biostation im cell-s1 / cell-s1-p - mvi-inc.com im cell-s1_s1-p software.pdf · i introduction...

Time Lapse Imaging System

BioStation IM

CELL-S1 / CELL-S1-P Instructions

<Application Software>

M412 E 07.11.NF.3 (2/2)

i

Introduction

Thank you for purchasing the Nikon products.

This instruction manual is written for the users of the application software of the Time lapse imaging system BioStation IM.

To ensure correct usage, read this manual carefully before operating the product.

• No part of this manual may be reproduced or transmitted in any form without prior written permission from Nikon.

• The contents of this manual are subject to change without notice.

• Although every effort has been made to ensure the accuracy of this manual, errors or inconsistencies may remain. If you note any points that are unclear or incorrect, please contact your nearest Nikon representative.

• Some of the equipment described in this manual may not be included in the set you have purchased.

• If you intend to use any other equipment with this product, read the manual for that equipment too.

• If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.

• Microsoft, Windows, and Internet Explorer are registered trademarks of Microsoft Corporation in the U.S. and other countries. Other product and company names mentioned in this manual are trademarks or registered trademarks of their respective owners.

ii

Contents Introduction ........................................................................................................................................................... i

Chapter 1 Common Function .......................................................................................................................... 1 1.1 Screen Switch Buttons................................................................................................................... 1 1.2 Environmental Conditions.............................................................................................................. 2

Chapter 2 Live Observation Screen ................................................................................................................ 3 2.1 Image Compensation Button and Image Capturing....................................................................... 4

2.1.1 Tone curve compensation ................................................................................................ 5 2.1.2 Checking saturation ......................................................................................................... 5 2.1.3 Brightness screening........................................................................................................ 6

2.2 Setting Observation Conditions (Filter, Magnification, Mode, Z position, and Save) ..................... 7 2.2.1 Saving observation condition ........................................................................................... 9

2.3 Setting Observation Conditions (Focus Mode, Automatic Exposure, Condition File Loading,

Light Intensity, Exposure Time, Gain, and Resolution) ................................................................ 10 2.3.1 Exposure compensation value fine adjustment .............................................................. 11

2.4 Observation Point and Focus Adjustment.................................................................................... 12

Chapter 3 New Time-Lapse Setting Screen.................................................................................................. 13 3.1 Live Image Screen....................................................................................................................... 13

3.1.1 Time-lapse start confirmation dialog box........................................................................ 15 3.1.2 Observation Point Verification ........................................................................................ 15 3.1.3 Time Lapse Experiment Scheme (Points Tab)............................................................... 16 3.1.4 Time Lapse Experiment Scheme (Time Tab)................................................................. 18 3.1.5 Time Lapse Experiment Scheme (Cell Name etc... Tab) ............................................... 20 3.1.6 Time Lapse Experiment Scheme (Z stack Tab) ............................................................. 21 3.1.7 Setting Observation Conditions (Observation Filter and Magnification) ......................... 23

3.2 Wide Field Screen ....................................................................................................................... 24 3.2.1 Observation Point Indication .......................................................................................... 25 3.2.2 Setting/Clearing a Browse Area of a Tiled Image........................................................... 26 3.2.3 Setting Observation Conditions and Moving the Stage .................................................. 28

Chapter 4 Time-Lapse Images in Process Screen....................................................................................... 29 4.1 Channels Display......................................................................................................................... 29

4.1.1 Image Magnification and Enlarge/Reduce Buttons ........................................................ 31 4.1.2 Scrolling an Enlarged Image .......................................................................................... 31 4.1.3 Setting a Clipping Range of the Displayed Image .......................................................... 32 4.1.4 Magnification button ....................................................................................................... 32 4.1.5 Images of Time-Lapse Experiment (Ph/FI1/FI2/Multi) .................................................... 33 4.1.6 Brightness Graph ........................................................................................................... 37 4.1.7 Brightness Graph Displaying Method............................................................................. 38 4.1.8 Time Line and Image Playback Control ......................................................................... 41 4.1.9 Time-Lapse Experiment Scheme (Points Tab)............................................................... 43 4.1.10 Time-Lapse Experiment Scheme (Time Tab)................................................................. 44 4.1.11 Time-Lapse Experiment Status Display ......................................................................... 45 4.1.12 Captured Image Printing ................................................................................................ 46 4.1.13 Captured Image Saving ................................................................................................. 47 4.1.14 Example of a Saved Image ............................................................................................ 54

Contents

iii

4.2 Points Display.............................................................................................................................. 55

Chapter 5 Time-Lapse Images Acquired Screen ......................................................................................... 56 5.1 Channels Display......................................................................................................................... 56

5.1.1 Saving a Time-Lapse Result File as an Alias File/Saving a Captured Image................. 58 5.2 Points Display.............................................................................................................................. 61

1

Common Function1 1

The items shown on the top screen of the software are common functions for all screens.

Figure 1.0-1 Live observation screen

1.1 Screen Switch Buttons Click these buttons to select each function of the BioStation IM.

Figure 1.1-1 Screen switch buttons

Table 1.1-1 Functions of screen switch buttons

Item Function Live observation Select this screen when the BioStation IM is used as a microscope. New time-lapse setting

Select this screen to perform time-lapse experiment. Select this screen to start time-lapse experiment, after setting observation point, observation condition, and so on.

Time-lapse image in process

This screen displays time-lapse experiment in progress. This screen appears automatically after time-lapse experiment is started on the New time-lapse setting screen. Also, this screen cannot be displayed by operating any buttons.

Time-lapse images Acquired

Select this screen to playback performed time-lapse images. This screen automatically appears after time-lapse experiment ends. On this screen, loading and reproducing saved file of time-lapse experiment results are available.

Screen switch buttons Environmental conditions

Chapter 1 Common Function

2

1.2 Environmental Conditions This area shows the current temperatures and the temperature settings of the culture chamber and the humidifier water tank. The outside air temperature is also shown here.

For the temperature settings of the culture chamber and the humidifier tank, see Step 3 in Section 2.1, “Start-up,” in the separate manual, “BioStation IM Instructions <System>.”

Figure 1.2-1 Temperatures and conditions

Table 1.2-1 Device condition items and their functions

Item Function Temp Click the Temp button to display the Temperature dialog box.

Display area Temperature detected with the temperature sensor of the culture chamber is displayed.

Chamber

Setting area Set temperature inside the culture chamber to be suitable for time-lapse experiment.

Display area Temperature detected with the temperature sensor of the humidifier water tank is displayed.

Water

Setting area Set water temperature inside the humidifier water tank to be suitable for time-lapse experiment.

Outside Current ambient temperature is displayed. If room temperature is below 18°C or above 28°C a warning symbol appears and “POWER” lamp of the LED indicator blinks rapidly.

Stable/Unstable When both the temperature inside the culture chamber and the water temperature inside the humidifier water tank reach the set value and stabilize, the status display changes from “Unstable” to “Stable.” And, time-lapse experiment becomes possible. Also, the “STABLE” lamp of the LED indicator on the microscope is lit.

Displaying graph of device conditions Click the Temp button to display the Temperature dialog box.

Figure 1.2-2 Temperature dialog box

Table 1.2-2 Temperature dialog box items and their functions

Item Function Range Select time range (horizontal axis) of temperature graph. Temperature graph

Changes in the temperature inside the culture chamber, changes in the temperature inside the humidifier water tank, and changes in ambient temperature are shown.

Close button Click this button to close the Temperature dialog box.

The time-lapse experiment is running under a stable condition.

An unstable condition is detected during the time-lapse experiment.

3

Live Observation Screen2 2

When the BioStation IM is used as a microscope, select this screen. You can check the specimen is suitable or not for time-lapse experiment by observing cell shape and the amount of fluorophore in cell.

Figure 2.0-1 Live observation screen

Table 2.0-1 Live observation screen items and their functions

Item Function (1) Live image

display Live image of the field of view is displayed. Click in this screen to move the stage until the clicked point locates at the center of this screen.

(2) Observation point

The red box shows the field of view. Its Live image is displayed. Click a point desired to display its Live image to move the stage to the clicked point and display the Live image.

(3) Image compensation button

Click these buttons to set tone compensation, saturation check, and brightness screening.

(4) Capture button

Press this button to capture a live image. The captured image is put into the thumbnail area. Display area

Captured thumbnail images are displayed here. Any thumbnail image can be enlarged and displayed on another window by clicking on the enlarge button (magnifier icon).

(5) Image capturing

Function Click the capture button to capture images and display thumbnail images.Images can be saved by highlighting the thumbnail images and clicking on the save button ( ). All highlighted images will be saved together.Click the trash box button to delete selected thumbnail images.

(6) Observation condition and observation point

The stage position, the focusing condition, and the observation conditions such as filters, magnification, light intensity, and exposure time can be adjusted here.

(2)

(5)

(1)

(6)

(3) (4)

Chapter 2 Live Observation Screen

4

2.1 Image Compensation Button and Image Capturing

Figure 2.1-1 Image compensation and capturing image

Table 2.1-1 Image compensation button functions and image capturing operations

Item Function Automatic tone curve compensation apply button

Click this button to automatically set the range. Display range of brightness is automatically set. The highest brightness (A) of the displayed image is set as the upper limit (C) and the lowest brightness (B) is set as the lower limit (D).

Figure 2.1-2 Brightness graph

Tone curve compensation setting button

Click this button to set tone curve compensation manually. The Histogram dialog box appears to set tone curve compensation.

Tone curve compensation cancel button

Click this button to cancel the applied tone curve compensation and return to the original screen.

Saturation check button (FL1 & FL2 only)

Click this button to display saturation point in red. And then, adjust the intensity and gain until there is no saturation point. To clear the saturation check condition, clicked this button again. This function can be used only for the fluorescent microscopy.

Brightness screening button (FL1 & FL2 only)

Click this button to display the Screening dialog box for setting brightness screening. This function can be used only for the fluorescent microscopy.

Capture button Click this button to capture the current Live image and register with thumbnail display. If brightness screening and image compensation are performed, the performed image is registered. With the single image capture switch of the ergonomic controller, this operation can be performed.

Trash box button Click this button to delete only selected thumbnail image. The frame of selected thumbnail image is displayed in blue.

Image save button

Click this button to save all thumbnail images in a file. Thumbnail image must be highlighted prior to saving. Select save format from TIFF, JPEG, BMP, and PNG. For TIFF format, image resolution is selectable between 16 bits and 8 bits.

Thumbnail display

Thumbnail of images captured with the capture button is displayed. Click the enlarge button (magnifier icon) at the bottom right of thumbnail image to display enlarged image on other screen. If number of thumbnail images becomes five or more, a scroll bar appears at the bottom of thumbnail display.


5

2.1.1 Tone curve compensation

Click the tone curve compensation process button ( ) to display the Histogram dialog box.

Figure 2.1-3 Histogram dialog box

Table 2.1-2 Tone curve compensation function

Item Function Apply button After setting tone curve compensation, click this button to apply the setting to Live

image display. Close button Click this button to close the histogram dialog box without applying setting.

2.1.2 Checking saturation

Click the saturation check button ( ) to display saturation point of the Live image in red.

And then, adjust the intensity and gain until there is no saturation point.

Figure 2.1-4 Displaying saturation point


6

2.1.3 Brightness screening

Click the brightness screening button ( ) to display the Screening dialog box.

Figure 2.1-5 Screening dialog box

Table 2.1-3 Brightness screening function

Item Function Image select box (yellow box)

Left click and drag to select an area for brightness screening. And then, enlarge, reduce, or move the area with a mouse pointer if necessary. Also, to delete the box, right click the box and display the Delete menu.

Image display Screening points (same brightness points) are displayed in blue. Histogram Brightness histogram of the image in the yellow box is displayed.

To adjust the brightness range, drag the ▼ and ▲ buttons from side to side. Apply button Click this button to apply setting of brightness screening to Live image display. Close button Click this button to close the Screening dialog box.

Image select box


7

2.2 Setting Observation Conditions (Filter, Magnification, Mode, Z position, and Save)

Some observation condition settings are different between the Ph microscopy and the fluorescent microscopy. Also, there are the Manual mode and the Simple mode for setting observation condition. Both modes are available for settings in this section.

Figure 2.2-1 Setting observation conditions (for fluorescent filter, left figure: Manual mode, right figure: Simple mode)

Table 2.2-1 Observation condition items and their functions (1/2)

Item Function Click these buttons to select filter for observation. The frame of selected filter button is displayed in blue. With the microscopy select switch of the ergonomic controller, this operation is also available.

Select this button for the Ph microscopy. The diascopic illumination of the red LED illuminator built in the microscope is used. Fluorescent filter is not used.

Filter button

Select these buttons for the fluorescent microscopy. The illumination of external mercury lamp is used and selected filter is used. This button is displayed only when the HG precentered optical fiber light source is connected and also the power is turned on.

Magnification button

Select magnification button to select magnification for observation. With the magnification selector switch (UP/DOWN) of the ergonomic controller, this operation is also available.

(Close) Excitation light shutter button

(Open)

Click this button to open or close the shutter for excitation light. This button is enabled only when fluorescent filter is selected. With the shutter open/close switch of the ergonomic controller, this operation can be also performed.

Simple button

Select observation condition from three conditions as follows: ● Bright = Select this option button to make a dark image bright. ● Middle = Select this option button to observe medium bright images. ● Dark = Select this option button to make a too bright image dark. Select one of those conditions for each combination of a filter (Ph, Fl1, or Fl2) and a magnification (three kinds).

Manual button

Click this button to manually set all observation conditions, or to set observation conditions by loading the already registered condition file. Register observation condition setting for each combination of filters and magnifications.


8

Table 2.2-1 Observation condition items and their functions (1/2)

Item Function Z Position Register Z position (focus position) appropriate for each selected filter.

For example, when inputting Z position appropriate for the fluorescent filter 1 (Fl1), click the Fix button to display the input value in red and memorize it. After that, even if selecting the fluorescent filter 2 (Fl2) and changing Z position, the memorized value is displayed when the fluorescent filter 1 (Fl1) is selected again. To clear the memorized value, click the Fix button again. Additionally, even after memorizing Z position, changing it is available. To display memorized Z position again, click the Go button. These settings can be performed on the Simple mode.

Save button

Click this button to save observation condition setting in a setting file or in a file for setting on the Simple mode.


9

2.2.1 Saving observation condition

Click the save button ( ) in the observation condition setting area to display the Save setting dialog box. And then, save observation condition setting in a setting file or the Bright, Middle, or Dark setting file on the Simple mode.

Figure 2.2-2 Save settings dialog box (Save into file)

Table 2.2-2 Observation condition storage functions (for “Save into file” option)

Item Function Save into file Select this check box to save observation condition in a setting file. Save button Click this button to display the Windows Save As dialog box.

Input a file name and save observation condition as a setting file. Close button Click this button to close the dialog box without saving observation condition setting.

Figure 2.2-3 Save settings dialog box (Save as “Simple Setting” for)

Table 2.2-3 Observation condition storage functions (for “Save as 'Simple Setting' for” option)

Item Function Save as “Simple Setting”for

Select this check box to save observation condition setting in a file for setting on the Simple mode. If selecting this check box, select “Bright Sample”, “Middle Sample”, or “Dark Sample” to save as.

Save button Click this button to display the save confirmation dialog box. To save setting, click the OK button. To return to the Save settings dialog box without saving, click the Cancel button.

Figure 2.2-4 Save confirmation dialog box

Close button Click this button to close the dialog box without saving observation condition setting.


10

2.3 Setting Observation Conditions (Focus Mode, Automatic Exposure, Condition File Loading, Light Intensity, Exposure Time, Gain, and Resolution)

Each setting in this section is available only on the Manual mode.

Figure 2.3-1 Setting observation condition on the Manual mode (left figure: fluorescent filter, right figure: Ph filter)

Table 2.3-1 Observation condition setting functions (1/2)

Item Function AE (Focus) button

Click this button only for the Manual mode. The exposure adjustment is performed with a focus priority mode. It is useful for the focus adjustment because the exposure time for the dark specimen is 1/6 second maximum. However, noise of the image increases because this operation increases gain. In this case, exposure time and gain are adjusted automatically. These conditions cannot be adjusted manually. Click this button again to clear focus priority mode. In this case, although gain value returns to its original value, exposure time remains automatically adjusted value. Therefore, exposure time must be set again.

AE button

Click this button only for the Manual mode. Automatic exposure is performed once. Exposure time and gain value used for the automatic exposure are displayed in each item box of observation condition.

Load settings Click this button to load the registered observation condition setting file.

Adjust intensity of each lamp with the slider and the right and left triangle buttons. Display changes depending on selected filter. With the illumination intensity adjustment (UP/DOWN) switch of the ergonomic controller, this operation can be also performed. EPI Lamp For fluorescent filter, set intensity of episcopic illumination (illumination of

external mercury lamp). For Ph filter, intensity of diascopic illumination (illumination of built-in LED illuminator) can be set.

Turned on

Intensity setting

DIA Lamp

Turned offBuilt-in LED illuminator can be turned on or off.


11

Table 2.3-1 Observation condition setting functions (2/2)

Item Function Exposure time

Select exposure time from the pull-down menu. Setting value can be increased or decreased by “1” using the ▼ and ▲ buttons.

Gain Click the compensation value displayed next to the Gain to display the Edit detailed gain dialog box. In the dialog box, set the compensation value by using the keyboard. The compensation value can also be set with the slider and the right and left triangle buttons displayed next to the value.

Resolution Select resolution from the pull-down menu. 800 x 600 normal resolution 800 x 600 Binning normal resolution and high sensitivity 1600 x 1200 high sensitivity

Resolution can be set for each channel separately. However, the following combinations are not applicable:

800 x 600 and 1600 x 1200 in combination 800 x 600 Binning and 1600 x 1200 in combination

2.3.1 Exposure compensation value fine adjustment

Click the compensation value displayed next to “Gain” to display the Edit detailed gain dialog box. Exposure compensation value can be adjusted finely.

Figure 2.3-2 Edit detailed gain dialog box

Table 2.3-2 Exposure compensation value fine adjustment functions

Item Function Compensation value setting

Set the compensation value by using the keyboard or the ▼ and ▲ buttons.

Apply button Click this button to apply selected compensation value and close the dialog box. Close button Click this button to cancel compensation value setting and close the dialog box.


12

2.4 Observation Point and Focus Adjustment

Figure 2.4-1 Setting observation point

Table 2.4-1 Observation point movement and focus adjustment items and their functions

Item Function

Coarse focus

Jog dial

Fine focus

Click each direction button of the jog dial to move stage. At the center part of the jog dial, X and Y coordinates of current stage position are displayed. With the X stage knob or the Y stage knob of the ergonomic controller, this operation is also available.

Coarse focus

Focus button

Fine focus

Click the up-arrow button or down-arrow button to move objective in the Z direction. With the focus knob of the ergonomic controller, this operation can be also performed.

Undo button

Click this button to return observation point to the previous focus position.

Slide bar Slide up and down the scroll bar to perform focusing.

Jog dial

13

New Time-Lapse Setting Screen 3 3

On this screen, set and save observation points and observation conditions, to perform time-lapse experiment. Two screens, “Live image” screen and “Wide field” screen, are provided to set observation points and observation conditions for time-lapse observation. On the Live image screen, the live image is used for the settings. On the Wide field screen, a captured tiled image is used for the settings.

3.1 Live Image Screen On this screen, set observation point and observation condition while checking the current Live image.

Figure 3.1-1 New time-lapse setting screen (Live image screen)

Table 3.1-1 Live image screen functions (1/2)

Item Function (1) Screen switch

button Click these buttons to switch between the Live image screen and the Wide field screen.

(2) Observation point

The red box shows the field of view, which is displayed on the Live image screen. Registered observation point is marked with the blue box. Click a point desired to display its Live image to move the stage to the clicked point and display the Live image.

(3) Live image display

Live image in the field of view is displayed. Click anywhere on the image and that point will move to the center of the screen.

(4) Image compensation button

Click these buttons to set tone compensation, saturation check, and brightness screening. Operation of each function is the same as that for the Live observation screen.

(5) Start time-lapse button

Click this button to start time-lapse experiment. Time-lapse start confirmation dialog box (Confirmation window) appears.

(1)

（3）

(5)

(4)

(6)

(7)

(8)

(2)

Chapter 3 New Time-Lapse Setting Screen

14

Table 3.1-1 Live image screen functions (2/2)

Function Detail Points tab Select this tab to display observation conditions (filter name,

exposure time, and magnification) for each observation point. Click an observation point indication to open the Point information dialog box. Observation conditions can be verified or deleted.

Time tab Select this tab to display capturing interval time, total observation time, and rounds for time-lapse experiment. Click time-lapse experiment time display to display the Timelapse dialog box. In the dialog box, changing and deleting time-lapse experiment time setting are available. Click the New button to add new time-lapse experiment time.

Cell name etc... tab

Select this tab to input accompanying information of time-lapse experiment. Inputting information in “Sample name”, “Cell name”, and “User name” is available. Click either of these names to display the Cell name etc... dialog box and input, change, or delete name in the dialog box.

(6) Time-lapse experiment scheme

Z stack tab To specify multiple observation points in the Z-direction for an observation point, use this function. Specify a travel amount and a travel count.

(7) Time-lapse experiment point registration button

Observation point and conditions are registered with time-lapse experiment scheme. If registered observation condition setting is changed, the setting is overwritten.

(8) Observation condition and observation point

Set observation conditions (filter selection, magnification selection, intensity adjustment, and exposure time setting) and move stage and perform focusing with the Jog dial. After all conditions are selected for a given field it can be entered by clicking on the time-lapse experiment point registration button.


15

3.1.1 Time-lapse start confirmation dialog box

Click the Start time-lapse button ( ) to display the following window.

Figure 3.1-2 Confirmation window

Table 3.1-2 Time-lapse start confirmation dialog box items and their functions

Item Function Start time-lapse button

Click this button to display the Windows Save As dialog box. Input a file name of time-lapse experiment result and click the Save button. And then, the Time-lapse image in process screen automatically appears and time-lapse experiment starts.

Back to setting button Click this button to cancel time-lapse experiment and return to the New time-lapse setting screen.

3.1.2 Observation Point Verification

Checking the registered observation points and current view point are available in the entire observation area. Click in the following screen to move the stage to the clicked point.

Figure 3.1-3 Observation point

Table 3.1-3 Observation point verification items and their functions

Item Function Registered observation point

Registered observation point is marked with the blue box. The number over the blue box is the same as the point number shown on the Point tab.

Live image display range

The red box shows the field of view, which is displayed on the Live image screen. X and Y coordinates of the center are displayed.

When two or more observation points are set at points very near each other or at a same point, the “...” symbol appears by the number. The example on the figure indicates observation points from 1 to 5 are set at the point.


16

3.1.3 Time Lapse Experiment Scheme (Points Tab)

Figure 3.1-4 Points tab

Table 3.1-4 Items and functions on the points tab

Item Function Observation point

Select the Points tab to display observation conditions (filter name, exposure time, and magnification) for each observation point. When time-lapse experiment is performed, only point selected with a check mark is observed. Click an observation point indication to open the Point information dialog box. Observation conditions can be verified or deleted.

Delete button Click this button to delete the selected observation point. Delete confirmation dialog box appears. To delete the selected observation point, click the OK button. To return to the Points tab without deleting, click the Cancel button.

Figure 3.1-5 Delete confirmation dialog box

Load button Click this button to load the registered time-lapse experiment condition file. Save button Click this button to save the registered observation point, observation condition, and

time-lapse experiment time to a time-lapse experiment condition file. Warning mark If time-lapse experiment time setting is disabled or hard disk space of control

PC is insufficient, this mark appears.


17

Checking or deleting observation condition Click an observation point on the Point tab to display the Point information dialog box. In the dialog box, check observation condition setting for each combination of filters and magnifications.

Figure 3.1-6 Point information dialog box

Table 3.1-5 Buttons and their functions on the Point information dialog box (checking or deleting an observation condition)

Item Function Go button Displayed settings are changed.

Click this button to change displayed observation condition settings to new settings. Delete button Click this button to delete the selected

observation point. Delete confirmation dialog box appears. To delete the selected observation point, click the OK button. To return to the Point information dialog box without deleting, click the Cancel button.

Figure 3.1-7 Delete confirmation dialog box

Close button Click this button to close the Point information dialog box.


18

3.1.4 Time Lapse Experiment Scheme (Time Tab)

Figure 3.1-8 Time tab

Table 3.1-6 Items and their functions on the Time tab

Item Function Time-lapse experiment time display

Click the Time tab to display capturing interval time, total observation time, and number of rounds for time-lapse experiment. Click time-lapse experiment time display to display the Timelapse dialog box (for setting change). In the dialog box, changing and deleting capturing interval time and total observation time are available.

Delete button Click this button to delete the selected time-lapse experiment time. Delete confirmation dialog box appears. To delete the selected time-lapse experiment time, click the OK button. To return to the Time tab without deleting, click the Cancel button.

Figure 3.1-9 Delete confirmation

dialog box

New button Click this button to set new time-lapse experiment time. The Timelapse dialog box (for new registration) appears.

Load button Click this button to load the registered time-lapse experiment condition file. Save button Click this button to save the registered observation point, observation condition, and

time-lapse experiment time to a time-lapse experiment condition file.

Information

Registering multiple time-lapse experiment times is available.

This software can register multiple time-lapse experiment times, which are composed of different total observation time and capturing interval time.

When a time-lapse experiment starts, the multiple time-lapse experiment times registered with the Time tab are automatically applied in descending order.

Additionally, even during the time-lapse experiment, adding a new time-lapse experiment time and changing or deleting unperformed time-lapse experiment time are available.



19

Changing/deleting time-lapse experiment time Click a time-lapse experiment time on the Time tab to display the Timelapse dialog box (condition modification).

Figure 3.1-11 Timelapse dialog box (condition modification)

Table 3.1-7 Time lapse setting items and their functions (changing or deleting a time-lapse condition)

Item Function Acquisition cycle Input capturing interval time for time-lapse experiment.

Select a unit from the pull-down menu next to the input box. Total time Input total observation time of time-lapse experiment.

Select a unit from the pull-down menu next to the input box. Rounds Round value is displayed.

This value is automatically calculated from total observation time and capturing interval time.

Apply button Input values are applied to time-lapse experiment time setting. Delete button

Selected time-lapse experiment time is deleted. Delete confirmation dialog box appears. To delete the selected time-lapse experiment time, click the OK button. To return to the Timelapse dialog box without deleting, click the Cancel button.


dialog box

Close button Time-lapse experiment time setting is cleared and the dialog box is closed.

Registering new time-lapse experiment time Click the New button of the Time tab to display the Timelapse dialog box (for new registration). Setting values of capturing interval time and total observation time are the same as those of the Timelapse dialog box (for setting change). After setting time-lapse experiment time, click the Add button to register the setting.

Figure 3.1-13 Timelapse dialog box (for new registration)


20

3.1.5 Time Lapse Experiment Scheme (Cell Name etc... Tab)

Figure 3.1-14 Cell name etc... tab

Table 3.1-8 Items and their functions on the Cell name etc... tab

Item Function Sample name

Cell name

User name

Input the specimen name, cell name, and observer name. Click either of these names to display the Cell name etc... dialog box and input, change, or delete name in the dialog box. Even if not inputting names, time-lapse experiment is possible. Also, displaying input information in the dialog box on saved image or Live image is available.

Load button Registered time-lapse experiment condition file is loaded. Save button Registered observation point, observation condition, and time-lapse experiment time

are saved to a time-lapse experiment condition file.

Inputting accompanying information of time-lapse experiment Click either of names on the Cell name etc... tab to display the Cell name etc... dialog box.

Figure 3.1-15 Cell name etc... dialog box

Table 3.1-9 Time-lapse experiment accompanying information items and their functions

Item Function Sample name Cell name User name

Input the specimen name, cell name, and observer name (letter string is arbitrary).

Apply button Click this button to register the input name. Close button Click this button to close the dialog box without registering the input name.


21

3.1.6 Time Lapse Experiment Scheme (Z stack Tab)

Use this function to set up multiple observation points in the Z-direction at a same X-Y coordinate. When the Z stack function is used, the setting information is saved and will be restored at the next time.

Figure 3.1-16 Z stack tab

Table 3.1-10 Z stack items and their functions

Item Function Z position Add Points Check the checkbox to enable the Z stack function.

Step: Specify the travel amount (step width) in the Z direction. Enter a value with the keyboard. (Minimum step : 0.05 µm) Besides, the value can be changed with the arrow buttons in 0.1 µm steps.

Steps Specify the travel count (step count) in the Z direction. Enter a value with the keyboard. (Minimum : 1 step) Besides, the value can be changed with the arrow buttons.

From/To Total travel amounts in the Z direction (plus and minus directions) appear. These values are calculated automatically from the step width and the step count.

Load button Registered time-lapse experiment condition file is loaded. Save button Observation point, observation condition, and time-lapse experiment time are

saved to a time-lapse experiment condition file. The following figure shows the Z stack function example for the Z stack tab settings above. Be aware that total of five observation points (not two) are set including the original observation point. When these settings are saved, five observation points are registered at a time.

Figure 3.1-17 Z stack setting explanation

Z directionY direction X direction

Step width (0.5 µm)

2nd step (+1.0 µm)

1st step (+0.5 µm)

1st step (−0.5 µm)

2nd step (−1.0 µm)

Original observation point (0 µm)


22

Caution on image reproduction

• The image of an observation point registered with the Z stack function cannot be reproduced successively with this application software.

Observation points registered with the Z stack function When observation points are registered with the Z stack function, they will appear as a group of same background color on the tab. Two colors, light blue and white, are used alternately. Observation points appear in order of Z position from the lower point to the higher point. The figure below shows an example where numbers 1 to 5 are in a same group.

Figure 3.1-18 Z stack group appearance

Z position display examples for observation points registered with the Z stack function The Point information dialog box for observation points registered with the Z stack function shows the Z position as a plus or minus value on a base of the original Z position.

Observation point in the minus direction from the original point

Original observation point Observation point in the plus direction from the original point

Figure 3.1-19 Z position examples


23

3.1.7 Setting Observation Conditions (Observation Filter and Magnification)

Figure 3.1-20 Setting or changing observation conditions

Table 3.1-11 Items and their functions for setting or changing observation conditions

Item Function Time-lapse experiment point registration button

Observation point and conditions are registered with time-lapse experiment scheme. If registered observation condition setting is changed, the setting is overwritten.

New point Select “New point” to register observation condition the with a new observation point.

New registration /setting change select menu

For point Select “For point” to change observation condition of the existing observation point.

Filter check box In the filter check box, select a filter for time-lapse experiment.

Select filter button to display its filter image. The frame of selected filter button is displayed in blue. With the microscopy select switch of the ergonomic controller, this operation is available.


Filter button

Select these buttons for the fluorescent microscopy. The illumination of external mercury lamp and selected filter is used. This button is displayed if the HG precentered optical fiber light source is connected and the power is turned on.

Magnification check box In the magnification check box, select a magnification for time-lapse experiment.

Magnification button

Select magnification button to select magnification for observation. With the magnification selector switch (UP/DOWN) of the ergonomic controller, this operation is also available.

Reference

Details about setting observation condition, moving observation point, and focusing

The following operations are the same as those of Live observation screen: setting observation condition, selecting observation point with the Jog dial, and focusing.

Reference: Chapter 2 “Live Observation Screen”

• 2.2 Setting Observation Conditions (Filter, Magnification, Mode, Z position, and save)

• 2.3 Setting Observation Conditions (Focus Mode, Automatic Exposure, Conditon File Loading, Light Intensity, Exposure Time, Gain, and Resolution)

• 2.4 Observation Point and Focus Adjustment


24

3.2 Wide Field Screen If a specimen is exposed to an excitation light during the observation point settings and the observation condition settings, the deterioration speed of the specimen becomes faster. To avoid it, use a captured tiled-image to set up observation points and conditions. On the Wide field screen, you cannot adjust the light intensity and the focusing condition. Therefore, adjust intensity and focus on the Live image screen in advance.

Figure 3.2-1 New time-lapse setting screen (Wide field screen･CELL-SI)

Table 3.2-1 Items and their functions of the wide field screen


buttons Click these buttons to switch between the Live image screen and the Wide field screen.

(2) Observation point The current pointer is marked with a red box. A registered observation point is marked with a box.

(3) Browse area set buttons

Click these buttons to set a browse area and capture an image or clear the setting.

(4) Tiled image screen A tiled image is displayed. The current pointer is marked with a yellow box. A registered observation point is marked with a blue box.

(5) Image compensation buttons

Click these buttons to set up tone curve compensation. Operation of these buttons is the same as that of the Live observation screen.

(6) Partial enlargement Click the magnifier button and point the mouse pointer on the tiled image screen to enlarge a part to the magnification selected in the pull-down menu.

(7) Start time-lapse button

Click this button to start a time-lapse experiment. A time-lapse start confirmation dialog box (Confirmation window) appears.


The tabs of this area have the same functions as the functions in the Live image screen.

(9) Observation condition and observation point settings

Select filters and a magnification to be used. When an enlarged view of the tiled image is displayed, the display area can be scrolled with the operation of these controls.

(1)

(4)

(7)

(3)

(2)

(8)

(9)

(6)(5)


25

Figure 3.2-2 New time-lapse setting screen (Wide field screen/CELL-S1-P)

3.2.1 Observation Point Indication

The current pointer location, the registered observation area locations, and the current image display area can be identified on the upper screen.

Figure 3.2-3 Observation point (magnification: left figure = Full/middle figure = 2x/right figure = 4x)

Table 3.2-2 Items on the observation point indication

Item Function Observation point A registered observation point is marked with a blue box.

The number over the blue box is the same as the point number shown on the Point tab.Current pointer The current pointer is marked with a red small box (on the tiled image, it is marked

with a yellow box) and X and Y coordinates of the center are provided. When a point is clicked with the mouse pointer, the current pointer moves to the position.

Image display area A red large box indicates the displayed area of the tiled image. When the magnification is “Full”, the red box does not appear because the entire tiled image is displayed.


26

3.2.2 Setting/Clearing a Browse Area of a Tiled Image

A tiled image is captured and set for the observation area.

Figure 3.2-4 Tiled image

Table 3.2-3 Items and their functions for setting or clearing the tiled image

Item Function

Click this button to clear the tiled image.

Select an area with the mouse pointer. Click the Browse button to capture an image and set the image into the area as a tiled image. (See the figure at the lower left.)

Browse area set/clear buttons

Click this button to set the entire image as a tiled image. (Refer to the right figure at the bottom.)

Blue box Registered observation point Yellow box Current pointer, which specifies an observation point Scroll bar Use this scroll bar to scroll the display when the tiled image is enlarged.

Figure 3.2-5 Tiled image setting (left figure = Browse/right figure = Browse all)


27

When browsing only the image within selected area

(1) Select browsing area. Left click and drag on the tiled image screen to select an area.

(2) Click the Browse button. Click the Browse button to start browsing image within the selected area. Display of the Browse button changes to “Browse cancel” while the image is browsed. To stop browsing the image, click the Browse cancel button.

Figure 3.2-6 Selecting browsing area

When browsing entire image of observation area

(1) Click the Browse all button. Click the Browse all button to start browsing entire image of observation area. Display of the Browse all button changes to “Browse cancel” while image is browsed. To stop browsing image, click the Browse cancel button.

Figure 3.2-7 Starting browsing image

(1)

(2)

(1)


28

3.2.3 Setting Observation Conditions and Moving the Stage

Figure 3.2-8 Setting observation condition

Table 3.2-4 Items and their functions for setting observation conditions and moving the stage

Item Function Time-lapse experiment point registration button

Click this button to register the observation point data and the observation conditions for the tiled image into the time-lapse experiment scheme.

Filter setting check boxes Check filters for the tiled image.

Click to select a filter button to display the filtered image. When two or more filters are selected, their images are overlapped and displayed. Switching display while loading an image is not available. If a filter button is not selected when capturing a tiling image, the filter button is disabled to select. The button of selected filter is marked with the blue frame. With the microscopy select switch of the ergonomic controller, this operation is also available.


Filter buttons

Select these buttons for the fluorescent microscopy. The illumination of external mercury lamp and selected filter is used. This button is displayed if the HG precentered optical fiber light source is connected and the power is turned on.

Selecting magnification

On the Wide field screen, magnification is fixed to 20x. Therefore, changing magnification is not available.

Image display magnification button

Select magnification of tiled image display with these buttons.Full: entire observation area is displayed. 2x: image is enlarged by twice. 4x: image is enlarged by four times. 20x: displayed image size is one sheet of a tiled image. 40x: image is enlarged by 40 times. 80x: image is enlarged by 80 times.

Coarse focus

Jog dial

Fine focus

When the entire tiled image is not displayed due to partial enlargement, the tiled image display can be scrolled. X and Y coordinates of current pointer are displayed at the center. With the X stage knob or the Y stage knob of the ergonomic controller, the image can be scrolled too.

Jog dial

29

Time-Lapse Images in Process Screen4 4

This screen shows process of time-lapse experiment. When time-lapse experiment is started, this screen automatically appears. Therefore, this screen cannot be displayed by operating any buttons. There are the Channels mode and the Points mode for the Time-lapse images in process screen.

Table 4.0-1 Channels display and Points display

Item Function Channels Four display areas are provided for one observation point. Three filter images, filters

overlapped image, and Ratio image can be displayed at the same time. Points One display area is provided for one observation point. Switch the display area to

display each filter image and overlapped image. Therefore, up to four observation points can be observed at the same time.

4.1 Channels Display

Figure 4.1-1 Time-lapse images in process screen (Channels)

Table 4.1-1 Items and their functions on the Time-lapse images in process screen (Channels) (1/3)


button Click these buttons to switch between the Channels screen and the Points screen.

(2) Image magnification and enlarge/reduce buttons

Magnification of the current image compared to the original image size (resolution) is displayed. Click the magnifier button to enlarge or reduce the image.

(3) Scroll mode button Click this button and drag the image to scroll an enlarged image.

(1)

(6) (7)

(8) (9)

(12)

(3） (4) (5)(2)

(13)

(14)

(15)

(16)

(10)

(11)

Chapter 4 Time-Lapse Images in Process Screen

30


Item Function (4) Clipping button This button is used to cut an image area. Click the button to enable or

disable clipping function. (5) Magnification

buttons Click this button to show the current magnification.

(6) Image of time-lapse experiment (Ph)

Ph microscopy image of time-lapse experiment is displayed. If Ph filter is not selected for observation condition, its image is not displayed.

(7) Image of time-lapse experiment (Fl1)

Fluorescent microscopy image of time-lapse experiment is displayed. If the fluorescent filter (Fl1) is not selected for observation condition, its image is not displayed.

(8) Image of time-lapse experiment (Fl2)

Fluorescent microscopy image of time-lapse experiment is displayed. If the fluorescent filter (Fl2) is not selected for observation condition, its image is not displayed.

When two or more filters are selected, their images are overlapped and displayed.

Click this button to display the Ratio image of time-lapse experiment.

(9) Image of time-lapse experiment (Multi Ch)

Click this button to display the Ratio setting dialog box and set the Ratio image.

(10) Brightness graph This graph shows changes in brightness during time-lapse experiment. The color of graph line is the same as that of each filter.

(11) Time line and image control

Use the slider to check process of time-lapse experiment. The ▲ button at the right and left of the slide bar is used to reproduce images step by step or reproduce images step by step in a reverse order. Also, use the image control buttons to pause, playback, forward fast, playback slowly, reverse slowly, repeat partially, and mark a point.

(12) Observation point Observation point of time-lapse experiment is marked with the yellow box. Registered observation point is marked with the blue box. Points tab Observation conditions (filter name, exposure time, and

magnification) are displayed for each observation point. Click an observation point to display the Point information dialog box. In the dialog box, checking, changing, and deleting detailed condition are available. Click the STOP button to stop the running time-lapse experiment.

Time tab Capturing interval time, total observation time, and number of rounds for time-lapse experiment are displayed. If time-lapse experiment time display is clicked, the Timelapse dialog box appears. In the dialog box, changing and deleting time-lapse experiment time setting are available. Click the New button to add new time-lapse experiment time.


Cell name etc... tab

Click this tab to input accompanying information of time-lapse experiment. Inputting information in “Sample name”, “Cell name”, and “User name” is available. Click either of these names to display the Cell name etc... dialog box and input, change, or delete name is available in the dialog box.

(14) Time-lapse experiment process

Start time and estimated end time of time-lapse experiment, number of rounds, and passage of time are displayed.

(15) Print button Click this button to print the image captured with time-lapse experiment. The Print dialog box appears.


31


Item Function Click this button to save images captured with the time-lapse experiment to an image file. When a clipping area is set, the save confirmation dialog box for the clipped image appears. Click the OK button in the dialog box.

(16) Save button

Then, the Save image dialog box appears. To cancel the save operation and return to the Time-lapse images in process screen, click the Cancel button. When a clipping area is not set, this dialog box does not appear, but the Save image dialog box appears.

Figure 4.1-2 Confirmation dialog box

4.1.1 Image Magnification and Enlarge/Reduce Buttons

Figure 4.1-3 Image magnification

Table 4.1-2 Items and their functions for image magnification

Item Function Magnification Magnification of the current image compared to the original image size (resolution) is

displayed. Enlarge/reduce buttons

These buttons are used to enlarge or reduce the image magnification by 5% steps.

Enlarge/reduce menu

The down arrow (▼) button at the right of the magnification shows image magnification list box. Click a magnification to enlarge or reduce the image magnification.

4.1.2 Scrolling an Enlarged Image

Figure 4.1-4 Scroll mode button

Table 4.1-3 Scroll function for an enlarged image

Item Function Scroll mode button

When this button is clicked and an enlarged image is dragged, the image can be scrolled. When the image is dragged without clicking the scroll mode button or the clipping button, a region of interest (ROI) area is drawn.


32

4.1.3 Setting a Clipping Range of the Displayed Image

Figure 4.1-5 Clipping button

Table 4.1-4 Clipping range function for the displayed image

Item Function Use this button to set a clipping range of the displayed image. Select (left click and drag) an area of the displayed image. A clipping range of the image can be set. If this operation is performed for one image, the same clipping area is set for other three images. However, when a clipping area is set for an image, only the set clipping area (clipping image) can be saved.

Clipping button

You must click the clipping button again to turn off the clipping function. To delete the set clipping area, display the delete confirmation dialog box by right clicking the clipping area and selecting Delete from the submenu, or selecting the clipping area and pressing the Delete key on the keyboard, and then click the OK button in the dialog box.


dialog box

Reference

Saving an image when a clipping area is set with the clipping function

If a clipping area is set, click the Save button to display the save confirmation dialog box for the clipping image and click the OK button to save the clipping image. If a clipping area is not set, the displayed image is saved.

Figure 4.1-7 Save confirmation dialog box

4.1.4 Magnification button

Figure 4.1-8 Magnification button

Table 4.1-5 Magnification buttons

Item Function Magnification button Magnification used for running time-lapse experiment is displayed.


33

4.1.5 Images of Time-Lapse Experiment (Ph/FI1/FI2/Multi)

Images of time-lapse experiment are displayed. If a filter (Ph/Fl) is not selected for time-lapse experiment scheme, its image cannot be displayed.

Figure 4.1-9 Image of time-lapse experiment

Table 4.1-6 Time-lapse observation image operation items and their functions (1/2)

Item Function Microscopy image Image of each microscopy is displayed on each display area.

Right-click the filter button to open the sub-menu. Click on the sub-menu. The Input Superpose Percent dialog box appears. The dialog box is used to adjust the ratio (or strength) of the phase contrast image against the overlaid image. If the phase contrast image is too strong and the Fl1 or Fl2 image is not visible, use this function.

Filter button

Filter name and color of filter button can be changed. Right-click the filter button to open the sub-menu. Click on the sub-menu. The Input filter name dialog box appears. The dialog box is used to change the filter name or to select an image color. Besides, the Input filter name dialog box is also used to set up the image color for fluorescence images and overlaid image.

Scale bar button

Click this button to display scale bar on the bottom left of observation image. The scale length is 10 µm and has three ticks at 0 µm, 5 µm, and 10 µm.

Automatic tone curve compensation apply button

Click this button to automatically set the range. The range of the brightness to be displayed is automatically set. The highest brightness (A) of the displayed image is set as the upper limit (C) and the lowest brightness (B) is set as the lower limit (D).


Ph microscopy image

Fluorescent microscopy image (1) Multi microscopy image

Fluorescent microscopy image (2)


34

Table 4.1-6 Time-lapse observation image operation items and their functions (2/2)

Item Function Tone curve compensation setting button

Click this button to set tone curve compensation manually. The Histogram dialog box appears to set tone curve compensation.

Tone curve compensation cancel button

Click this button to cancel tone curve compensation and return to the original screen.

Saturation check button

Click this button to display saturation point in red. To clear the saturation check condition, clicked this button again. This function can be used only for the fluorescent microscopy.

Brightness screening button

Click this button to display the Screening dialog box for setting brightness screening. This function can be used only for the fluorescent microscopy.

Pseudocolor button

Use this button to assign a pseudocolor for fluorescence images. For overlaid images, this button is available only when a fluorescence image is used. When this button is clicked, the saturation check button and the brightness screening button are disabled. On the other hand, when the saturation check button or the brightness screening button is clicked, the pseudocolor button is disabled.

Enlarge button

Enlarge/reduce buttons

Reduce button

Click the enlarge button to enlarge the image. The display of the enlarge button changes to the reduce button after the image is enlarged. Click the reduce button to reduce the image to the original size.

Overlapping button

Click this button to display selected filters overlapped image or display Ratio image.

Ratio setting button

Click this button to set Ratio image. The Ratio setting dialog box appears.

Phase contrast image ratio (strength) adjustment Right-click the Ph filter button to open the sub-menu, Input percent. Click on the sub-menu. The Input Superpose Percent dialog box appears.

Figure 4.1-11 Input Superpose Percent dialog box

Table 4.1-7 Phase contrast image ratio adjustment items and their functions

Item Function Percent Specify the phase contrast image strength (%). Enter the value with the keyboard or

use the ▲▼ buttons. Apply Click the button to apply the settings. The dialog box closes. Close Click the button to close the dialog box without applying settings.


35

Changing filter name and display color Right-click the Fl1 or Fl2 filter button to open the sub-menu, . Click on the sub-menu. The Input filter name dialog box appears.

Figure 4.1-12 Input filter name dialog box

Table 4.1-8 Filter name, display color, and overlaid image color setting items and functions

Item Function Filter The selected filter name appears here. Name Enter a filter name (up to four alphanumeric characters). Color Select a color from these buttons to apply its color to filter button displayed image

color and graph line. Click a button to select a color. A check symbol appears.

Use the custom color to overlap the image

Check here to enable the custom color function for fluorescence images and overlaid images.

Custom color setting button

Click the button to open the Color dialog box. You can create an arbitrary color. The color can be used for the pseudocolor of fluorescence images and for the display color of overlaid images.

Figure 4.1-13 Color dialog box

Apply button Click this button to apply settings of file name and color. The dialog box is closed. Close button Click this button to close the dialog box without applying settings.

Custom color settingbutton


36

Setting Ratio image Click the Ratio setting button ( ) of the time-lapse experiment image (Multi Ch) to display the Ratio Setting dialog box.

Figure 4.1-14 Ratio Setting dialog box

Table 4.1-9 Ratio image setting items and their functions

Item Function Ratio Channel Select “Ratio Channel” from the pull-down menu. Ratio Range Input “Ratio Range.” Apply button Click this button to register Ratio settings. Close button Click this button to close the dialog box without applying Ratio settings.

In this area, set the lower limit of the brightness for ratio setting.


37

4.1.6 Brightness Graph


Table 4.1-10 Brightness graph items and their functions

Item Function Time Range Select the time range (horizontal axis) of one-page graph from the pull-down menu.

Fit: total observation time fits in the time range of one-page graph. 1x, 2x, 3x, and 4x: Values of Time Range Time per scale and time range of one-page graph are calculated from minimum interval time, value of Time Range, and coefficient. Time per scale = (4)/(value of Time Range) x (minimum interval time) Time range of one-page graph = (time per scale) x 7 Example: when value of Time Range is 2x and minimum interval time is 10 minutes: (4/2) x 10 = 20 minutes (time per scale) 20 x 7 = 140 minutes (time range of one-page graph) If total observation time cannot fit in one page, scroll bar appears at the bottom of graph.

Intensity Range Select a brightness range (horizontal axis) of one-page graph from the pull-down menu. Fit: full range of measured brightness fits in one-page graph. 1x, 2x, 3x, and 4x: Values of Time Range When one unit = maximum brightness (measurement result)/4 Brightness range of one-page graph= (4/value of Intensity Range) x one unit Example: when value of Intensity Range is 2x and one unit is 600

(brightness: 0 to 2400): (4/2) x 600 = 1200 (brightness range for one-page graph) When value of Intensity Range is 2x or more, scroll bar appears at the right of graph.

Grid button Click this button to display or hide grid line on graph. Calculate button Click this button to calculate brightness value.

If this button is clicked after an area of an observation image is selected, the brightness graph for the selected area is displayed. See Section 4.1.7, “ Brightness Graph Displaying Method.”


38

4.1.7 Brightness Graph Displaying Method

Brightness graph of an image can be displayed during a time-lapse experiment or by selecting an area (by specifying an area using the ROI symbol) in the display area of a time-lapse result file.

The procedure to show the brightness graph for the Channels view is different from that for the Points view.

Channels view

1. Select an area of an image for brightness graph Select an area to display its brightness graph by left clicking and dragging. When this operation is performed in one display area, the same operation is performed in other three display areas.

Note

Image area setting for a brightness graph

When only the phase contrast (Ph) filter is selected for the microscopy, no image area can be set for a brightness graph because the brightness analysis is not necessary.

Figure 4.1-16 Selecting an area of an image for brightness graph

Note on setting the ROI symbol

• In the scroll mode or the clipping mode, no ROI symbol can be drawn on the image area. Therefore, cancel the scroll mode or the clipping mode to draw an ROI symbol.

2. Display the brightness graph. Click the Calculate button. The brightness graph of the selected area is displayed in the brightness graph display area.

When the brightness graph is displayed, the selected area frame changes from yellow to red.

Display area of a brightness graph

For information on the time (horizontal axis) and the brightness (vertical axis) of the brightness graph, see Section 4.1.6, “Brightness Graph.”

Figure 4.1-17 Calculate button

Figure 4.1-18 Displaying brightness graph

ROI symbol


39

Points view 1. Select an area of an image for displaying the

brightness graph. Select an area to display its brightness graph by left clicking and dragging.

Different observation point and range can be set to display a brightness graph.

Note

Image area setting for a brightness graph

When only the phase contrast (Ph) filter is selected for the microscopy, no image area can be set for a brightness graph because the brightness analysis is not necessary.

Figure 4.1-19 Selecting an area of an image to display the brightness graph

Note on setting the ROI symbol

• In the scroll mode or the clipping mode, no ROI symbol can be drawn on the image area. Therefore, cancel the scroll mode or the clipping mode to draw an ROI symbol.

2. Select an observation point to display a brightness graph. Click an observation point of an image to display its brightness graph.

The point number of the selected observation image is marked with a blue box.

Figure 4.1-20 Selecting an area of an image to display the brightness graph


40

3. Display the brightness graph. Click the Calculate button.

The brightness graph of the selected area is displayed in the brightness graph display area.

When the brightness graph of a selected area is displayed, its frame color changes from yellow to red The frame color of other selected areas remains yellow.

Display area of a brightness graph

For information on the time (horizontal axis) and the brightness (vertical axis) of the brightness graph, see Section 4.1.6, “Brightness Graph.”

Figure 4.1-21 Calculate button

Figure 4.1-22 Displaying the brightness graph


41

4.1.8 Time Line and Image Playback Control

Figure 4.1-23 Time line and image control

Table 4.1-11 Time line display and image playback operation items and their functions (1/2)

Item Function A to B repeating mark

This mark is displayed on the repeating section selected with the A to B playback button.

Number of rounds

The number of rounds of the playback slider's current position is displayed.

Playback slider

This slider shows the current playback point and elapsed time, and moves in synchronization with the playback image. To play back the image from the point, move this slider to a point with a mouse pointer or the right and left arrow buttons.

Frame-by-frame button

Click this button to play back the image forward or backward frame by frame.

Pause button

Click this button to pause playback of the image.

Slow playback button

Click these buttons to slowly play back the image forward or backward. (One to three images per second. The reproduction speed changes every time the button is clicked. The circle symbol of the button changes in accordance with the reproduction speed.)

Playback button

Click this button to play back the image. (Five images per second)

Fast-forward button

Click this button to fast-forward the image. (10 to 15 images per second. The reproduction speed changes every time the button is clicked. The circle symbol of the button changes in accordance with the reproduction speed.)

Marked image playback button

Click this button to play back ten or more images before and after the user mark or the cell stimulus mark. When the playback slider is located on either mark, images before and after the mark will be playback. When the playback slider is not located on either mark, images before and after the next mark will be playback.

A to B playback button

Click this button to select and repeat a repeating section, and clear the selected repeating section. The A to B repeating mark is displayed on the selected repeating section. Every time this button is clicked, the function of this button changes as follows: set A point → set B point → repeat from A to B → clear repeating section.

User mark setting button

Click this button to set the user mark. The user mark is displayed at the current playback point or observation point.


42

Table 4.1-11 Time line display and image playback operation items and their functions (2/2)

Item Function Click this button to set the cell stimulus mark. The cell stimulus mark is displayed at the current playback point or observation point.

Cell stimulus mark setting button

Right click this button to display the submenu for assigning keys. Assignable keys are “Space”, “Return”, “Shift”, and “Tab.” Select one from these four keys. And then, the selected key can be used instead of the cell stimulus mark setting button. If the None key is selected, no key is assigned.

Figure 4.1-24 submenu

This mark is displayed at the point set with the user mark setting button. Right click the user mark to display the submenu.


Select Delete from the submenu to delete comments. Select Edit to display the Mark Comment Input dialog box with which you can input, confirm, or change comments.

User mark

Click the Apply button in the Mark Comment Input dialog box to apply the edited comments. Left click the user mark to move the playback slider to the user mark position.

Figure 4.1-26 Mark Comment Input dialog box

This mark is displayed at the point set with the cell stimulus mark setting button. Right click the cell stimulus mark to display the submenu.


Select Delete from the submenu to delete comments. Select Edit to display the Mark Comment Input dialog box with which you can input, confirm, or change comments.

Cell stimulus mark

Click the Apply button in the Mark Comment Input dialog box to apply the edited comments. Left click the cell stimulus mark to move the playback slider to the cell stimulus mark position.

Figure 4.1-28 Mark Comment Input dialog box


43

4.1.9 Time-Lapse Experiment Scheme (Points Tab)

Figure 4.1-29 Points tab

Table 4.1-12 Items and their functions on the Points tab

Item Function Observation point Observation conditions (filter name, exposure time, and magnification) are

displayed for each observation point. When time-lapse experiment is performed, only the point selected with a check mark is observed. Click an observation point to display the Point information dialog box. In the dialog box, checking, changing, and deleting detailed condition are available.

Stop button Click this button to stop the running time-lapse experiment. The stop confirmation dialog box appears. To stop time-lapse experiment, click the OK button.

Figure 4.1-30 Stop confirmation dialog box

Resume button When time-lapse experiment is stopped, the Stop button is changed to the Resume button. Click the Resume button to restart time-lapse experiment.

Checking observation condition Check the observation condition in the following Point information dialog box.

Figure 4.1-31 Point information dialog box

Table 4.1-13 Button function on the observation condition check dialog box

Item Function Close button Click this button to close the Point information dialog box.


44

4.1.10 Time-Lapse Experiment Scheme (Time Tab)


Table 4.1-14 Items and their functions on the Time tab

Item Function Time-lapse experiment time

Capturing interval time, total observation time, and rounds for time-lapse experiment are displayed. Click a time-lapse experiment time to display the Timelapse dialog box. In the dialog box, changing and deleting capturing interval time setting and total observation time setting are available.

Delete button Click this button to delete the selected time-lapse experiment time. Delete confirmation dialog box appears. To delete the selected time-lapse experiment time, click the OK button. To return to the Time tab without deleting, click the Cancel button.


dialog box

Red dot The red dot shows the interval time of running time-lapse experiment. New button Click this button to set new time-lapse experiment time.

The TImelapse dialog box appears for new registration of time-lapse experiment time.

Reference

Details on the Timelapse dialog box

Operation of the Timelapse dialog box is the same as that of the New time-lapse setting screen.

Reference: Chapter 3 “New Time-Lapse Setting Screen”

3.1.4 Time Lapse Experiment Scheme (Time Tab) Changing/deleting time-lapse experiment time Registering new time-lapse experiment time

Figure 4.1-34 For setting change

Figure 4.1-35 For new registration


45

4.1.11 Time-Lapse Experiment Status Display

Figure 4.1-36 Displaying time-lapse experiment process

Table 4.1-15 Items and their functions on the time-lapse experiment status display

Item Function Start time/ estimated end time

Start time and estimated end time of time-lapse experiment are displayed. (month/date/time)

Time line Process of time-lapse experiment is displayed with the bar graph. Number of rounds Number of rounds of running time-lapse experiment is displayed. (current number of

rounds/total number of rounds) Passage of time Passage of time-lapse experiment time is displayed (current passage of time/total

observation time). Temperature log button

Click this button to check changes in temperature during time-lapse experiment.The Temperature dialog box appears.

Pause button Click this button to pause the running time-lapse experiment. Stop button Click this button to stop the running time-lapse experiment.

Displaying graph for device conditions

Click the Temperature log button ( ) in the time-lapse experiment process area to display the following dialog box.

Figure 4.1-37 Temperature dialog box

Table 4.1-16 Temperature dialog box items and their functions

Item Function Range Select time range (horizontal axis) of temperature graph. Temperature graph

Changes in the temperature inside the culture chamber, changes in the temperature inside the humidifier water tank, and changes in ambient temperature are shown.

Close button Click this button to close the Temperature dialog box.

Start time of time-lapse experiment

Estimated end time of time-lapse experiment

The time-lapse experiment is running under a stable condition.

An unstable condition is detected during the time-lapse experiment.


46

4.1.12 Captured Image Printing

Click the Print button ( ) to display the Print dialog box.

The Print dialog box changes depending on the observation image conditions (Channels display or Points display).

Use this dialog box to print the images captured with time-lapse experiment.

Print dialog box for Channels display Print dialog box for Points display

Figure 4.1-38 Print dialog box

Table 4.1-17 Items and their functions for printing images

Item Function Select channel Select the image to be printed. For Channels display, select a filter. For Points

display, select an observation point. The filters that are not specified for the time-lapse experiment condition are disabled.

Print button Click this button to print an image. Cancel button Click this button to stop printing and close the dialog box.


47

4.1.13 Captured Image Saving

Click the Save button ( ) to display the Save image dialog box.

If a clipping area is set, the save confirmation dialog box for the clipped image appears. Click the OK button in the dialog box. Then, the Save image dialog box appears.

Figure 4.1-39 Save image dialog box

Table 4.1-18 Button functions for saving images

Item Function Save as...(BioStation IM format) button

During time-lapse experiment, this button is disabled because the file is already saved at the beginning of the experiment.

Export images and data (Single point) button

Time-lapse images captured at a point are saved to image files. The Export images and data (Single point) dialog box appears.

Export images and data (Multi points) button

Time-lapse images captured at multiple points are saved to image files. The Export images and data (Multi points) dialog box appears.

Cancel button Click this button to close the dialog box.


48

Image data save settings for single observation point (1/2) Click the Export images and data (Single point) button of the Save image dialog box to display the Export images and data (Single point) dialog box.

Figure 4.1-40 Export images and data (Single point) dialog box

Table 4.1-19 Items and their functions on the Export images and data (Single point) dialog box (1/2)

Item Function Export images and data of

Select the observation point and magnification for saving the image from the pull-down menu. All Select “All” to save all images of the selected

observation point under the selected magnification in a file.

One shot of current time Select “One shot of current time” to save only the current displayed image in a file.

Neighbors of current time Select “Neighbors of current time” to save 11 images in total: current displayed image, five images before the displayed image, five images after the display image.

Concatenate all marks Select “Concatenate all marks” to link all images captured at the user mark and the cell stimulus mark of the Time Line, five images before these marks, and five images after these marks, and save the linked images in a file. If neither user mark nor cell stimulus mark is selected, this item is disabled.

Save time range

Input time range Select “Input time range” to save images captured within input time in a file.


49

Table 4.1-19 Items and their functions on the Export images and data (Single point) dialog box (2/2)

Item Function Ph Check here to save the Ph channel data into a file. Fl1 Check here to save the Fl1 channel data into a file. Fl2 Check here to save the Fl2 channel data into a file.

Multi ch Check here to save overlaid image composed of any of the Ph, Fl1, or Fl2 channel.

Intensity data Check here to save the brightness data into a file. This option is enabled only when the brightness is calculated.

Time-lapse-log Check here to save the time-lapse experiment log into a file.

Save channel

Mark comment

Check here to save the comments of the user mark and cell stimulus marks. This option is enabled only when the user mark or the cell stimulus mark is used.

Next button Click this button to display the Save images dialog box. In the dialog box, specify the destination to save image and input its file name and file format.

Back button Click this button to cancel saving and close the dialog box.


50

Image data save settings for single observation point (2/2) Click the Next button of the Export images and data (Single point) dialog box to display the Save images (Single point) dialog box.

Figure 4.1-41 Save images (Single point) dialog box

Table 4.1-20 Items and their functions on the Save images (Single point) dialog box (1/2)

Item Function Folder Select a folder to save images.

Click the button to display the Brows For Folder dialog box and specify the destination to save.

Figure 4.1-42 Brows For Folder dialog box


51

Table 4.1-20 Items and their functions on the Save images (Single point) dialog box (2/2)

Item Function Format Select a format from six formats to save images.

TIFF (Gray: 16bit Color: 24bit),TIFF (Gray: 8bit Color: 24bit),BMP (Gray: 8bit Color: 24bit),PNG (Gray: 8bit Color: 24bit), JPEG (Gray: 8bit Color: 24bit), and AVI

File name Input a file name to save images. Channel names will be added to the end of file names automatically. Ph：_ch1, Fl1：_ch2, Fl2：_ch2, Multi：_ch4

Apply tone curve processing

Select this check box to save the image that underwent tone curve compensation.

Write time into mages

Select this check box to write the number of rounds, capturing date, and passage of capturing time into images.

Write current ROI into images

Select this check box to write the ROI mark into images for the brightness graph.

Save image

Write observation setting into images

Select this check box to write observation conditions such as exposure time, gain, and resolution on the image.

Format Select a format from the pull-down menu to save the brightness graph.

Save Intensity data

File name Input a file name to save the brightness graph. The suffix, “_intensity”, is added to the end of the file name automatically.

Time-lapse log Input a file name to save a log file (text file) of time-lapse experiment. The suffix, “_timelapse”, is added to the end of the file name automatically.

Mark comment Input a file name to save comments of the user marks and cell stimulus marks. The suffix, “_mark”, is added to the end of the file name automatically.

Save button Click this button to save the image into a file according to setting. After saving is succeeded, the dialog box appears. Click the OK button to close the dialog box.

Figure 4.1-43 Save succeeded dialog box

Back button Click this button to return to the Export images and data dialog box.

Reference

How to input information during capturing images:

If “TIFF (Gray: 16bit Color: 24bit)” is selected in the Format, selected information is invalidated.

To input information in the save file of images, select “Gray8bit” in the Format.

Figure 4.1-44 Save images (Single point) dialog box


52

Image data save settings for multiple observation point (1/2) Click the Export images and data (Multi points) button of the Save image dialog box to display the Export images and data (Multi points) dialog box.

Figure 4.1-45 Export images and data (Multi points) dialog box

Table 4.1-21 Items and their functions on the Export images and data (Multi point) dialog box

Item Function Select All Points To select all observation points, check here. Select export

data Point1 to Point3 All registered observation points appear here. To select desired observation points, uncheck the “Select All Points” checkbox and check desired observation points.

All Select “All” to save all images of the selected observation point under the selected magnification in a file.

One shot of current time

Select “One shot of current time” to save only the current displayed image in a file.

Neighbors of current time

Select “Neighbors of current time” to save the current displayed image, the five preceding images, and the five following images (total of 11 images) into a file.

Concatenate all marks

Select “Concatenate all marks” to save all concatenated images into a file. The concatenated images consist of images of all marks including user marks and cell stimulate marks added in the Time Line, five preceding images of each mark, and five following images of each mark.

Save time range


Ph Check here to save the Ph channel data into a file. Fl1 Check here to save the Fl1 channel data into a file. Fl2 Check here to save the Fl2 channel data into a file.

Save channel

Multi ch Check here to save overlaid image into a file with the displayed condition.


53

Image data save settings for multiple observation point (2/2) Click the Next button of the Export images and data (Multi points) dialog box to display the Save images (Multi points) dialog box.

Figure 4.1-46 Save images (Multi points) dialog box

Table 4.1-22 Items and their functions on the Save images (Multi point) dialog box (1/2)

Item Function Folder Select a folder to save images.



Format Select a format from six formats to save images. TIFF (Gray: 16bit Color: 24bit),TIFF (Gray: 8bit Color: 24bit), BMP (Gray: 8bit Color: 24bit),PNG (Gray: 8bit Color: 24bit), JPEG (Gray: 8bit Color: 24bit), and AVI

File name Input a file name to save images. Channel names will be added to the end of file names automatically. Ph：_ch1, Fl1：_ch2, Fl2：_ch2, Multi：_ch4

Apply tone curve processing

Select this check box to save the image that underwent tone curve compensation.

Write time into mages

Select this check box to write the number of rounds, capturing date, and passage of capturing time into images.

Write current ROI into images

Select this check box to write the ROI mark into images for the brightness graph.

Save image

Write observation setting into images

Select this check box to write observation conditions such as exposure time, gain, and resolution on the image.


54

Table 4.1-22 Items and their functions on the Save images (Multi point) dialog box (2/2)

Item Function Save button

Click this button to save the image into a file according to setting. After saving is succeeded, the dialog box appears.Click the OK button to close the dialog box.

Figure 4.1-48 Save succeeded dialog box

Back button Click this button to return to the Export images and data dialog box.

4.1.14 Example of a Saved Image

Figure 4.1-49 Example of a saved image

Observation conditions

ROI mark

Displaying the number of rounds, photo date,and passage of photo time

Scale

Input information on the Cell name etc... tab


55

4.2 Points Display This screen shows process of time-lapse experiment. One display area is provided for one observation point. Switch the display area to display each filter image and overlapped image. Therefore, up to four observation points can be observed at the same time. Functions other than the way to display images are the same as those for the Time-lapse images in process Screen (Channels).

Figure 4.2-1 Time-lapse images in process screen (Points)

Table 4.2-1 Items and their functions on the Time-lapse images in process screen (Points)


button Select filters for reference by clicking those buttons. A check mark is placed and an image of the selected filter is displayed. Multiple filters can be selected. When multiple filters are selected, the selected filters overlapped image is displayed.

(2) Observation image display

Images at different points can be displayed on the four observation image displays.Additionally, the ROI for brightness analysis can be set for each observation point. Click in the observation image display to select an observation point. The point number at the upper left of the observation image display is marked with a blue box.

(3) Selecting observation points

Only the image at the checked observation point is displayed on the observation image display. Up to four observation points can be displayed at a time.

(4) Calculate button

Click this button to calculate brightness value. If this button is clicked after an area of an observation image is selected, the brightness graph for the selected area is displayed.

(5) ROI mark The ROI mark is normally marked with a yellow box, and the ROI mark when a brightness graph is displayed is marked with a red box.

(1)

（2）

(3)

（2）

（2）（2）

(5)

(4)

56

Time-Lapse Images Acquired Screen5 5

This screen automatically appears as initial screen of software or at the end of time-lapse experiment. On this screen, loading and reproducing the saved file of time-lapse experiment results are available. There are the Channels mode and the Points mode for the Time-lapse images Acquired screen. A part of functions is different from those of the Time-lapse images in process screen. This section indicates only the different part.

5.1 Channels Display

Figure 5.1-1 Time-lapse images Acquired screen

Table 5.1-1 Items and their functions on the Time-lapse images Acquired screen (Channels) (1/2)

Item Function (1) File load button Click this button to load the saved file of time-lapse experiment results. (2) Observation point

and file information Position of observation point, its X and Y coordinates, loaded file name of time-lapse experiment results, sample name, and cell name are displayed.

(3) Time-lapse experiment result

Total time/start time/end time of time-lapse experiment, number of rounds, logarithmic graph of temperature changes, and maximum and minimum values of temperature change are displayed.

(1)

(2)

(3)

(5)

(4)

Chapter 5 Time-Lapse Images Acquired Screen

57

Table 5.1-1 Items and their functions on the Time-lapse images Acquired screen (Channels) (2/2)

Item Function Click this button to save a time-lapse results file as an alias file, or to save an image captured with the time-lapse experiment into an image file. If a clipping area is set, the save confirmation dialog box for the clipped image appears. Click the OK button in the dialog box. Then, the Save image dialog box appears. To cancel the save operation and return to the Time-lapse images Acquired screen, click the Cancel button.

Figure 5.1-2 Save confirmation

dialog box

(4) Save button

If a clipping area is not set, this dialog box does not appear, but the Save image dialog box appears.

(5) Fluctuation correction button

Click this button to measure the fluctuation amount of the stage from a captured image and correct the image by shifting it in the X and Y directions. In principle, the image size is reduced by the shift amount. The area shifted is marked with a black frame. Corrected image data is saved in a new file.


58

5.1.1 Saving a Time-Lapse Result File as an Alias File/Saving a Captured Image

Click the save button ( ) to display the Save image dialog box.

When a clipping area is set and the OK button is clicked on the save confirmation dialog box for a clipping image, the Save image dialog box appears.

Figure 5.1-3 Save image dialog box

Table 5.1-2 Items and their functions on the Save image dialog box

Item Function Save as... (BioStation IM format) button

Click this button to save the time-lapse experiment result as a new file. The Save into BioStation IM format (ics/ids) dialog box appears.

Export images and data (Single point) button

Time-lapse images captured at a point are saved to image files. The Export images and data (Single point) dialog box appears.

Export images and data (Multi points) button

Time-lapse images captured at multiple points are saved to image files. The Export images and data (Multi points) dialog box appears.

Cancel button Click this button to close the dialog box.

Reference

Export images and data (Single point) dialog box and Export images and data (Multi points) dialog box

Operations of the two Export images and data dialog boxes are the same as the Time-lapse images in process screen.

Refer to the following sections: Chapter 4 “Time-Lapse Images in Process Screen”

4.1.13 Captured Image Saving Image data save settings for single observation point (1/2) and (2/2) Image data save settings for multiple observation point (1/2) and (2/2)

Figure 5.1-4 Export images and data

(Single point) dialog box


59

Saving into the BioStation IM format Click the Save into BioStation IM format (ics/ids) button of the Save image dialog box to display the Save into BioStation IM format (ics/ids) dialog box.

Figure 5.1-5 Save into BioStation IM format (ics/ids) dialog box

Table 5.1-3 Items and their functions on the Save into BioStation IM format dialog box (1/2)

Item Function Folder Select a folder to save an image.



Filename Input a file name for time-lapse experiment results.


60

Table 5.1-3 Items and their functions on the Save into BioStation IM format dialog box (2/2)

Item Function All Select “All” to save all images of the selected

observation point under the selected magnification into a file.

One shot of current time Select “One shot of current time” to save only the current displayed image into a file.

Neighbors of current time Select “Neighbors of current time” to save the current displayed image, the five preceding images, and the five following images (total of 11 images) into a file.

Concatenate all marks Select “Concatenate all marks” to save all concatenated images into a file. The concatenated images consist of images of all marks including user marks and cell stimulate marks added in the Time Line, five preceding images of each mark, and five following images of each mark.

Save time range


Save points Select an observation point to save. Save tone curve processing parameter

Select this check box to save the tone curve processing parameter. The image to which the tone curve processing applied itself is not saved. Only tone curve processing parameter is saved.

Save ROI information Select this check box to save positions of the ROI set for each observation point.Save button

Click this button to save images into a file according to settings. When the file is saved successfully, a dialog box appears. Click the OK button to close the dialog box.

Figure 5.1-7 Save succeeded

dialog box

Back button Click this button to cancel saving and close the dialog box.


61

5.2 Points Display On this screen, one observation point occupies one display area. A filtered image or an overlapped filtered image of the observation point can be displayed on the area when specified. Up to four observation points can be observed at the same time. Functions other than the way to display images are the same as those for the Time-lapse images in process Screen (Channels).

Figure 5.2-1 Time-lapse images Acquired screen (Points)

Table 5.2-1 Items and their functions on the Time-lapse images Acquired screen (Points)


button Select filters for reference by clicking those buttons. A check mark is placed and an image of the selected filter is displayed. Multiple filters can be selected. When multiple filters are selected, the selected filters overlapped image is displayed.

(2) Observation image display

Images at different points can be displayed on the four observation image displays.Additionally, the ROI for brightness analysis can be set for each observation point. Click in the observation image display to select an observation point. The point number at the upper left of the observation image display is marked with a blue box.

(3) Selecting observation points

Only the image at the checked observation point is displayed on the observation image display. Up to four observation points can be displayed at a time.

(4) Calculate button

Click this button to calculate brightness value. If this button is clicked after an area of an observation image is selected, the brightness graph for the selected area is displayed.

(5) ROI mark The ROI mark is normally marked with a yellow box, and the ROI mark when a brightness graph is displayed is marked with a red box.

(1)

(2)

(3)

(2)

(2) (2)

(5)

(4)

biostation im cell-s1 / cell-s1-p - mvi-inc.com im cell-s1_s1-p software.pdf · i introduction...

Documents