window is selected, three data from camera1, camera2 and camera1-camera2 can be shown on the canvas.

1. Additions in analysis functions - Available for MiCAM ULTIMA, MiCAM02, MiCAM01 data

(1-1) [Together by step num]

● The functions for reducing number of total frames can be executed from new menu, [Compression of number of frames], in the popup (right click) menu. "Sum mode" is newly implemented in the [Together by step num] function, which can be selected from [Compression of number of frames] menu.

● The window appears as below by selecting the [Compression of number of frames]-[Together by step num] menu from the popup (right click) menu. The [average num is not changed] checkbox is newly added on this window. With this [average num is not changed] checkbox, the method for compressing number of total frames can be selected, average or sum.

● When the [average num is not changed] checkbox is NOT selected, the total frame number is reduced by averaging some every frames selected in the [Step Num]. This is the same way as old versions. The software replaces averaging times in a raw data with (averaging times)*[Stem Num]. Each differential value is calculated by (1) summing differential values of every specified frames and (2) dividing them by (number of averaging times)*[Stem Num]. This means each value is averaged.

● When the [average num is not changed] checkbox is selected, the total frame number is reduced by summing some every frames selected in the [Step Num]. Each differential value is calculated by (1) summing differential values of some every frames and (2) dividing them by number of averaging times. This means each value is summed.


(1-2) [MaxMin by step num]

● The functions for reducing number of total frame can be executed from new menu, [Compression of number of frames], in the popup (right click) menu. This menu has new sub menu [MaxMin by step num]. With this [MaxMin by step num] function, number of total frame can be compressed by extracting maximum or minimum pixel values from some every frames and then, making one frame.

● The window appears as below by selecting data on the canvas and executing the [Compression of number of frames]-[MaxMin by step num] from the popup (right click) menu.

● Setting parameter Input numbers of start frame and end frame into the [Start Frm No.] and [End Frm No.], respectively. When the checkbox on the left of the [Start Frm No.] box is OFF, the first frame of data is used as the start frame for this calculation. When the checkbox on the left of the [End Frm No.] box is OFF, the end frame of data is regarded as the end frame. Input number into (or select from) the [Step Num] combobox. Frame number are compressed at every frames of the specified number. Select either "MAX" or "MIN" and then, push the [OK] button.

● Newly created data After pushing the [OK] button, new data is created and shown on the canvas. The background image is the same as the original data.

- Number of total frames = total number in selected range divided by step number
- Sampling time = (original sampling time) * (step number)


(1-3) [Activation Map]

● New function for showing / exporting number of pixels that meet conditions specified in the [Activation Map] window. The [pixel graph num] checkbox was added on the [Activation Map Show] window, that can be opened from the [Analyze]-[Display Window for Activation Map] menu.

● The graph like the left image will be drawn when the [pixel graph num] checkbox is ON. The horizontal axis has activation time and the vertical axis has number of pixels that meet conditions.

● This data can be saved in a CSV file from the [CSV Export] button.


(1-4) Detailed setting for analog signal wave display - Update

● The detailed setting for analog signal waveform display can be changed on the sub window like below, this window can be opened by selecting the [detail] checkbox next to the [Ana] scroll bar and then, pushing the [Set] button.

● The [Apply All] button was added on this sub window. This button allows the analog wave settings such as gain and offset to become same all over the canvas.

● After inputting value into the [pre-amplifier gain] box, push the [set] button so that the setting can be changed.


(1-5) [APD for single]

● The function for analyzing one action potential was changed from the [Display Window for APD ] to the [Display Window for APD (single heart beat)] . Use the [Display Window for APD (multi heart beat)] for analyzing a series of action potentials.

● The [Histogram] checkbox was added in the [APD Map Option] area on the [Display Window for APD (single heart beat)]. By selecting the [Histogram] box, the [APD Histogram] sub window can be displayed as below. The horizontal bar indicates "APD (action potential duration; msec)" and the vertical bar indicates the total number of pixels.

● To zoom in a selected scale, drag a mouse from left to right. To undo zoomed scale, drag a mouse from right to left.

● Click on a histogram so that APD time and the pixel number at a clicked point can be displayed with red color.

● The [Save CSV] button allows user to export information about the historgram in a CSV format.


(1-6) [APD for multi]

● With this function, a series of action potentials on one pixel are overlapped for comparison and averaged APDs (action potential durations) and standard deviation are calculated.

After selecting data on the canvas, execute the [Cardio]-[Display Window for APD (multi heart beat )] (or [Analyze]-[Display Window for APD (multi heart beat )]) from the main menu or push button.

Please note the following should be done before using this function.
(1) Correct deviation of waveform baselines (e.g. caused by some photobleaching of fluorescence dye) by using the [Bleaching] or [Drift Remove] on the [Calculation] page.
(2) Execute software filters such as the [Spatial Filter] or [HPF/LPF] from the calculation page in order to fully remove noises from an image .
(3) Adjust image threshold suitably and select suitable frame number. A pixel with color overlaid can be used for APD analysis.

● The [Show APD multi] window and a map drawn with a default setting can appear as below.

● In the following steps, user can make a map with different settings.

(0) (A) area shows a waveform on one pixel selected in an image. Can change gain of this waveform display by scrolling the ①[Gain] vertical bar below the [Select wave gain & start frm for raw wave].
In default setting, the [Start Frm] in the [Control] page is used as the start frame. This start frame can be changed with the ②[Start Frm] scroll bar.

(1) (B) area shows a waveform processed with first derivative calculation. Can change gain of this waveform display by scrolling ③[Gain] scroll bar. The position of green dotted horizontal line can be adjusted with the ④[threshold]. This [threshold] bar ranges from 1 to 100, this value indicates percentage against maximum value of a waveform.
Green dotted vertical lines indicate maximum points on a first derivative waveform. By arranging each waveform of action potentials along these dotted vertical line, some waveforms are overlaid on the (C) area.
(C) area in the above example shows ovelaid 9 cardiac action potentials.

(2) Base line can be set with the [Select base line setting]. When the [Auto] is selected in the ⑤ area, minimum value in all area is used for baseline.
It is also possible to limit the range from which minimum value is extracted by using the [Manual setting of base line range] in the ⑥.
Gray rectangle will be displayed on waveforms in the (C) area. Drag start vertical line and end vertical line with a mouse in order to decide a range. When the [Min] is selected in the ⑥, minimum value in selected area is used for baseline. When the [Mean] is selected in the ⑥, average value in selected area is used for baseline.

(3) Select percentage of repolarization from the [Repolarization Time(%)] in the ⑦ area.
Repolarization time is calculated with the following expression.

repolarization time = (peak value-base line) x (100-β) /100 + base line
(β: selected percentage of repolarization)

Positions of each colored vertical line in the (C) area indicates repolarization time

In the (D) area, mean APD time (Mean) / standard deviation (Std) and histogram showing APD time and pixle number are displayed.

(4) Color map images show mean APD time (left) and standard deviation (right) regarding all of valid pixels. Waveforms and map images can be redrawn with the settings in the (1)-(3) area by pushing the [Recalc & Redraw APD Map] button in the ⑧ area.

- It is possible to change details for map images by selecting the checkboxes in the [APD Map Option] area.

⑨ The [Contour line] checkbox displays contour line on map images.

⑩ The [Cursor On] checkbox displays cursor points on two maps. These points are for waveform display.

⑪ The [Color] checkbox makes map image with 256 gradation color. This color gradation is the same as that used on the main canvas. When unchecking the [Color] checkbox, map image is drawn with 256 gradation of black/white color.

⑫ CSV file that defines 256 color gradation can be changed by the [Load CSV File] button.

⑬ Can change the magnification of map image with With the [Zoom] combobox.

⑭ Save a created map image from the [Save APD Map] button.

    File extention -- BMP/JPG : save a map image in bmp/jpg format.

    File extention -- CSV : save value data in csv format.
    (Values of activation time/repolarization time/APD time regarding all pixels can be exported. Exported values are "number of frame". Time can be calculated by multiplying those values by sampling time.)

(5) After changing settings on the [Control] page, such as start/end frame and threshold etc., push the ⑮[Canvas setting reload]button so that settings can be reflected on data on the [Show APD multi] window.

(1-7) [Drift Remove2]

● This function removes drifting curve, which may be caused by breaching or LED illuminations, using exponential curve F(t) = k*(1-1/exp(At))^N.
Select the data on the canvas, and click the [Drift Remove2] button on [Calculation] tab page.The [Drift Remove2] box as shown below is displayed.

(1) Drag four [Reference Point] on map to regions of interest, then averaged wave is created automatically.

(2) Drag horizontal and vertical lines on the averaged wave so that F(t) curve fits on the wave.

The maximum point of the curve may be good position for the horizontal line.
Almost 63% of the maximum of the wave may be good position for the vertical line.

The multiplier for F(t) is valiable. The default number is "1".
The scroll bar on the side of [Dim of F(t)] changes the multiplier from 0.1 to 2.0 by 0.1.
The multiplier can be input to the Multiplier BOX, then click [Set] to set it.
When the multiplier chnages, the shape of upstroke of wave changes.

(3) When [Fitting=ON], a blue line appears on the "Preview Wave".
This blue line should be on the maximum point, so that "k" is set automatically to make same the maximum of wave.

When "Fitting=OFF", "k" is set according to the ratio of background intensity on the "Preview Point".

(4) Push the [Preview] button to show the result of this processing. The result is shown by red wave.
The [Preview] button should be pushed after any setting in above 1, 2, 3 sections.

(5) When the result shown by [Preview] is OK, cpush the [Drift OK] button. Data on the canvas is processed by the setting.


(1-8) [Setup display option] sub window

● The [Setup display option] sub window opens by pushing the [Show Option] button on the [Control] page. The following functions are available from this version.

● Can show a threshold value with percentage of change in light intensity only after data has been normalized by [dF/F]. Can also input number into the ① area and push the [Set] button in order to change a threshold value directly.

Below data on the main canas, can display total number of pixels that are over threshold level (displayed as "valid pixel number/all pixel number") when the ② checkbox is selected. When the [interpolation] checkbox is "ON" or the [real] checkbox is "ON" on the [control] tab page, ② checkbox becomes disable.

Execute the [Export function]-[Effective pixel num csv export] from the popup (right click) menu when exporting number of pixels (that are over threshold) along all frames.


(1-9) [Remove by SD]

● This function is for removing noises with Ave (average value) and SD (standard deviation). Values ranging in Ave±(SD x N) are regarded as noises and removed. To open the below window, select the [Remove by SD] menu from the bottom part of the popup (right click) menu.

(1) Drag two points on the image in [Remove By SD] window to show sample waves.

(2) In order to specify base area, drag start/end line of gray rectangle shown on sample waves. This can be also done by directly inputting frame number into the [Start Frm]/[End Frm] boxes and pushing the [Set] button.

Average (Ave) and standard deviation (SD) of sample waves in gray rectangle area are shown below each wave.
Pink dotted horizontal lines indicate the range of Ave±SD. Break line shows [Average +/- SD].
% shows difference from the intensity of background image.

(3) Select [All points] or [Each point].
In case of [All points], differential value (%) can be set for threshold. Pink line shows the thresold level.
In case of [Each point], magnification for SD can be set.
Pink line shows "average +/- N-times SD" as threshold level.

(4) After setting as above, click the [Create new data] button.
New data, which difference under set threshold is set to "0", is shown on canvas.
Save data after checking newly created data.
Click the [Cancel] button to quit this function after saving new data.


(ATTENTION)

★ The [Remove By SD] window shows single pixel value even when the [Wave filter] is set on the [Control] tab page.
★A denominator is divided by N (not by N-1) in the calculation of SD. This is the same as the "STDEVP" function in Microsoft (R) Excel.

(1-10) [Stripe]

● New function was added to the [Analyze]-[Display Window for Stripe].

● Can manually specify coordinates of start/end points for a line. After inputting values, push the [Set] button to apply the setting to the right image.

● Push the [Save Setup] button to save the setting in a file. This can save line formats [One Line/Bezier Curve/3 Bezier Curve] of each stripe No., coordinates of start/end points and settings on the (B) stripe window.

● Push the [Load Setup] button to load saved settings from a file.

● The [display wave chart at a frame(time)] checkbox was newly added on the (B) [Stripe map] window. The (C) sub window opens by selecting this checkbox.

(1) The graph on the (C) sub window shows differential values on a stripe line at current selected frame. The left/right end of horizontal axis indicate the start/end points of a stripe line, respectively. The vertical axis indicate differential values.
Length of horizonal axis is the same as vertical axis (height) of an image on the (B) [Stripe map] window. (This height of an image on the (B) [Stripe map] window can be changed, depending on the [Zoom] magnification in the (A) window).

(2) Each stripe map is displayed on each separate windows.
When window is closed by pushing the [x] button, turn the [display wave chart at a frame(time)] off first and then, select the [display wave chart at a frame(time)] so that the window can be opened again.

(3) The [Wave Filter] on the [Control] page is not applied to this stripe map. Use the [Spatial filter] on the [Calculation] page if some noise reduction processes are needed.

(4) Select the [Start Zero] checkbox on the (C) window so that start point (red small rectangle) on the graph can be moved to zero.

(5) Push the [CSV Export] button in order to export values of this graph in a CSV file, which includes coordinates on all pixels, light intensity values of background image and differential values. Exported differential values are values devided by averaging times.

(6) When the "Rotate"=0 and "View"=90 on the (B) window, dotted blue vertical line is placed on the stripe map. This lines indicate current frame, and this line can be also dragged sideways for changing frame position. Depending on dragging the vertical line, graph on the (C) window is redrawn.

(7) When the "Rotate"=0 and "View"=90 on the (B) window, (pink / yellow-green / yellow) points are shown on the (A) window and the corresponding dotted horizontal lines can be shown on the (B) window when the [Image Point] checkboxes are selected. At same time, the corresponding dotted vertical lines is also indicated on the (C) window. When the [Bezier Curve]/[3 Bezier Curve] is selected, these lines may not correspond exactly due to error in calculation.


(1-11) [BV_Thumbnail]

● The following 2 functions are added to the "BV_Thumbnail" tool, which can open with the [BV Thumbnail] button in the [File List] window.

① Show basic information of data such as frame number, samplig time on the bottom part of the BV_Thumbnail window when a mouse cursor is over a thumbnail image.
② Can open data on the BV_Ana by double-clicking data on the BV_Thumbnail.

(1-12) [Apply all for wave point &overlay]

● Overlap waveforms, which are from same coordinates of different data, on a selected image. This is valid for data selected (checked) in the data list on the left upper part of the BV_Ana.

(ATTENTION)
★ Waveforms from one data have same color. (For example, waveforms from data 1 are red, data 2 are blue, data 3 are green.)

★ After data, waveform and observation point on an image are moved to other position, select the [Apply all for wave point & overlay] again.

(Example)


(1-13) [Image export by specifying frame No.]

● The functions for exporting data in various types of image/CSV files were put in the [Export function] menu, which can be selected from the pop-up (right-click) menu. In this menu, the [Image export by specifying frame No.] was newly added. This function is for exporting selected frames in BMP/JPG files by specifying frame number.

● Select the [Export function]-[Image export by specifying frame No.] from the pop-up (right-click) menu.

Manually input frame numbers that should be exported. Each line has to have one frame number.

Select magnification of exported images from [x1] - [x8].

Select the [interpolation] checkbox to make images smooth when magnified.

Select a file format from either BMP or JPG.

Select options for displaying additional information from NONE / Frame No. / Time (msec).

When is not [NONE], select display position from four options.

Select folder for saving images by opening the button.

Push the [OK] button to execute export process after the above setting is completed.


(1-14) [Effective pixel num csv export]

● The functions for exporting data in various types of image files and CSV file were gathered in the [Export function], which can be selected from the pop-up (right-click) menu. In this [Export function] menu, the [Effective pixel num csv export] was added. This function is for exporting number of pixels that are over threshold in CSV file format.

● Select the [Export function]-[Effective pixel num csv export] from the pop-up (right-click) menu.
Input a file name and push the [Save] button on the file save window.

(ATTENTION)
This menu becomes disable either when the [interpolation] checkbox (to make images smooth) is on or the [real] checkbox is on.

(Example of a CSV file )
The following is an example that an exported CSV file is opened in Microsoft Excel.


2. Additions in Acquisition Function (Available only for MiCAM02)

(2-1) [NoStim-Stim] mode

● New acquisition mode, [NoStim.- Stim], was added to the [Page mode] in the [Acquisition] page.
In the [NoStim] acquisition, the MiCAM02 processor does not output any signal from the "stim" connector on the front panel.
In the [Stim] acquisition, the MiCAM02 processor outputs from the "stim" connector with a setting in the [Digital Ouput] on the [Acquisition] page.
With this [NoStim.-Stim.] mode, the cycle of acquisition without stimulation and acquisition with stimulation are repeated and, one data is created from (data with no stimulation) - (data with stimulation).

(2-2) [Option] button on the [Acquisition] page

The optional setting of digital I/O output on the back of MiCAM02 processor was added from this version.
EXD2, which is the center of upper line of D-sub 9-pin, can output stimulation pulse channel-2, vertical readout synchronization pulse, or odd number of frames information pulse.
Click "option" at the bottom of "Acquisition" tab to open digital I/O setting window.

Select output from the list. The output pulse is 3.3V.

0 Stimulation Ch-2 This output can be designed at "Digital Output " setting on "Acquisition" tab.
1 V-Sync This output vertical readout frame timing all the time.
2 V-Sync & Acq This output vertical readout frame timing same as "1" but only during acquisition.
3 Odd frames & Acq The voltage is high during odd number of frames only in acquisition.

Click "OK".If it sets it once, it is set by the automatic operation at next start.


(2-3) Display light saturation percentage in a selected region on the monitor image.

● The [ROI Monitor] checkbox is added on the [Acquisition] page, opened from the [Open Acquisition Window] button.
By selecting the [ROI Monitor] checkbox, the [ROI Monitor] window opens and on this window, light saturation percentage (maximum, minimum and average value) and total number of pixels in a selected region (rectangle/circle) during monitoring. To close this window, set this checkbox off.

● Select shape of ROI (region of interest) from either the [Rectangle] or [Circle].

[Rectangle]
To move a rectangle, drag the upper left corner or the bottom right corner of a green-bordered rectangle on a sample monitor image by mouse.
Or, input X-Y coordinates into the [Left-Top] box and the [[Right-Bottom] box. After that, push the [Set] button.

[Circle]
To move a circle, drag the center of the green-bordered circle by mouse.
Or, input X-Y coordinates of the center of green-bordered circle into the [Center] box and input a radius into the [Radius] box. After that, push the [Set] button.

In the area, the information regarding light intensity is shown.
    min : minimum saturation percentage of light intensity in a region
    max : maximum saturation percentage of light intensity in a region
    ave : average saturation percentage of light intensity in a region
    pix total : total number of pixels in a region

Saturation percentage of light intensity is an actual light intensity value (0-16384) divided by maximum light intensity (16,384).
Only in monitoring, these values are displayed. At every refresh of monitoring image, these values are refreshed as well.