Living up to Life

STELLARIS 5 WLL
LAS X
Basic
20211124_EN

Confidential - Company Proprietary

I. STELLARIS

1

II. Start up

2

III. Acquire Image

5

1. The Beam Path Setting

6

2. Objective Selection

6

3. Configure Beam Path Settings

7

1. Setup a New Experiment7

2. Selecting Fluorescent Dye with Drag and Drop

9

3. Assigning Fluorescent Dyes to a Detector

94. Assigning Laser to a Setting

10

5. Deleting Fluorescent Dyes

10

6. Adjusting the Excitation Wavelength10

7. Setting Detector and Detection Range

11

8. Save and load settings

11

9. Configure Pseudo color look-up table12

10. Selecting the acquisition mode: Line Sequence, Frame Sequence, Stack Sequence
13
11. Restore the image acquire settings

14

4. Define the parameters and Acquire image

15

1. Live Scan and Fast Live Scan (Preview scan)15

2. Control Panel

16

3. Optimizing image contrast

16

4. Acquire image or image series

17

5. Configuration of Image Settings

18

1. Configure image format, scan speed, zoom18

2. The Nyquist Oversampling Theorem and Pixel Size

22

3. Smooth Rendering

22

4. The difference of HyD S, HyD X and HyD R

23

5. About HyD (Hybrid Detector)

24

6. Z stack (XYZ)

26

7. Selecting the z-actuator (z-Galvo, z-Wide)27

8. Time Series (XYT, XYZt etc.)

28

9. BF (Bright Field), DIC (Differential Interference Contrast), PH (Phase Difference) image
29

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IV. TauSense

31

1. TauContrast

32

2. TauGating

33

4. TauScan

34

5. TauSeparation

35

V. Viewer37

1. Change acquired image Pseudo color, adjust scaling37

2. Function in the context menu

38

3. Functions in the Viewer

38

4. Creating Maximum Projection in the Process step

41

VI. Quantify

42VII. Data Handling43

1. Tool Bar

43

2. Save data

43

3. Export image

44

4. Organization of the data in projects

45

5. Free viewer software LAS X Small (Win10)

46

VIII. Online Help46

IX. Shut down

47

X. How to use DMi8

49

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I. STELLARIS description and technical specifications
STELLARIS is a point scanning confocal microscope with a super resolution LIGHTNING.

Scan mode
・FOV (Field Of View) Scanner
A FOV scanner is an assembly of galvanometric scanning mirrors used in single-point confocal
microscope that offer the correct optical recording of a large field sizes. The galvanometricscanners are controllable in speed and position, allowing zoom and pan functions as well as tuning
the scan frequency. Maximum format is 8192 x 8192 pixels.
・Resonant Scanner ＊Optional
A resonant scanner is a type of galvanometric mirror scanner that allows fast image acquisition withsingle point scanning microscopes. High acquisition speed is required to track fast processes,
especially in living samples. It has the additional benefit of providing a better fluorescence signal
and reduced photobleaching. It works at a constant scan speed that you cannot change (8 kHz or
12 kHz).
You can switch the FOV and Resonant scanner during a software session.

Prism - based tunable multiband spectral detection
The spectral detector splits light into up to 5 spectral bands. The bands are independent andcontinuously tunable within the full visible spectrum. The light in each band I s detected by a HyD(Ref.: p23, 24). The arrangement of prism and mirror-slit cascaded detectors is virtually
polarization-independent and highly transparent. Therefore. is features a very high photon
efficiency which is needed to collect as much as possible of the fluorescence photons by applying
illumination with as low as intensity as possible.

1

Confidential - Company ProprietaryII. Start up

5.

Side

ON OFF
2.
1.

3.
4.

＊Equipment appearance changes depend on the specification
1. Turn ON the PC*Automatically log in to “LAS X User”
2. Turn ON the main power
3. Turn ON the laser power
4. Turn clockwise the laser key switch
5. Turn ON the Hg Lamp for eyepiece (If required)
LED: Default setting is ON, skip this stepPower switch
Hg Lamp: Turn On the power switch, if required.
*When turn ON Hg lamp again, you need to wait for cooling it
It takes 15- 20 min.

Shutter Swich*Shutter switch default setting is “Remote”.

2

Confidential - Company Proprietary6. Launch the desktop app “LAS X”
7. Bellow window will be displayed.
Select “machine.xlhw” for the Configuration and “DMi8” for the Microscope.
(The “simulator.xlhw” is used to view and analyze image and not acquisition.)
Click OK to start the initialization of the LAS X

DMi8

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9. Turning ON the Lasers
Click on the “Configuration” tab and the “Laser Config” to open the main laser
control window

Turn ON the laser(s) with the appropriate laser line(s) that will excite the fluorophores you are using

WLL(White Light Laser)のDo
not change WLL (White Light Laser) %パワーは変更しない*Do Never change WLL %*OPSL is single wavelength CW laser*WLL is wavelength tunable pulse laser*Equipment appearance changes depend on the specification
Laser Wavelength (nm)405
WLL: 485-685

Fluorescent dye/protein
Blue Ex. (DAPI, Hoechst etc.)
Green -Infrared Ex.
Green Ex. FITC, Alexa488, Cy2, GFP etc.
Yellow Ex. YFP etc.
Red Ex. TRITC, Alexa555, Cy3, RFP, DsRed, mCherry etc.
Infrared Ex. Alexa633, Cy5 etc.

OPSL

448

Cyan Ex. CFP, Lucifer Yellow etc.

488

Green Ex. FITC, Alexa488, Cy2, GFP etc.

514

Yellow Ex. YFP etc.

561

Red Ex. TRITC, Alexa555, Cy3, RFP, DsRed, mCherry etc.

638

Infrared Ex. Alexa633, Cy5 etc.

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III. Acquire Image

Configure various

Configure theProvideProvides various

2D image

hardware andsettings forvarious tools

tools foranalyses andsoftwareimage acquisition

for imagequantitative image

image processing

editting

evaluation

component

The beam Path settings：(Ref. next page)
Configure the settings for lasers, detectors,

Viewer and its control

objective and AOBS (filter cube)(Ref. 37)

Buttons for Image Acquisition
Acquisition/Open Projects：Preview scan (Ref. page 15)

switching Tab＊The image is not stored

Acquisition：The basic setting for：Acquires a frame of the selected channel

initial image acquisition.
Projects：Organization of the dataof the current stage position. The images are stored in

All images and image series areproject directory.

collected and shown in a directory

Navigator: The acquisition region is marked by a white box.

structure.

：Starts the experiment including z-stacks, timeseries and tile scan if there are defined. The images are
stored in project directory. During acquisition, the markingof the button change to “Stop”. You can click the button to
sop acquisition.
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Confidential - Company ProprietaryIII-1. The Beam Path Setting
There are all buttons and options that you will require for configuring and carrying out yourexperiments. You can select and set the fluorescent dyes, the lasers, the system component along
the beam path and the detectors for image acquisition.
Control Panel Configuration
Change objective
Transmitted Light setting

Notch Filter

AOBS (Filter cube)

settingsetting

Fluorescent Dye Search bar
Selecting Fluorescent Dye

Emission Range (nm)*Notch Filter Lines (Optional) attenuate the laser reflection.
*Creating a Transmitted Light Image (Ref. page 29)*Control Panel
You can configure detector gain, laser power, Zoom, Rotation, Pinhole size and Z position (Ref. page18 -).

III-2. Objective Selection
Single click the objective name field for display the objective selection. A list of all available
objective opens. Select the objective from the list. The list close again, and the objective
automatically changed.
*Equipment appearance changes depend on thespecification

6

Confidential - Company ProprietaryIII-3. Configure Beam Path Settings
III-3-1. Setup a New Experiment
To set up a new experiment with the aid of the Dye Assistant, proceed as follows:
1. Click the Open “Dye Assistant” button ○a in the middle area of the beam path settings.

The following dialog opens:

2. Click the “...” button ○b . A list box of the emission spectra stored in the system opens.
3. Select a fluorescent dye. The selected fluorescentdye is listed on the left side. Another line in which you
can select an additional fluorescent dye appears below
the selected fluorescent dye.
As soon as you have selected two fluorescent dyes,
various configuration options for the acquisition of thespecimen used are suggested in the lower area of the
dialog, and the detection range overlap is displayed
graphically.
The displayed color bars for “Yield” illustrate the yield of
the individual fluorescent dyes.
“Crosstalk” illustrates the intensity of the crosstalk into other channels.
4. Follow the same procedure for all other fluorescent dyes contained in your specimen.
5. Depending on the system configuration, a specificdetector type or acquisition mode can be assigned toeach fluorescent dye. To do so, click the selection fieldand select the desired detector type or “Selecting
acquisition modes for experiment configuration” (Ref.
page 23, 24) from the dropdown menu (see Right).
It is possible that individual configurations are not available. Example: If two fluorescent dyes in
use are assigned to a hybrid x-detector but the system is only equipped with one hybrid x-detector,
this necessarily results in a configuration in stack-by-stack or frame-by-frame sequential mode
(Ref. page 13).

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6. The system assigns a color look-up table toeach dye automatically during the selection ofmultiple fluorescent dyes. The suggested colors
do not follow the spectral properties of the dye
but are intended to achieve a high contrast sothat the fluorescent dyes are highly distinct inoverlay images.
Click the box next to the detector selection inorder to select a different color look-up table from a list as necessary.
7. If one of the suggested configurations
appeals to you, click the corresponding“Apply” button. The Dye Assistant is closed,
and the configuration is incorporated into thebeam path settings.
8. Select one of the suggested instrument configurations for your application and click thecorresponding “Edit” button if subsequent optimization is still required. An additional editing
window opens.

9. Adjust the settings for laser line ○c ordetection range ○d as necessary.
Depending on the instrument configuration, the conventional laser lines available are displayed as
short gray lines that the excitation laser line locks onto.
A gray bar is displayed for instrument configurations with a white light laser; the laser line can be
moved to any point on this bar.
You can change the size and position of the detection ranges ○d.
10. Apply the adjustments:
Apply: Adjusts the hardware for image acquisition and closes the Dye Assistant.
OK: Applies the settings to the main window of the Dye Assistant.

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Confidential - Company ProprietaryIII-3-2. Selecting Fluorescent Dye with Drag and Drop
The fluorescent dyes are dragged and dropped into the settings. You can use the fluorescent dyes
from your favorites bar or from the overall list.

Click the desired fluorescent dye and drag it (a) to the desired setting while holding down the left
mouse button. While dragging the fluorescent dye, the available detectors are displayed andhighlighted when you hover the mouse over them. You can drop the fluorescent dye over adetector of your choice. If you do not select a certain detector and drop the fluorescent dye into the"empty" space above the detectors, the software ensures the allocation to the most suitabledetector.
As soon as you let go of the mouse button, the spectral properties of the selected fluorescent dye
are displayed as a curve (b). Although the emission spectrum is displayed by default, you can also
have the excitation spectrum or both displayed, see Beam Path Settings: Displaying theExcitation/Emission Spectrum.
The software analyzes the spectral properties of the fluorescent dye and automatically suggests a
suitable excitation laser line and a detector with a corresponding spectral range (c).
In the Yield/Crosstalk field, the colored bars give you immediate information about the strength ofthe optical crosstalk to other channels and the intensity yield of the individual fluorescent dyes.

III-3-3. Assigning Fluorescent Dyes to a Detector
If you want to assign a fluorescent dye to a detector manually:
1. Click the desired detector to call up its configuration area.
2. Select the desired fluorescent dye from the drop down menu.
The corresponding emission curve is loaded. Adjust the excitation line and the detection range asnecessary.

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Confidential - Company ProprietaryIII-3-4. Assigning Laser to a Setting
1. You can insert the laser lines using the

button. Click the button, hold the left

mouse button down and drag into the desired setting onto the excitation range.
2. Set the detector and detection range (Ref. next page, Setting Detector and Detection Range,
page)

III-3-5. Deleting Fluorescent Dyes
Click the curve of the fluorescent dye you want to delete and then click the recycle bin icon or use
the Delete key on your keyboard.
Alternatively, you can click the fluorescent dye, hold the left mouse button and drag the fluorescentdye out of the setting.
The fluorescent dye is deleted from the setting, the associated detector is switched off, and theYield/Crosstalk display is updated accordingly.

III-3-6. Adjusting the Excitation Wavelength
1. Click the excitation laser line (○g ) that the software has suggested on the basis of the spectral
properties of the dyes. The excitation line is displayed as active and in white. Below the setting,the configuration area expands. The shutter has been opened already ON.

2. Set the desired laser intensity (AOTF setting) by pulling on the Intensity slider (○h ). Alternatively,
you can also enter the intensity directly. Click the displayed value and change it in the input
window.
You can adjust the power also using the mouse wheel or the control panel.
You can accordingly configure the control panel in the USB Control Panel dialog. If the parameter
Smart Channel Intensity has been assigned to the control panel, the corresponding excitation line isdetected automatically as soon as you select an image in the viewer.
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Confidential - Company ProprietaryIII-3-7. Setting Detector and Detection Range
1. Click the detector (○i ) that thesoftware has suggested based on
the spectral properties of the dyes.
The detector is displayed as active
and in white. Below the setting, theconfiguration area expands. The
detector has been activated already (ON).
2. Adjust the detection range as necessary. The detection range can be shifted using the mouse.
You can also change the size of the detection range by dragging one of its ends or entering exact
values in the entry window (double-click on the detection range).
Alternatively, you can double-click the detection range to open an entry window in which you canenter values directly.
3. Set the detector sensitivity by pulling at the Gain slider (○j ). The default setting is 100%.
Alternatively, you can also enter the sensitivity directly. Click the displayed value and change it in
the input window. The value can also be set from the control panel.

III-3-8. Save and load settings

Save setting
You can save the settings for image acquisition for later experiments with the same or a similar
specimen. The complete sequential configuration you have created is saved. This can consist of
individual setting or a sequence of multiple settings.
1. Click the

button. The file browser is opened.

2. Enter a file name and confirm with “Save” to store the setting.
Load setting
If you have saved setting or sequence at an earlier time, you can load these to work under
reproducible acquisition conditions.
1. Click the

button. The file browser is opened.

2. Select the desired setting and click “OK”.
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Confidential - Company ProprietaryIII-3-9. Configure Pseudo color look-up table
In the configuration for the respective detector, single click the color field for the display of the lookup table. A list of all available look-up tables opens. Select a look-up table from the displayed listby double-clicking. The list close again, and the look-up table is assigned to the detector.

Pseudo color of look-up table

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Confidential - Company ProprietaryIII-3-10. Selecting the acquisition mode: Line Sequence, Frame Sequence, Stack
Sequence

Line Sequence

Stack Sequence
Frame Sequence
Line Sequence

The method is particularly advantageous for specimens

For each row, the systemthat change their position (e.g. living cells).

switches from the parameters of*In this mode, the following parameters in the setting

one setting to the parameters of

can differ:

next.

Laser setting, Detector status (ON/OFF)* The results in virtually*When configuring the sequence, detectors are not shutsimultaneous creation of theoff electronically when switching between the individual

frames.

setting. Therefore, do not select detection range for thedetectors that is under the excitation line of anothersetting.

Frame SequenceFirst, one image is acquired with the parameters fromThe system switches to the next

the setting: then, the parameters from the next setting

setting after scanning complete

are configured and the next image is acquired.

image.

*In this mode, the following parameters in the setting* Additional application options

can differ:include scanning a specimen

Laser intensity, Frame Accumulation, Line/Frame

stained multi times with a single

Average, Pinhole and motCorr setting, Detector property

detector.

(Detection range, Gain, Tau Mode, Operation Mode),
Shutter status (ON/OFF)

Stack SequenceFirst, the complete image series is acquired with the

The system switches to the next

parameters from the setting: then, the parameters fromsetting after scanning entire

the next setting are configured and the next image series

image series (z-stack, timeis acquired.

series, spectral series).

*In this mode, the following parameters in the setting* Additional application options

can differ:include scanning a specimen

Laser intensity, Frame Accumulation, Line/Frame

stained multi times with a single

Average, Pinhole and motCorr setting, Detector property

detector.

(Detection range, Gain, Tau Mode, Operation Mode),
Shutter status (ON/OFF)

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Confidential - Company ProprietaryIII-3-11. Restore the image acquire settings
You can restore some instrument parameters regarding the selected file.

Apply image settings

Open the dialog
You can check all information and instrument parameters regarding the selected file from thecontext menu (Right mouse button).

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Confidential - Company ProprietaryIII-4. Define the parameters and Acquire image
III-4-1. Live Scan and Fast Live Scan (Preview scan)

You can configure the setting for a Fast Live mode. The Fast Live mode enables you to toggle
quickly back and force between two settings.
Fast Live mode: scan with bellow setting, e.g. fast navigation
Live mode: scan with defined parameters for image acquisition, e.g. define the parameters foracquisition

Move the mouse over the right area of the button.

The configuration symbol isdisplayed.
The Fast Scan Setting dialog opens. Depending on which scanner your system has available (FOV
and/or resonant scanner), following parameters can be set.

＊Active Sequence Only(Between Lines Mode): When the function is activated, Fast Scan Livemode is carried out only for the sequence currently in progress (default setting).

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Confidential - Company ProprietaryIII-4-2. Control Panel
By clocking the “Control Panel configuration” button,

will display control panel dialog.

Default setting is bellow:

①

②

③

④

⑤

⑥

①Smart Gain：Configure selected channel Gain
②Smart Intensity：Configure selected channel laser intensity③Smart Wavelength：Configure selected channel laser wavelength (Only WLL)
④Pinhole：Configure selected sequence Pinhole size
⑤ Zoom：Configure Zoom rate
⑥Z position：Configure the Z position
z-Galvo: Configure super Z stage (Galvano stage)
z-Wide: Configure motorized stage (inverted microscope: objective revolver, Upright microscope:
specimen stage)
Difference of z-Galvo and z-Wide (Ref. page 27)

III-4-3. Optimizing image contrast
By clicking the “Quick Look-up table”

button, make it possible to change the look-up table

quickly.
Pseudo Color →Grow Over/Under →Gray →Pseudo Color
Grow Over/Under: Highlight grayscale values the top edge of the look-up table in “Blue” andgrayscale values the low edge of the look-up table in “Green”.
*In default setting “Blue” indicate saturation and “Green” indicate No signal.
255

4095

Blue (Saturation or Top edge of look-up table)

・

・

White

・

・

Brown

・

・

Black

0

0

Green (No signal or Low edge of loo-up table)

8 Bit

12 Bit

Blue: Saturated pixels or
Top edge of look-up table
Green: No signal pixels or
Low edge of look-up table
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Confidential - Company ProprietaryIII-4-4. Acquire image or image series
Buttons for Image Acquisition: Preview scan (Ref. page 15)*The image is not stored: Acquires a frame of the selected channel of the current stage position. The
images are stored in project directory.
Navigator: The acquisition region is marked by a white box.

: Starts the experiment including z-stacks, time series and tile scan if there are defined.
The images are stored in project directory. During acquisition, the marking of the button change to“Stop”. You can click the button to sop acquisition.

17

Confidential - Company ProprietaryIII-5. Configuration of Image Settings
III-5-1. Configure image format, scan speed, zoom
1 Format：Number of pixels in the image○

Default setting is 512x512 Pixels. By selecting the

①
2
○
3
○
④

preset format from the pulldown menu, the format+ , you can set thewas applied. By clicking the ○desired format.
2 Speed：Scan Speed(Hz)○Default setting is 400 Hz. By selecting the preset
scan speed from the pulldown menu, the scan
+ , you can set
speed was applied. By clicking the ○5○

the desired scan speed.
＊The zoom factor change if the scan speed is highthan 601 Hz (The zoom factor is depend on the scan
speed)＊The slower scan speed, the higher S/N but easier⑥

bleach.
＊Resonant scanner works at a constant scan
speed that you cannot change (8 kHz or 12 kHz).

3 Bidirectional X：<2x faster acquisition (need to adjust phase)○

The default scan mode is unidirectional where the beam scan in the X-direction, is blanked on
return and again scans in the X-direction for the set number of lines. bidirectional scanning
patters do not blank the beam while returning. This may reduce scan time, but may introduce
some scan artifacts that must be corrected by “Phase”.
Unidirectional

Bidirectional

4 Zoom：Adjust optical Zoom factor○

Adjust zoom factor various ways by following:-Adjusting slider, -Entering specific zoom factor, -Activating “Zoom In” and -drawing ROI in image, Zoom knob on Control panel (Ref. p16)
＊The zoom factor change if the scan speed is high than 601 Hz (The zoom factor is depend on thescan speed)
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5 Line/Frame Average/Accumulation○

Averaging and Accumulation remove noise
In general, less averaging required with a lower gain setting for the detectors.
Frame Average/Accumulation: The specific setting is valid for currently selected setting. Whencheckbox is enable, the setting is taken over for all settings in a sequence.
⑥Rotation：Rotate image if necessary (Optional)
You can use the image field rotation to rotate the display +/- 100° in the viewer during the live
image acquisition.
Rotation knob on control panel (Ref. p16)
Functions of adjusting the image display are arranged on the left edge of the viewer
Generally, the selection of a suitable look-up table for a specific use depends on the user’s own
judgement and needs.

Auto Scale
The look up table automatically adapted to
the gray scale values of the acquisition.
This makes it possible to extend the

The configured value isdynamic range and, to work with entire

shown in an entry window

bandwidth of the color depth. Automatic

below and above thescaling is applied to all channels, for z-

look-up table and can bestacks, to the entire series.

also entered here directly.

Above and bellow the color control, themaximum measurable photon count/pixels
are displayed can be detected theconfigured bit depth

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Confidential - Company Proprietary1 XY format optimization
○

①

2
○

Select optimize xy format to set the optimal
number of pixels correctly over sample (Ref. page
22).
＊Resolution depends on NA of objective andpinhole size.
2 Switch the function FOV Scanner and Resonant○

3○

Scanner
Gray: FOV Scanner
Red: Resonant Scanner＊Resonant scanner works at a constant scan
speed that you cannot change (8 kHz or 12 kHz).
＊For resonant scanner, you can not use HyD S
analog mode.
⑤Dynamic Signal Enhancement(DSE)：DSE is an④

image optimization method. It is available for time⑤

lapse series with the acquisition mode xyt, xyzt, etc.
Enable: Switch the function ON and OFF here
Frames: Set the number of individual images to beaverage here (max. 65). The number must be odd
so that, during image acquisition, the averaging calculation always includes the same number offrames before and after the current image.
Weight: You can adjust how strongly the neighboring frames are weighted in the average (value range
from 0 to 2). The higher the value the stronger the stronger the weighting of the more remote images.
＊If you have set the Counting mode for activate detector, no fluorescence intensities are averaged;
instead of counting photons are accumulated though the number of frames that are set.

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Confidential - Company ProprietaryDSE is “Exponential weighted moving average” and improve S/N without reducing FPS
In a left Time lapse series, 14 frames are being captured (from left toright). For frames, the number 7 is set. The graphic indicates whichframes are being used to calculate the averaged images.
The first raw indicates the weighted averaging for the 7th frame (Blue highlighting). Three frames
before and after the 7th frame are indicated in the calculation for the averaging (red box). The
frames outside the red box are not considered in the averaging. In the second row, the averaging
for 8th frame is performed is performed and in the third row, for the 9th frame.
In acquisition mode during image acquisition (Start): For the fist and last
images, only one half of the specific frames are averaged. The total number ofimages remains unchanged. Both raw data and the averaged image are
saved. The calculation starts during the acquisition, as soon as sufficient data
is available.
In live mode: Here the averaging aggregates up to the specific number
Frames. In the example, the 7 most recently acquired frames areaveraged if “7 frames” is set. No data is saved in live mode.

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Confidential - Company ProprietaryIII-5-2. The Nyquist Oversampling Theorem and Pixel Size
The Nyquist-Shannon sampling thorem describes the fact that an analog signal of any form can always
be restored if the sampling rate in digitizing is more than twice as high as the maximum frequency
included in the signal.
This means that the pixel size must be set in such a way that sufficient pixels are available forrepresentting the resolved structure.
-

The sampling spacing is good if the pixel size equals one half of the opical resolution-

If the pixel size is no longer than one half of the optical resolution of the microscope.

Adjusting Pixel Size
Sampling space is equal

Sampling space is one half

Intensity

analog signal
was restored

Analog signal
was not

60

restorednm

Display in pixel

III-5-3. Smooth Rendering
Switches the smoothing the images shown in the viewer on or off. This is exclusively an image
processing function and has no effect on the raw data. This function is ENABLED by DEFAULT.

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Confidential - Company ProprietaryIII-5-4. The difference of HyD S, HyD X and HyD R (Ref: Volker Schweikhard et al.,Nature Method 2020)
Select the detectors while referencing below figure that show the Photon Detection Efficiency(PDE) of HyD S, HyD X and HyD R.

-

HyD S

HyD S is silicon-based Multi-Pixel Photon Counter (MPPC) detector. This detector is the result ofyears of engineering at Leica. The result is enhanced image quality that sets new standards inconfocal imaging.
*HyD S is available for TauSense and -STED, but not for FLIM and FCS.
HyD S has 3 Operation mode: Analog, Counting and Reflection (Ref. Next page)23

Confidential - Company ProprietaryIII-5-5. About HyD (Hybrid Detector)
Operation mode
Depending on the detector, following mode are available.
-Counting: The counting mode makes it possible to carry out a quantitative image acquisition bydisplaying the image based on the number of photons detected per pixel. The number of photons
is displayed in a look-up table, whose gray-scale value in this case corresponds exactly to thenumber of photons (e.g. Gray-scale value: 15 corresponds to 15 photons).
*It is used, for example, for ratio imaging, FRET or image correction.
*The Gain function is deactivated in this mode.
*If the number of detected photons per pixel is very large, the depiction capacity of the color depth
can be exceeded e.g. 255 at 8 bit. In this case, switch to higher color depth, such as 12 bit or 16 bit(Ref. next page).
*For evaluation in other program, you can export the images as ASCII files (Ref. page 44 ).
-Analog (HyD S): This operating mode can be used for the regular image acquisition. To adapt theimage acquisition to the light intensity of the specimen, you can set the gain of the light signal.
-Reflection(HyD S): This mode is primarily intended for reflection image or extremely high signal
intensity. This mode should be used in special case only.

Acquire image using Counting mode
-Adjust laser power with Analog/Digital mode (Gain 50 - 100).
-Change to Counting mode
-Adjust image intensity with Scan peed and/or Accumulation(Not adjust laser power)
Lower scan speed, longer pixel dwell time, higher photon
number.
*Resonant scanner cannot change scan speed.
In counting mode, look-up table corresponds exactly thephoton number. Then Line/Frame Accumulation make itpossible to show high photon number image.
*To decide the accumulation number, check the gray scalevalue with “Auto Scale” (Ref. 19). Then, set the accumulation
number not to exceed the color depth coding.
Or switch color depth 8 Bit to 12 or 16 Bit (Ref. Next page).

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Switch the color depth
Switch to the “Configuration”

step

Select “Hardware”
In the dialog field “Hardware Settings” in Resolution, select the suitable color depth in the dropdown
menu “Bit Depth”.

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Confidential - Company ProprietaryIII-5-6. Z stack (XYZ)
1. Select the appropriate “Acquisition Mode”,
XYZ or XYZT①

2. Use the control panel or Smart move to movethe focus position to begging position of the
2
○

stack
1 “Begin” to set the start position
3. Click ○4. Use the control panel or Smart move to movethe focus position to end position of the stack3○

1 “End” to set the start position
5. Click ○1 Use arrows to move to the set Begin or End○

position
4○5○

2 Delete the Begin and End positions○
3 Z Size show the distance from the Begin to○End.
4 Stack Direction (Z): Change direction of○

6○

acquisition of the stack⑤ Select “z-Galvo” to indicate the focus driveof the microscope stage will be used to control z
position during the stack (Use only Control
panel to define the begin and end, Ref. nextpage).
6 Define the number of the z-stack○

Nr. of Steps: Manually define the number ofimages of z-stack
Z-Step Size: Manually define the distance between images
System Optimized: Optimized automatically
6. Acquire the image
Click the “Start” to acquire the z-stack image

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Confidential - Company ProprietaryIII-5-7. Selecting the z-actuator (z-Galvo, z-Wide)
Enable the suitable z-actuator for moving specimen in a vertical detection.
■z-Galvo(Optional)
Galvanometer-driven fine focusing stage. It is used for a low stack
thickness with high resolution in the z-direction.
Maximum travel distance: 500 um (Restrict Range) or 1500 um(Whole Range)
Configure with Control panel (if z-Galvo was selected)-Control panel and LAS X window show the current Z position, but there do not recognize themotor-driven focus position. Then, do not change the Z position using Focus knob or Smart Move,after definition of the z-stack positions. If you change it, acquiring are was moved from desired
position■z-Widemotor-driven focus of the microscope stand. It is suitable for very thick stacks that exceed thepositioning range of the focus. However, the resolution in the z-direction is lower.
Maximum positioning range: Corresponding to the free working distance of the objective used
Configure with Focus knob, Smart Move and/or control panel (if z-wide was selected)-Control panel (Ref. 16)
You can configure both z-Galvo and z-Wide, but NOT same time

Z position-Smart Move (Ref. page 49)

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Confidential - Company ProprietaryIII-5-8. Time Series (XYT, XYZt etc.)
1. Select the appropriate “Acquisition Mode” that include time (t), XYT or XYZT
xyt: Timelapse image of XY
xyzt: Timelapse image of XY or XYZ
Bellow display will open
Left: xyt
Right: xyzt

xyt

xyzt

1
○

1
○

2
○

2
○

3
○

3
○

1 Set the “Time Interval”
2. ○For no delay between images, select “Minimize”
2 ○3 Define the parameter for acquisition to stop
3. ○4. Acquire the image
Click the “Start” to acquire the z-stack image

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Confidential - Company ProprietaryIII-5- 9. BF (Bright Field), DIC (Differential Interference Contrast), PH (Phase
Difference) image
First, configure the settings for image acquisition. Click the detector bar for “Trans PMT”. The
configuration area is expanded.

Transmitted light Detector ON/OFF

Transmitted light Detector Gain

Transmitted light Detector Offset

Select the contrast type
Scan-BF: To make images in bright field mode
Scan DIC: To make DIC image, the polarizer and the analyzer are required for DIC image acquisition.

Differential Interference Contrast (DIC) image
1. Select “Scan-DIC” or “Scan-DIC-Pol”
2. You can set the prism combination (Prism) and DIC
prism position (Bias) here.
By moving the Bias slider to the left or the right, you can find the optimum prism position for yourexperiment. Depending on the prism position, the image appears with greater or less contrast.
3. If necessary, adjust laser power and/or gain value.
*Proper Prism for the objective is required, if not “Scan-DIC” and “Scan-DIC-Pol” were notdisplayed.
PH (Phase difference) image
1. select “Scan-PH”
2. If necessary, adjust laser power and/or gain

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Confidential - Company ProprietaryTips for Transmitted Light Imaging
Specimens that alter polarized light, such as birefringent specimens and plastic culture dishes, arenot suitable for DIC. Phase contrast is still the best method for examining cells in plastic culture
dishes. The excitation laser power and wavelength affect on the brightness of these image.
Köhler illumination alignment is required for all transmitted light imaging.
The difference of the Scan-DIC and Scan-DIC-POL
Scan-DIC consist of 1 polarizer and 2 prisms, whereas Scan-DIC-POL consist of 2 polarizer and 2
prisms. Insertion of these components to the optical path reduce the fluorescence signal and thefluorescence image resolution.
*Confocal microscope uses coherent laser as excitation, so 1 polarizer is enough for DIC image
acquisition.

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IV. TauSense (Ref: M Julia Roberti et al., Nature Research 2020)
TauSense analyze the photon arrival time and provide image independent of intensity.
This allows you to remove unwanted signals, such as reflections, from images and separates
different species in your specimen based on their fluorescence lifetimes.
＊A pulsed laser is required (e.g. WLL)
Click the detector, configuration
area expands①②①TauContrast: Ref. Next page
②TauModeIn the detector configuration area, the left image shown modes areavailable. Click theicon to open the TauMode dialog.

None: Only if TauContrast function is switched ON. The viewer only
displays the lifetime image and not intensity image of the specimen.
Intensity: Without gating. The viewer displays the full intensity image ofthe specimen. Default setting.
TauGating: Ref. page 33
TauScan: Ref. page 34
TauSeparation: Ref. page 35

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Confidential - Company ProprietaryIV-1. TauContrast
The “TauContrast” function is used to measure the average photon arrival time per pixel in thedetector and display is as a colored lifetime image. Two images are acquired per detector anddisplayed in the viewer: The first image the fluorescence intensity is shown through the brightness
and the second image additionally uses colors to represent the average photon arrival time.
＊TauSense does not require additional laser irradiation
Start your experiment by clicking Start or Capture.

Auto Scale
Histogram Show/Hide

TauContrast image

Intensity image

Average Photon arrival time

Scale for Average Photon arrival time

Histogram of Average photon arrival time
Average photon arrival time ofthe pixel @ the cursor

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Confidential - Company ProprietaryIV-2. TauGating
You can set the number of gates per channels as you please. Every channel is displayed in a
separate partition of the viewer.
Using TauGating mode, you can create multiple channels for one detector to eliminate, for example,
unwanted signals such as reflections or background noise from the acquisition. You can set
number of gates per channel and the number of channels as you please.
Three channels○
a with one time gate○b each are preset. Each line represents one channel, eachdetection range one time gate. You can add both more channels and time gates per channel.
○
a : Each channel is represented in one line. Every channel is displayed in a separate partition of
the viewer.
○b : Each time gate is displayed as a detection range. You can use the mouse to adjust the size ofthe time gates and to move them with ↔. In this way, you can define the beginning and end of theimage acquisition (based on the laser pulse). If a channel has multiple time gates, the viewer
collects them in one partition.
○d : Using the “Plus” and “Minus” buttons on the right margin, you can add more time gates to therespective channel or delete the currently selected time gate. A maximum of three time gates canbe configured per channels.
○e : Time scale. Photon arrival time after the laser pulse in nanoseconds. Here you can read thedefined time gates.
Add Channel: In doing so, you add another channel below.
Remove Channel: By doing this, you delete the currently selected channel.
Start your experiment by clicking Start or Capture.

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Confidential - Company ProprietaryIV-3. TauScan
You can obtain a diagram of the fluorescence lifetime components. This curve indicates how
frequently lifetime components appear in the entire image. With the aid of the diagram, thelifetime components in the different species of the specimen can be determined. In this way, you
can also determine whether a dye contains more than one lifetime component, for example.

In this dialog, you can break down the entire range of detectable lifetime components into aselectable number of sections. One channel is assigned to each section. The channel isdisplayed as a separate intensity image in a viewer partition. With the aid of this, you can assign
the different species in your specimen to individual lifetime components. In addition, you candetermine whether species contains more than one lifetime component. You can use the
TauSeparation (Ref. next page) method to separate the various species in your specimen if
necessary.
○a : Lifetime diversity curve Graphical display of the frequency of lifetime components (lifetime
measured in ns) in the entire image. Each peak indicates a lifetime component present in theimage.
○b : Set the number of channels or images here. This number corresponds to the breakdown of theentire range of detectable lifetime components in ○c.
○c : Entire range of detectable lifetime components.
○
d : Time scale Lifetime in nanoseconds, broken down to the number of images set under ○b . Thedouble arrow marks a section of detectable lifetime components.
Start your experiment by clicking Start or Capture.

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Confidential - Company ProprietaryIV-4. TauSeparation
You can carry out a separation (e.g. dye separation) of species present in the specimen based on
different fluorescent lifetime that you have determined, e.g. in a preceding TauScan (Ref. Previouspage). For each lifetime component, an intensity image is generated in a separate channel anddisplayed in a viewer partition.

You can use the TauSeparation method to separate species present in the specimen based on theirfluorescence lifetimes and acquire the resulting in separate channels. If a species has multiple
lifetime components, you can also detect them separately. Configure the setting for species
separation in this dialog.
○a : Lifetime diversity curve Graphical display of the frequency of lifetime components (lifetime
measured in ns) in the entire image. Each peak indicates a lifetime component present in theimage.
○
b : Each line represents a channel. Each species is assigned a separate channel.
○
c : The line represents a lifetime component for the respective channel. Move this line below a peak
to assign a lifetime component to a channel and, consequently, to a species.
○d : You can adjust the color for the channel display. Double-clicking the color field opens a dialog
for selection of a LUT.
○
e : Using + and – buttons, you can add further channels or species and remove them.
○f : By clicking “Find”, the analysis is carried out for all configured channels, and optimum setting forthe lifetimes are determined.
Start your experiment by clicking Start or Capture.

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Tips
It will be easier to separate lifetime component in bellow conditions.
・There are big lifetime difference
・There are no intensity difference
・The lifetime components localization are big different
TauSeparation: There are cases in which divided images are saturated, even in entire image is not
saturated. Then, you can change the bit color depth to 12 bit or 16 bit (Ref. page 25).

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V. Viewer
V-1. Change acquired image Pseudo color, adjust scaling
If a time or z series is

Change Pseudo color:

displayed in the viewer,

Double clicking the color

a control element bar

bar. A list of all

appears in the lower oravailable look-up tableright edge of the viewer

opens. Double click towith a slider used to

select a different look-up

navigate through thetable from the left area.

various acquisition

Adjusting the Scaling

times.

Range: You can adjust
the scaling. This makes
it possible to extend the
dynamic range and,
therefore, to work withthe entire bandwidth ofShow the Acquisition mode, format andthe memory size
*Show units for the image size. By

the color depth. this isclicking

button, you can switch

primary helpful inbetween “ｍ” and “pixel”

evaluating dark images

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Confidential - Company ProprietaryV-2. Function in the context menu

ROI

Save: Open the file browser for selection of a storage location. All ROIs are

Reference ROI

saved to a location defined by you with (ROI) or without (Reference ROI) the
information on the coordinates.
Load: Open the file browser. in which you can select the storage location of
the desired ROI. ROI: The loaded ROI replace the existing ROI, Reference
ROI: The loaded ROI added in the center of the viewer.

Snapshot

An image file is created from the image you have right-clicked. ROIs andannotations of images are saved, where appropriated.

Snapshot All

If multiple images are displayed in the viewer: An image file is created fromthe image you have right-clicked. ROIs and annotations of images are saved,
where appropriated.

Properties

Open the shape properties: For each plotted annotation, you can open a dialog
field for displaying the properties.
General: Adjust the designation and set the size
Color: Define the color
Font: Configure the literal and select font, fontsize and styleV-3. Functions in the Viewer(Ref. Advanced Manual)Adds the functions for image explanation to the list of buttons.
Select single ROI
Clear selected ROIs

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Draws a scale bar by means of holding down the left button and dragging (unit inm).
You can enter exact bar size and change color etc. from right-click on thescale bar to open the bellow context menu, “Properties”

Fit to display size: Adjust the displayed image to the size of the viewer. Next to it,on the right, the current image size is displayed in %.
Change Look Up Table quickly: By clicking the button, look up table are displayedin bellow sequence.
Pseudo color → Grow Over/Under → Gray

Pseudo color

Grow Over/Under
Blue: Above the color control

Green: Bellow the color control

Gray Scale

Show one partition per image ↔ Show multiple partitions per image: Switch
between the view of one or more image channels in the viewer. By doubleclicking the partition, switching the per image ↔ multiple image

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Show overlay image of selected channels: For multi-channel acquisitions, switches
the overlay view on and off. This allows you to superimpose the color channels of
an image on one another or show them separately.
Maximum projection
Calculates a maximum projection for z-series andpresents it in a viewer.
*The image is just preview, you cannot save it.
*To save maximum projection image Ref. Next page

Orthogonal Sectioning
Creates a display of the z-stack in xy, xz and yz-direction. Using the crosshairs,
precise positions canbe reached in all three
dimensions within the

XY
YZ

stack

＊The image is just

XZ

preview, you cannot
save it.
＊Saving an orthogonal
section in the xz or zy-direction: Right-click on the XZ/ZY-direction of the z-stack
to open the context menu, “Snapshot”.
Open in 3D viewer
opens the 3D viewer for 3D image processing
Show/Hide Image Channel information: Indicate the number of the associatedsetting and information on the detector and/or detection channel and lifetime (forTau modes) and the dye with designation and color of the detection range on eachpartition insofar as this information is present on the image
Example: Setting No. I Detector name or gate or lifetime I Fluorescent Name I Dye
Pseudo color*You can rename the “Setting name”. Double-click the setting name in Beampath and enter desired name.

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Confidential - Company ProprietaryV-4. Creating Maximum Projection in the Process step

1. Proceed to step “Process” menu.
2. Under “open projects”, select the image series you want to view as the maximum projection.
3. Select the “Projection” option in ProcessTools.
4. At the bottom edge of the window, click “Apply” to create a maximum projection using the current
settings.
It is then displayed in the both the preview and the viewer. The created projection is also listed in
the project directory.

＊In Process menu, TauContrast image were consist of 2 channels: I and AAT.
“I” has intensity information and “AAT” has average photon arrival time information.
Left image is example. Capture 2 channel images, Channel 1: Intensity image,
Channel 2: TauContrast image. Then TauContrast image divided into I2 andAAT2.
＊In older versions LAS X Small, TauContrast image divided into just channels not I and AAT.

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VI. Quantify

The values measured in the ROI are displayed asgraphs on the “Graph” tab and as tables on the“Statistics” tab.
Line Profile: Measure gray scale value in images and in the individual imagesof image series along linear ROI.
Stack Profile: Determine the mean gray scale values in the individual images ofimage series based on ROI. If you have not drawn any ROI, the mean grayscale value of the entire individual image (Frame) are measured.
Histogram: Determine the distribution of gray scale values in images and in theindividual images of image series based on ROI.
Select Channels and ROIs: You can select ROI and color channels
for the quantitative evaluation of images and image series.
ROI/Frame: For ROI to be available for selection here, you have todraw it in the viewer. Select the desired ROI for the quantitativeevaluation.
Channel: It provides you with ROI-Channel combination. Thisfunction is available only if you combine multi-channel acquisition in
the viewer.
Sort charts by Channels and ROIs: You can define how the result of the quantitative evaluation ofimages and image series are displayed.
Sort Channels: The results are sorted by color channels
Sort ROIs: The results are sorted by color ROI
All in One: The results are combined into one display
Report: To save the evaluation results as a report further use. The report contains the evaluatedimage, including the ROI used. The evaluation
results displayed as graph and in table form in thereport. Furthermore, the report contains
information about the detailed information about theimage or image series and the systems settings
used during image acquisition.

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VII. Data HandlingVII-1. Tool Bar
All of images are displayed in the “Open Project”
1 Changes to the tree and
○

1 ○
2 ○
3 ○
4
○

5 ○
6
○

7
○

directory structure of “Work in
Folders”. You can use button to
return to the project structure.

2 Create the new project○3 Open the file manager for selecting an existing project○
4 Save ALL opened project○
5 Applies the instrument parameters of the highlighted file (Ref. page 14).
○
6 You can use this button to call up the project browser dialog. The file browser searches and○browses for project data created using LAS X and displays a preview of the included images in aviewer.
7 Show/Hide a gallery with thumbnails of all images in a folder.
○VII-2. Save data
All images and image series you have acquired are collected in a project and shown in a directory
structure. When saving the project data, a corresponding folder structure is created in the project
directory on the hard drive.
Project: Data record containing all of the data for experiment.
Image series: Can be a 2D single image or a multi-dimensional image series
Metadata: Every data object can have metadata where hardware setting, LUT, image display
settings. project settings, ROIs etc. are saved.
All of acquired images and image series are temporarily stored in project.

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Confidential - Company ProprietaryVI-3. Export image
Right-click on the desired image to open the context menu, “Export
image”.

① Image: Exporting image acquisitions
Movie: Exporting videos② Destination Folder: Select the storage location for
the exported images/videos③ Store in separate folders: This option is available
only if you have selected multiple elements.
Checkbox enable: The project is saved with thecorresponding folders and subfolders.
④ Select the export format:
Image：TIFF、ImageJ TIFF、JPEG、ASCII (Export
intensity value)、BMP、PNG
Movie：Avi、QuickTime、MPEG-4、WMV⑤ Lossless compression/Quality: TIFF: Losslessly
compress the images for the export
JPEG/Quicktime: Set the Desired compression rateusing the Quality⑥ Export LUT: The color look-up table (LUT) of theselected object is saved in the corresponding
metadata subfolder.
⑦ Select Channels: In the case of multi-channel
acquisitions, you have the option of exporting
individual channels. By clicking the desired button,
you can include the corresponding channel in the export (Red button) or exclude it (Gray button).
⑧ Export Channels: When the checkbox is enabled, each selected channel is saved as a separate
image.
Raw image: The images are saved without having been processed. Burn-in options are notavailable. (Options for TIFF/ImageJ TIFF)
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Scaled Viewer Image: Available only for multi-channel acquisition. The images are saved as ashown in the viewer. (Options for TIFF/ImageJ TIFF)⑨ Export Overlay as scaled Viewer Image: When the checkbox is enable, all selected channels aresuperimposed and saved in one file.
⑩ Save Multipage: Option for TIFF images with more than two dimensions. When the checkbox isenabled, each image is saved in a searchable, multi-page file. Otherwise, each image is saved in a
separate file.
⑪Reduce Tilescan Size: You can use this slider to reduce the relative size of a composed mosaic
acquisition (as a percent). Otherwise, the composed mosaic acquisition is exported in its full size.
⑫Burn in to scaled Viewer Image: In this dialog, you can configure the visualization of additional
image information that is displayed in the exported images or videos. When the respective
checkbox is clicked, the desired image information will also be exported. The

button opens

another dialog with setting options for the desired image information.
⑬Save: When “Save” is clicked, the export is started with the configured settings and exported
images/movies are saved at the specified path.

VII-4. Organization of the data in projects
All images and image series are collected in Projects and shown in a directory structure, referred toin the following as the project directory.
Close Project: Close the selected project
Save Project: Save the selected project
Save All: Saves all opened projects and files in the configured format
Save as: Opens the save dialog for selecting storage location and storage
formatDelete: Delete the selected file completely. Cannot be restored.
＊Not “Hide”
Rename: Makes it possible to rename a selected file
Cut: Cuts the selected file and places it in the clipboard
Copy: Copies the selected file into the clipboard
Export: Opens the Export dialog (Ref. previous page)
Properties: Opens the Properties dialog (Ref. page 14)
Open in new viewer: Opens the selected file in a new viewer
Open file location: Open the storage location of the selected file in windows explorer
Apply image settings: The instrument parameters of selected image are restored (Ref. page 14)
Close All: Close all open projects and files

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VII-5. Free viewer software LAS X Small (Win10)
This module has full image viewing capabilities, including annotations, image overlay and image
comparison.
Search for “Leica LAS X Core” and download the installer from ”Download free LAS X Core Offline
version for Life Science”.

*ImageJ, FIJI and Imaris are available to open the lif file.
*ImageJ requires the plugin, “Bio-Format”.

VIII. Online Help
Click on the help symbol

located in the top right corner of every LAS X dialog field.

The online help opens in your default web browser. An overview of the dialog topic is displayed,
where you will find links to all relevant topics.

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IX. Shut down
1. Laser OFF
SelectConfiguration menu and Laser Config

Turn off all lasers with single click*Do never change WLL%Do Never change WLL％

2. Shut down the LAS X software
3. Turn off the Hg lamp

5. Turn counter-clockwise Laser security key switch
ON OFF
6. Turn off the laser switch

76

7. Turn off the main power switch

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How to Clean Immersion Objectives
It is important to remove the immersions after use
Never use kimwipes on objective
Oil Objective
1. Just simply wipe the oil off the lens with lens cleaning paper.
2. Put 100% ethanol on lens paper and wipe of remaining oil.
3. Repeat step 2 several times with fresh part of lens cleaning paper.
Water Objective
1. Just simply wipe the water off the lens with lens cleaning paper.
2. Put 100% ethanol on lens paper and wipe of remaining water.
3. Repeat step 2 several times with fresh part of lens cleaning paper.
Glycerol Objective
1. Just simply wipe the glycerol off the lens with lens cleaning paper.
2. Put 100% ethanol on lens paper and wipe of remaining glycerol.
3. Repeat step 2 several times with fresh part of lens cleaning paper.
Confirm the chiller solution amount
When the “LOW FLUID INDICATOR” lamp is ON, add the below liquid.

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X. How to use DMi8
1. Movement in XY-direction and Focusing

Focusing (Motor driven)
Inner: Fine

outer: Coarse

Smart Move

Motor driven focus(Optional)

Clockwise: Up
Counterclockwise: Down

Movement in XY direction

Switching Coarse/Fine button

Microscope; Upper: Y, Lower: X

Left side: XY movement

PC monitor: Upper: X, Lower: Y

Right side: Z movement Z

Front side: Coarse, Back side: Fine

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Confidential - Company ProprietaryX-２. Change Objective using Touch Screen①PressAll available objective is shown. The droplet
mark indicatesimmersion

objective. Selecting objective were turn red.
②Press desired objective
If it is different type immersion objective, the icon
flashes. Touch again automatically change andturn red.

X-3. Setting the Transmitted Light using Touch Screen① Press② Press BF (Bright Field)③ Close/Open the shutter with “TL-Shutter” button*The shutter control is available from microscope left
side (See below)④The brightness control from the knob in
microscope left side (See below)

Adjusting the Brightness

Motorized shutter control

TL (Transmitted Light) or FLUO (Fluorescence)

TL (Transmitted Light) or FLUO (Fluorescece)

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You can confirm the brightness (FIM) from touch
screenPress*FIM can be adjusted 5 steps: 10, 17, 30, 55,
100%

X-4. Setting the Fluorescence using Touch Screen①Press②Press “FLUO”③All available filter were shown, press desired filter.
④Close/Open the shutter with “IL-Shutter” button*The shutter control is available from microscope left
side (Same as TL, See previous page)⑤The brightness control from the knob in microscope left side (Same as TL, See previous page)
Specification
Filter Name

Fluorescence

Ex. Range

Ex. Filter

Dichroic

Em. Filter

DAPI

DAPI

UV

BP 350/50

400

BP 460/50

FITC

FITC

Blue

BP 480/40

505

BP 527/30

FITC LP

FITC

Blue

BP 470/40

510

LP 515RHOD

RhodamineGreenBP 515-560

580

LP 590*BP: Band-Path, LP: Long-Path

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53

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