Olympus FV3000 User manual

FV3000
Operation Manual

Contents
-----------------Image Acquisition---------------
Starting the System ――――――――――――――――― 4
Visual observation with microscope ―――――――――― 5
XY Image Acquisition ――――――――――――――――― 6
XYZ Image Acquisition ―――――――――――――――― 8
XYT Image Acquisition ―――――――――――――――― 9
4D（XYZT）Image Acquisition ―――――――――――― 10
XYT Image Acquisition（with Z drift compensation） ―― 11
Multi Area Time Lapse ―――――――――――――――― 12
Multi Area Time Lapse using Map Image（１）――――― 14
Tips of Image Acquisition ――――――――――――――― 17
Exiting the system ―――――――――――――――――― 18
Configuration ―――――――――――――――――――― 19
------------------Processing Image ----------------
2D view and operation ――――――――――――――― 21
3D view and operation ――――――――――――――― 22
Creating the movies ――――――――――――――――― 23
Processing: Projection ―――――――――――――――― 24
Exporting the image ―――――――――――――――― 25
Reloading and saving Observation Methods ―――――― 26
------------------Spectral Imaging------------------
Lambda Series（acquiring by single channels）―――― 28
Lambda Series（acquiring by multiple channels）――― 30
Processing: Unmixing（１） ――――――――――――― 32
Processing: Unmixing（２） ――――――――――――― 33
Processing: Unmixing（３） ――――――――――――― 34

Image Acquisition

Starting the system
Turning ON the power
① Set the main switch of the PC to ON
② Switch on the central power..
Laser controller
③ Switch on the main power of each
laser. And Rotate the start key.
Touch Panel Controller（TPC)
④ Press the main switch of the touch
panel controller to set it to ON.
Mercury burner for the light source
⑤ Press the lamp switch on the front side
of the light source.
Logon Windows
⑥ Logon Windows with your own user ID
and password.
Starting software for FV3000
⑦ Double-click the [FV31S-SW] icon on
the desktop.
※Start the software after “Start operation”
is displayed in TPC.
⑦ The stage setting dialog box appears.
Set each checkbox and click [OK].
4
Factory Default ID & password
Factory User ID：olympus
Password：olympus
Switch on the
main power
Rotate
start key
③Laser Combiner
TPC main
switch
④Touch Panel Controller（TPC)
ON/OFF
switch
To be blue
⑤Mercury burner
②Central
power
①PC
switch
Selection whether or not to initialize the
resonant scanner
XY stage type
Selection whether or not to control the XY
stage by the software
Selection whether or not to perform the
centering in XY direction

Visual observation with microscope
★Fluorescence observation
*Tap the button of the TPC to select
the objective for observation.
① Tap the light path selection button
of the touch panel controller.
(or select “Ocular” in [Ocular] Tool
Window. )
② Tap the button.
③ Tap the [EPI(or EPI1) tab.
④ Select the mirror unit suitable for the
fluorescent probe for observation.
⑤ Open the shutter of the reflected
illumination light on the touch panel
controller.
⑥ Select the ND filters to adjust the
brightness.
※Brightness can be adjusted by rotating
the light volume adjustment dial of
the light source.
Rotate the light volume
adjustment dial
⑦ Adjust the focus
using the focus
knob.
★Transmitted DIC observation
*Tap the button on the TPC to
select the objective for observation.
① Tap the light path selection button
of the touch panel controller.
② Tap the button.
③ Tap the [DIA] tab.
④ Tap the [Elements] tab
and check whether
the [Polarizer] is “IN”.
⑤ Insert the DIC prism
slider.
⑥ Open the shutter of the transmission
illumination light on the touch panel
controller.
⑦ If necessary, tap the [Brightness] tab
and adjust the brightness.
⑧ Adjust the focus and contrast.
④
①
②
⑤
④
③
⑥
3
①
②
⑦
③
⑥
Adjust the DIC contrast
by rotating the knob.

XY Image Acquisition(1)
① Select “VBF” in the [PMT Setting] Tool Window.
Assigning the detector to channel
② Press the button on [PMT Setting]
Tool Window to open the [Dye & Detector Select]
dialog box.
③ Press the button to reset the Assigned
Dye.
④ Double-click the name of the fluorescence dye to
observe.
⑤ When registering a fluorescence dye, TD channel
is registered automatically. If you don’t need,
double-click the TD channel to cancel it.
⑥ If you want to observe with multiple phases using
the virtual channel scan, press the button
to add the phase. And drag&drop the selected
“Dye” to the observation channel list of the phase
you want to add.
⑦ After setting all channels, press the [OK] button.
Adjusting the live image
⑧ If you want to acquire multiple channel image,
select Sequential scan “Line” in the [PMT Setting]
Tool Window.
⑨ Press the button on the [Live] window.
Adjust focus and set Laser Intensity(%),
Sensitivity(HV), Gain and Offset on [PMT setting]
Tool Window.
⑩ If you use virtual channel scan, press
to switch phases. On both phases, adjust focus
and set Laser Intensity(%), Sensitivity(HV), Gain
and Offset.
Virtual Channel Scan
This mode allows to acquire multiple CH image
whose channel number is larger than the
number of detector.
①
Adjusting Confocal
Aperture.
Reducing noises by
Averaging.
②
③
④
⑤
⑦
⑧
⑨
Laser Intensity
Sensitivity
4
⑩
【Hi-Lo Mode】
pixels of intensity 4095 are shown in red.
Pixels of intensity 0 are shown in red.
u5

Slide 6
u5 確認（英語のほう）
元島 ゆき; 03.02.2017

XY Image Acquisition(２)
Setting the scanner
⑩ On [LSM Imaging] Tool Window, select the
scan method in [Type] and [Mode].
※See 23 pages for details.
⑪ Set scanning speed.
⑫ Specify “Image Size” to set the scan area.
⑬ Set the zoom, rotation and clip scan if
needed.
Starting Acquisition
⑭Select [Normal] tab in [Acquire] Tool Window.
Press the button to open the dialog
box, and select the folder to save the images.
*The acquired images are saved automatically.
Serial number is added at the end of file name
like “***_0001” and “***_0002”.
⑮Press button to start acquiring the
image.
5
DIC observation
1. Insert the DIC slider to microscope.
2. Register the TD channel.
3. Set the laser to be used for observation.
4. Adjust the sensitivity(HV).
5. Adjust the contrast by rotating the knob of
the DIC slider.
(See page 3 ⑤ in left side. )
Attention
Check [LSM] in [Series] Tool Window whether it
is selected “OFF” [Z] and [Time] .
⑩
⑪
⑫
⑬
⑮
⑭
4.
3.

XYZ Image Acquisition
*Before starting the following procedure, make
adjustments for XY imaging.
(refer to page 4-5.)
Setting Z series
① Select “ON” in [Z] on [Series] Tool Window.
② Select “Start/End” in [Motor] on [Z section].
Register start/end position
③ Change the Z position by rotating the
focusing knob or pressing the
button. Press the button in [Start]
at the Z position to start acquiring the
image.
④ Change the Z position and go on to press
the button in [End] at the Z position
to end acquiring the image.
⑤ Enter a value in [Slices] or [Step Size].
Setting one will set other automatically.
Pressing the button, both numerical
values “Slices” and “Step Size” is optimized.
Starting Acquisition
⑥ Select [Normal] tab in [Acquire] Tool
Window. Press the button to display the
dialog box, and select the folder to save the
images.
Press the button to start acquiring
the image.
Finishing Acquisition
⑧ Finishing acquisition, buttons blink.
Press the button to complete the
image acquisition. If you want to Additional
images under the same condition, enter the
number of additional acquisition and press
the button. After the image is
acquired, press the button.
①
③
⑤
②
④
⑥
⑦
⑧
6
:Each click shifts the position by the value
entered for Step Size
:Each click shifts the position by ½ the value
entered for Step Size.
Pressing the button, current
position is registered as origin.
u6
u7

Slide 8
u6 新しく追加。要チェック。
元島 ゆき; 08.02.2017
u7
Step Size
にしたけど大丈夫だろうか？
内容・英語ともに。
元島 ゆき; 08.02.2017

XYT Image Acquisition
*Before starting the following procedure,
make adjustments for XY imaging. (refer to
page 4-5.)
Setting Time Series
① Select “ON” in [Time] on [Series] Tool
Window.
② Set the interval to acquire the image in
[Interval] and [Cycle] on [Time Lapse].
Starting Acquisition
③ Select [Normal] tab in [Acquire] Tool
Window. Press the button to display
the dialog box, and select the folder to
save the images.
*The acquire images are saved
automatically. Serial number is added at the
end of file name like “***_0001” and
“***_0002”.
④ Press the button to start
acquiring the image.
Finishing Acquisition
⑦ Finishing acquisition, buttons
blink.
Press the button to complete
the image acquisition. If you want to
Additional images under the same
condition, enter the number of additional
acquisition and press the button.
After the image is acquired, press the
button.
If you attempt to set the shorter than the time
displayed in [Scan] in [Interval], “FreeRun”
appears. In this case, the interval to acquire
the image is the time displayed in [scan].
7
①
②
③
④
⑤

4D Image Acquisition
*Before starting the following procedure,
make adjustments for XY imaging.
(refer to page 4-5.)
① Select “ON” in both [Z] and [Time] on
[Series] Tool Window.
Setting Z series
② Changing the Z position, register the Z
position of Start/End.
③ Set either [Slices] or [Step Size].
（See p.6 for details.）
Setting Time series
④ Set the interval to acquire the image
in [Interval] and [Cycle] on [Time
Lapse].
Starting Acquisition
⑤ Select [Normal] tab in [Acquire] Tool
Window. Press the button to display
the dialog box, and select the folder to
save the images.
*The acquire images are saved
automatically. Serial number is added
at the end of file name like “***_0001”
and “***_0002”.
⑥ Press the button to start
acquiring the image.
Finishing Acquisition
⑦ Finishing acquisition,
buttons blink.
Press the button to complete
the image acquisition. If you want to
Additional images under the same
condition, enter the number of
additional acquisition and press
the
button.
After the image is acquired, press the
button.
①
③
②
⑤
⑥
⑦
⑧
8

XYT Image Acquisition
(with the Z drift compensation）
＊In advance, prepare to use ZDC.
（See page 22 for details. ）
＊Set for acquiring the series image.
Setting Near limit
③ Select [Microscope] in [Tool Window] menu.
Press the button to
set the Near limit to all objective lenses in
[Z limit Setting].
④ Pressing the button to current
position as 0 position.
ZDC Setting
⑤ Set [ZDC DM] in [ZDC Control] on
[Microscope] Tool Window to “In”.
⑥ Specify the search zone of the coverslip
position in [Upper Limit] / [Lower Limit] in
[Search Zone] .
⑦ Set [Z Drift Compensation in series scan] in
[ZDC Control] in [Microscope] Tool Window
to "ON".
⑧ Adjust focus and press the
button of [Coverslip position]. The coverslip
top surface position is acquired.
When the Z drift compensation is
successful, the buzzer sounds beeps once
only. If the Z drift compensation is not
successful, the buzzer sound beeps three
times.
⑧ When performing the Z drift compensation
continuously during series scan, press the
button in [ZDC Control] in
[Microscope] Tool Window. As this function
performs the drift compensation in real-
time during series scan, the focusing can
be at a high-speed.
⑨ Press in [Normal] tab in [Acquire]
Tool Window to start acquisition.
・High-speed scanning →Use continuous AF
・If Rest time is longer then 30 seconds
→You had better not use Continuous AF
：Don’t operate ⑦.
⑩
③
⑤
⑥
⑦
⑧
【Attention】
If [Rest] is shorter than 31 seconds, Z drift
correction works only first scanning. For
every time working, set the [Interval] so
that the [Rest] to be 31sec or longer.
④
◆ZDC◆
9

Multi Area Time Lapse
Imaging Acquisition
*Before starting the following procedure,
make adjustments for XY imaging.
① Press the button in [Live] Window.
② Select the [Map] sub pane.
Registering the group of MATL
③ Move the specimen to a desired
position and adjust the live image. If
you want to acquiring the XYZ image,
make adjustments for Z series
additionally.
④ Press the button to register the
position and its image acquisition
condition.
⑤ Repeat the operation of ③ and ④ to
register for multi area timelapse.
⑥ Set the interval to acquire the image in
[Interval] and [Cycle] on [Repeat
Setting].
⑦ Select [MATL] tab in [Acquire] Tool
Window.
⑧ Press the button to display the
dialog box, and select the folder to
save the images.
⑨ Press button to start acquiring the
image.
①
②
④
⑦
⑧
⑨
⑤
10
【Attention】
All acquiring conditions(XY position, focus
position, laser intensity, sensitivity, series
setting, and on. ) are registered when
you press the button. So, Register
after you finish adjusting all conditions.
◆motorized stage◆

Multi Area Time Lapse
（with the Z drift compensation）
*Before starting the following procedure,
make adjustments for XY imaging.
ZDC Setting (See page 10 for details. )
① In [▼Z Limit Setting], press the
button to set the
Near limit to all objective lenses.
② In [▼Z Section] in [Series] Tool Window,
press the to set current position 0.
⑤ Set [ZDC DM] in [▼ZDC Control] in
[Microscope] Tool Window to “In” and
place the dichroic mirror of ZDC in the
light path.
⑥ Specify the search zone of the coverslip
position in [Upper Limit] / [Lower Limit]
in [Search Zone] using the Origin
coordinate of Z as a base point.
⑦ Set [Z Drift Compensation in series scan]
in [ZDC Control] in [Microscope] Tool
Window to "ON".
⑧ Adjust focus and Press the
button of [Coverslip position]. The
coverslip top surface position is acquired.
When the Z drift compensation is
successful, the buzzer sounds beeps
once only. If the Z drift compensation is
not successful, the buzzer sound beeps
three times.
⑨ Press the button to register areas.
⑩ set [Cycle interval] and [Cycle].
Starting Multi Area Time Lapse
⑪ Select [MATL] tab in [Acquire] Tool
Window.
⑫ Press the button to display the dialog
box, and select the folder to save the
images.
⑬ Press button to start acquiring the
image.
◆ZDC◆
11
⑦
⑩⑪
⑫
⑬
◆motorized stage◆

Multi Area Time lapse
using Map Image（１）
*Before starting the following
procedure, make adjustments for XY
imaging.
① Press the button in [Live
Window], the sub pane appears.
② Select [Map] tab.
Create the map
③ Bring the image into focus and
adjust acquiring parameters using
the low magnification objective lens.
④ Press the button to register the
position and acquiring parameters.
⑤ Scroll to the right in registered are
List and Ticking [OverlayMap].
Map image acquisition
⑥ Select [Acquire] Tool Window in
[Tool Window] menu and select
[MATL] tab.
③ Press the button to display the
dialog box, and select the folder to
save the images.
④ Press button to start acquiring
the image.
Finishing the acquisition, map image
is displayed in [Map] tab.
Attention
All parameters register by pressing the
button. When acquiring a map image, Check
[LSM] in [Series] Tool Window whether it is
selected “OFF” [Z] and [Time] .
①
②
③
④
右へスクロール
⑤
⑨
◆motorized stage◆
12
⑥
⑧
⑦

Multi Area Time lapse
using Map Image（２）
Registering the multiple area
⑨ Set the higher magnification objective
lens. double-clicking on the map image,
the stage is moved to at the center of the
map.
⑩ Press the to register the position and
acquiring parameters.
⑪ Repeat ⑨⑩, register the multi areas
that you want to acquire images.
⑫ Check the registered area List whether
it is ticked to ”Enable” at the area
which you want acquire images.
⑬ If necessary, set [Cycle interval] and
[Cycle].
⑭ Select [MATL] tab in [Acquire] Tool
Window.
⑮ Press the button to display the
dialog box, and select the folder to
save the images.
⑯ Press button to start acquiring the
image.
Finishing the acquisition, map image
is displayed in [Map] tab.
Clear the Map Image
Delete registered area
⑨
⑩
⑫
If you want to see the image on map in
real-time, tick the “OverlayMap”.
13
⑬
【Attention】
All acquiring conditions(XY position, focus
position, laser intensity, sensitivity, series
setting, and on. ) are registered when
you press the button. So, Register
after you finish adjusting all conditions.
◆motorized stage◆

Acquiring the stitched image
using Map Image
① Refer to page 12, acquiring the
map image.
Define the area of acquisition
② Move the specimen to desired
position using the map image as
reference. Double-clicking on the
map, the motorized stage moves to
the position where double-clicked.
③ Press the or or button to
register the area.
④ Select [MATL] tab in [Acquire] Tool
Window. Press button to start
acquiring the image.
Processing to stitch
⑤ Select the image tab acquired and
press the button.
⑥ After the image is displayed in the
dialog box, press the button.
③
⑤
⑥
*If you ticked the [Stitching] in
[▼Registered Area List] before acquiring
the image, process to stitch automatically.
3 types
:Registering an arbitrary matrix area
:Registering a rectangular ROI in the
map image
display area.
:Registering a polygonal ROI in the
map image display area.
14
【Attention】
All acquiring conditions(XY position, focus
position, laser intensity, sensitivity, series
setting, and on. ) are registered when
you press the button. So, Register
after you finish adjusting all conditions.

★High voltage correction: This mode is the
function that the sensitivity is adjusted
automatically to keep brightness when
scan speed is changed.
Tips for Image Acquisition
■For the High speed scanning■
*Check whether [Scan Speed] is set “2.0μs/pixel”.
１）Changing the scanner
＊Use the Resonant scanner
＊Use the Roundtrip Mode
２）Selecting the smaller image size
３）Reducing the scanning area
＊Use “Clip scan” to limit the acquiring area.
■To make image more brighter■
４）Increasing laser intensity /
Increasing sensitivity(HV)
※If sufficient brightness cannot be obtained
even when the sensitivity(HV) increased to 600V or higher,
combine another method in additional to this method.
５）Increasing the C.A.
※When increasing the C.A., the Z-axis resolution gets worse.
６）Slowing down the scan speed
※Select “OFF” in [High voltage correction].
■To lower the noise level■
７）Using Average
This method averages specified number of images during image acquisition.
Select the “Line” or “Frame” and
enter the number of image to be averaged.
８）Slowing down the scan speed
※Select “ON” in [High voltage correction].
15
When using High speed scanner, the signal gets weaker and the noise increases. So,
refer to [■For brighter imaging■] and [■For the lower noises■] and combine them.
7)
Scanning time is displayed in this line.
Pixel/Line/Frame 1)
2)
3)
＊
6)
8)
4)
5)

Exiting the system
Exiting the software and PC
① Select [Exit] in the [File] menu to
exit the software.
② Select the “Shut down” in Windows
Start Menu.
Turning OFF the power
Touch Panel Controller（TPC)
③ Tap the “OFF” on display of TPC.
Then press the TPC main switch.
*Do not long-press the main switch.
Laser controller
⑤ Turn the laser combiner to OFF.
※Rotate start key of the Power
Supply and set the switch to OFF.
Mercury burner for the light source
⑥ Long-press(2 seconds) the lamp
switch on the front side of the light
source to turn OFF the Mercury
burner.
Central power
⑦ Switch off the central power.
*When using the immersion oil, clean
the objective lens.
Rotate the Key
Turn to
OFF
⑤Laser combiner
④TPC main
switch
Touch Panel Controller（TPC)
③Touch
the ”OFF”
ON/OFF
switch
turn off the
blue light
⑥the mercury lamp
16
①
②
⑦Central
power