Living up to LifeTCS SP8
FSU
Summery Manual
LAS X 3.1

20170703

01Contents
Ｉ. Start up 2
ＩＩ. Image acquisition
III. Viewer

620

IV. MaxProjection

23

V. Quantify menu

24

VI. Data saving 25
VII. Shut down 30
VIII. Microscope operation 32
IX. HyVolution (Cofocal super resolution method) 36

2

I. Start up

Main Switches①

Indicator lamp ofimaging lasers②③④①Switch for PC①②②Power switch
for the Scanner

3④Key switch of imaging
laser③Main power supply of
imaging lasers and cooling① Switch on the PC.
② Switch on the Scanner.
③ Switch on the main power supply of imaging lasers.
④ Switch on the key switch of the imaging lasers (yellow indicator lamp will be on )⑤ Switch on the HG lamp.
Note) Do not switch on/off frequently and wait about 5min when restart.

⑥ Switch on the microscope.

⑦ Log in as “TCS User”⑧ Double click on the “LAS X” on the desktop.

4

⑨ The following window comes up.
Select modes/switches and click “OK” and wait until the interface opens without operation
including microscope.
Configuration： machine.xlhw → Imaging through the microscope.
SimulatorSP8.xlhw → Starting up without connected hardware(Simulator mode). Turn on PC
only.
Microscope： DMi8

Select the ScannerOFF：
FOV scanner；High resolution
ON：Option
Resonant scanner；High speed⑩ Switch on the lasers
Open the Configuration tab, click the Laser icon.

Activate each laser by single click (Do not double click.).

-

Set Ar laser power as 20-30%.
WLL laser power will be set as 70% automatically, do not change the power.
5

⑪ Change the bit depth

Click the Configutation menu
And select an appropriate number of bit depth.

,

and click the Hardware,⑫ If using Resonant mode; Define the zoom
factorWith Resonant mode, the zoom factor set asbigger when the software opened (zoom factor
depends on the 8/12kHz.). Define the zoom
factor to minimum before starting acquisition.
（Default zoom factor with FOV scanner is 1.0
and minimum is 0.75）⑬ if using Resonant mode; set the Live averaging
In order to get higher image quality with resonant mode under Live scan, set the Live” Line
Average during Live Acquisition” in the “Hardware” in the Configuration tab
The live image will be averaged by setting number for Line average factor. (See II. Image
Acquisition)

6.

II. Image Acquisition

Configuration：
Set up menu for
microscope, lasers
and systems

Acquire：Main menu for
imaging

Process：
Image processing
menu

Quantify：
Quantify menu formeasuring andanalyses

Open Acquire Menu

Acquire menu

Wavelength settings:
Selecting lasers, detectors, emission wavelength,
objective lenses. To simplify the operation, load thedefined settings.

Viewer：
Display area. see IV.Viewer for
detail.

3buttons
for用のボタン
scanning
Scan
Scan
用のボタン

Switching Acquisition/Projects tab
Acquisition ： Definition of acquireparameters.
Pixels, zoom factor, Z stack settings,
etc.

3 buttons for scanning: for preview/adjust button. Data will not be
stocked.
: for single image acquisition.

Projects： Data container
Acquired image data using by Capture
Image button or Start button are: for multiple image acquisition.

7

Wavelength settings
Settings

forsingle color

Laser wavelength
selection and power
setting

Objective setting

Panel box, notch filter,
Dye assistant settings
detectors

Detection setting（PMT or HyD）

(Prism-slit)

Transmission detector(Bright field)8

①Select objective
Select objective and observe specimen, find the position and the focus.

① Load the fluorescence setting For Single staining observation
Select the light path setting from the pull-down menu of “Leica Settings” of“Load/Save single settings”. *For the multi staining, go to ③.
DAPI： UV ex. Blue fluorescence observation
FITC： Blue ex. Green fluorescence observation（Alexa488, Cy2, GFP etc）
TRITC： Green ex. Red fluorescence observation(Alexa555,568, Cy3, DsRed etc)
Cy5： Red ex. Far Red fluorescence observation(Alexa633,647, TOTO3, TOPRO3 etc)
Transmission: bright field② Load the fluorescence setting For multiple staining observation③ －１ Activate “SEQ”button in the “Acquisition mode” panel. “Sequential Scan” panel will open.

③－２ Click the “Load”button on the “Sequential scan” panel and select the sequential setting forthe multiple staining.

9③－３ Several “Seq.” buttons are displayed depend on the number of fluorophores.
Activate each “Seq”button and define for the each staining (laser power, gain).
Refer ④ and after how to define these parameters.

＊Sequential mode
between lines

Each excitation changes in line by line.
Suitable for live imaging since it is almost
simultaneous scanning, time lag is only aline period.Necessary to have same
number of detectors with dyes.

between frames

Image acquisition executes each frame byframe in different excitation. It means there
is a time lag of a frame period.

between stacks

Each excitation changes in stack by stack.
This method achieves the fastest
completion time even the time lag between
the dyes becomes bigger

10

Loading the settings from the acquired/saved data
By using the Apply button on the Experiments tab, the settings of the acquired/saved data can beloading (reproducible).Activate the raw image data on the Projects tab and click Apply.
All the parameters of wavelength(Excitation, emission), detectors will be reproduced.

Apply button

Activate certain data on the Project tab and click Apply button,
wavelength settings will restore according to that data.

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④Image acquisition
Click the Live button

at the lower left on the monitor.

Scan starts and align the parameters as follows.
Notes: Live button expose lasers your specimens. So it should be stopped often in order toavoid the bleaching.
■ Defining the laser output
Define the laser output so that it may be optimized to the specimen by using slider or imputing thenumber on the laser panel.
Switching WLL and Ar laser, 405nm laser.
Displays switch by clicking the vertical buttons “Switch to Whitelight”, ”Switch to Conventional”

Defining laser power by using slider or inputting numbers.

Ar.laser, 405nm laser setting

WLL laser settings* Activate corresponding Seq button whenever define each laser power.

First activate corresponding Seq
button and change the laser power.

12

Defining the focus, and the detector gains
Adjust focus and the detector gains and offsets by using each dial of the control panel (option) as
below.
For the multistaining, the “Smart Gain” and the “Smart Offset”dials can adjust each staining byactivating channel image respectively on the monitor.
- default settings of gains as a standard → PMT：800V HyD：100％

Smart Gain
Gain for the detectors

Smart Offset (only for PMT)
background setting

Z position
Focus adjustment- point
The less Gain achieves the higher S/N. If higher gain is used, use “Average” function in order toreduce noises.
PMT（conventional）
HyD（Hybrid detector）
Gain（the voltage of detector）
0-1250V
10-500％
Default Gain as a standard
800V
100％
Offset（background setting）
Possible
Photoncounting modepossible

For multicolor imaging, with Panel Box, you can change each channel Gain/Offset by using Smart
Gain/Smart Offset dial, just click certain (singlecolor) image on the monitor,

CH1

CH2

Click single color image on themonitorandoperateSmart
Gain/Smart Offset dial.

CH3

13

Quick Look Up Table (Q LUT) button
For easy tuning of signal intensity, click Q LUT button and the look up table (image
display color) changes to “Grow Over/Under”.
Image display color changes like follows by clicking this button.
Original （pseudo color like green, red, etc.）→Grow Over/Under （for checking）→Gray （black white）→Original （pseudo color like green, red, etc.）
Image can be taken with 8/12/16bit. For instance 8bit; it has 256 intensity levels (greyscale/gradation), and “Grow Over/Under” color shows this 256 level in different colors likefollows.
>255
255
・
・
・
0
0<

blue（saturation = overflow）
blue
white
brown
black
green
green

Blue color means the signal saturation. Readjust laser power and/or gain to avoid
saturation.
Green color means the darkest signal. Too low offset makes enlarged green area andsome weak signals may be failed to be detected.
Green:
Background
intensity = 0.
Blue:saturation.

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⑤ Other parameters（format, scan speed, zoom, etc）
If necessary, Format (pixel numbers of the acquired image), Speed(scan speed), Zoom
factor(optical zoom) can be also changed. The slower the scan speed, the smoother the image
quality.
Please note, the bleaching effects becomes bigger with the higher the zoom factor.
Format：Pixel numbers of the image

Speed：scan speed
Zoom：minimum is 0.75.
Zoom In button enables you to define theregion of interest on the image.
Control panel also can be used.

Rotation(option)
Scanning rotation

Zoom
Zoom factor

Average：averaging for reducing noise.

Rotation（option）：scanning rotation⑥ Average function“Average” enables to get higher signal to noise (image quality) ratio of the image.
■ Set higher number of average for higher detector Gain.
■ Higher number of average does not necessary for lower Gain.
⑦ Acquisition of the Single image
Click “Capture Image button”
for single slice image.
Image data will be kept in the Project tab as the name of “Image001” temporarily.
The number of Average will be activated by these buttons during the acquisition.

15

⑧ Z stack acquisition
Define start position with “Begin” button and end position with “End” button while scanning by“Live”button. Focus can be moved by Z position dial of the Control panel.

Z position
Adjust z position/focus⑧－１
Define Begin/End
position
DeletedefinedBegin/End
positionThickness by defined
Begin/End⑧－２
Define number of frames or
step size.

Activate either ofthemGuideline for z-step size:
About the twice of thenumber of “System
Optimized”

16

⑨ Acquisition of the Zstack
Data acquisition starts by clicking following button.
XYZ (or for several continuous image acquisition：
click “Start” button.
Image data will be kept in the Project tab as a name of “Series001”temporarily.
Note: keep away from the microscope and the anti-vibration table while scanning in order to avoid
the vibration noise to the images.

Parameters of the acquired image data
Right mouse click the data on the Experiments tab and select “Properties”.
“Experiment Data” will open and able to confirm the parameters of the image data.

17

HyD（Hybrid Detector *option）
HyD has higher sensitivity, higher S/N, and extremely low noises. Photoncounting mode can beselected.

Caution:- Please separate mobile items (cellar phone and other items which send electric wave) from
HyD about 1m. If they are close to locate each other, HyD would detect noises from them.
-

If HyD detects extremely high intensity, its shutter closes automatically. It means too much
emission fluorescence by high excitation power. Redefine lower laser power.

The difference from PMT- HyD Gain is variable from 10 to 500％ whose PMT is variable from 0V to 1250V.
- NO Offset function for HyD.
- There are three kinds of modes with HyD.
Detection mode for HyD- Standard: Normal mode as PMT.
- Counting: Please refer next page.
- BrightR: Mode for imaging with wider dynamic range a little bit.

Selection of HyD modes

18

Counting mode (Photon Counting mode)This is a mode to count and accumulate photons in each pixel. In this case, the intensity means
the number of photons. Detector noises are almost zero and the S/N and the quantitativity
improves quite a lot.
Theoretically it is not possible to use Gain with Counting mode, the image shows less bright thanStandard mode, to get brighter image with Counting mode, please refer following settings.
＜points of using Counting mode＞■ Gain cannot be used in theoretically. Only the number of photons are shown as a image.
■ brightness adjustment can be done with following method.
① take slower scanning speed. （only with FOV scanner）
slower scanning speed can get more photons per pixel which means brighter image.

② take Acculmulation
For example, 2 times Accumulation made about twice as bright image.

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⑩ timelapse⑩－１．Select mode
Select “xyｔ” or “xyzｔ” from the Acquisition Mode.
ｘｙｔ ： XY timelapse imaging without Z stackｘｙｚｔ ： XYZ timelapse imaging with Z stack.

⑩－２．Following panel will open according to the selected mode.
Define the “Duration”(whole acquisition time) and “Time interval”
Whole acquisition
time

Time interval

Frames/Stacks will show depend on the mode(xyt/xyzt). If the xyzt mode is selected,
this column will be Stacks and Z settings should be defined on the Z stack panel.

⑩－３．Timelapse acquisition
Define other settings（image adjustment, zoom, Z stack, etc.） and click “Start” button
for scanning.

20III.Viewer
Viewer：Window which is shown the acquired images. There are function buttons around theimages.

The colors of theimage can bechanged by clicking
this LUT bar.

Tiled/full display can bechanged by double clicking
the image.
Tiled display

Full display

21

Icons of the viewer
Select tool
Delete selected/activated item
Insert scale.
Activate the scale by using
button and right mouse click,select “Properties” then define the length, angle, etc.

Display scale.
On/Off this button and the image changes 100% display or shrink orzoom depend on the image resolution and the display resolution.
Quick Look Up Table
The color of the image changes by clicking this button each time,
Grow Over/Under→Gray→Original.

Original color

Grow over/under
Blue：indicates saturation

Gray

22

Overlay
Display or not display “overlay image” of the multiple channels by
activating/not activating this button.
Maximum projection
Create Maximum projection image
from the XYZ stack data
temporarily.
Themaximum
intensity in the z stack for eachpixel will be shown as a single 2D
image.

Orthogonal Sectioning
Show XZ and YZ section images from the XYZ image data.
YZ
XY

XZ

3D
3D Viewer launches with this button. 3D view will be shown from the
XYZ data.

23

IV. MaxProjection
Create Maximum Projection image data from the Z stack image.

Open the “Process” menu and select the “Projection” in the Process Tools.
Activate the Z stack data on the Experiments tab and click “Apply” button.
Maximum Projection image data will create/add in the Experiment tab.

24

V. Quantify menu
Functions to measure lengths, intensities, areas, histograms, ratio, etc.
There are three mode as follows.

Line

Stack

Lengths and intensities and other statistical data are shown bydrawing line on the images. This function is supposed to use forsingle image.
Intensities, areas and other statistical data of defined ROIs（Regionof Interest） are shown. This function is supposed to use for XYZ ortimelapse data.
Histogram and the other statistical data are shown by defined
ROIs(Region of interest). This function is supposed to use for single
image.

Histogram
Result export by “Report” button
Result sheet can be export by “Report” button.

Saved data by this button will be .xml, jpg, csv files. Graph data will be imported by Excel, as well.

25

VI. Data saving
All image data in the single file will be saved as a .lif file.
Open
Projects tab
Example）
Here, there are three
image data in the single
file “Project001”. They
will be saved as asingle .lif file.
Each single
file on the
LAS X.

Project Tool bar
New： Create new Project file.
Open： Open saved data.
Save： Save all data on the Projects tab.
Apply： Restore parameters of which activated data. Selection of applying
parameters can be defined from following mene.
Configuration > Instrument Parameter Settings (IPS)
Browse： Browsing saved data.
Shows： Show the first image data on the Projects tab.
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①Right mouse click the data and select”Save As”.
Image data in the same file will be saved as a single file as explained above.

All image data in the same file will
be saved as a single .lif file.

To rename each data
( Series001,Image001, etc）, rightmouse click and select “Rename”
or just double clicking the data.

② Select the path to save the data.

27

③ Overwrite saving (refresh saving )Adding/editing data after saving .lif file, data shouldbe overwritten by executing “Save Project”④ Acquiring new data in the new file
To create new file, click “New” button, new data will be acquired in the activated new file.

New button

Created new file
Activate this file and acquire new data, the data
will be input in the new file.
＊data will be able to copy/move by click anddrop/copy and paste to the other file.
Note)
If the new data are acquired without
activating new file, it will be acquired in the(another) activated file.

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⑤ Exporting file as “.Tiff”, “.Jpg” format
Right mouse click the data and select ”Export” and select ”As Tiff...” or ”As JPEG..”.

Then following dialog will open.
Select the path and click save.

Destination Folder
by clicking Browse button to select the path
Overlay Channels
For multicolor staining, select “Overlay” or
not for saving.

Select to inputting scale bar, timestamp,
etc.

29

LAS X Download site
1. Access to following site.
http://www.leica-microsystems.com/products/microscope-software/software-for-life-science-resea
rch/las-x/
2. There is a link at the most end of the page, click to download.

① For
SP8
②
For

①
②

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VII.Shut down
1. Switch off the lasers on the software.
Open the Configuration tab-

and click “Laser Config”.

Ar laser: Decrease the laser power down to 0% and switch off.
WLL laser: Switch off while keeping laser power 70%

2. Lens cleaning
Using lens paper/ cotton-tipped stick and lens cleaner, clean up liquid immersion lens.
Note) Do not use Kim wipe or other papers for lens cleaning.
3. Close LAS X and Windows.
Save all data and close the LAS X and shut down the Windows.
4. Switch off the mercury lamp .
Note; Wait about 5min. for restart.

31

5.Switch off the Microscope.

6.Switch off main switches① If the Windows is already finished, switch off the PC.
② Switch off the scanner③ (If Ar laser is equipped) after the cooling fan was stopped (about 5min after deactivate
Ar laser on the software), switch off the key switch.
④ Switch off the laser power supply.

①

②

④
③

① Switch for PC② Power switch
for the Scanner32③ Key switch of imaging
lasers.

④ Main power supply of imaging
lasers and cooling fan of Ar.laser

VIII. Microscope operation
１. Focusing and XY Stage control
Focus the image by turning the focus dials on the left and right sides of the stand. Alternatively, rearrotatory knob on the Leica Smart Move control element can also be used. XY stage can be controlled by
Leica Smart Move control element.
Outside dial： Coarse focus adjustment
Inside dial： Fine focus adjustment

Leica Smart Move control
XY
Z
movement

Z focusBlack buttons
Right side：
Z focus: Fine –Coarse
selection button.
Left side：XY stage :Precise- Fast
selection button

２．Changing objective nose pieces
２－１ Changing①

objective by using

the Touch Screen① Use thetab to switch to theMagnification
memu.
Currently
selected objective is displayed in red.
② To switch between objectives, press
the corresponding objective key from
Objective Nosepiece Mode. Immersion
objectives are marked with drop②

symbol. When you change

the objective between IMM and Dry,
you need to touch the objective key
two times. The objective key will brink by the first touch and the objective will be changed by
the second touch.
Notre）If the immersion liquid is attached to dry lens, clean the dry lens certainly. Otherwise it causes thedecrease of image quality.
33

２－２ Changing objective on the software.
Click ‘Objective’ in the software and select the appropriate lens from the pull-down menu.

※ The message is shown when changing between dry lens and immersion lens.
“Immersion of current objective(OIL/IMM/DRY) is different from selected objective(OIL/IMM/DRY).
Do you want to turn turret automatically? Yes No ”
→ Crick Yes to change the objective lens automatically
In changing from Immersion lens to Dry lens, wipe off the Immersion from the specimen to prevent
Immersion forming on the objective lens.
※ Immersion (IMM) Objectives
For immersion objectives use the appropriate immersion medium.
OIL: Use optical immersion oil from Leica only
W: Water immersion
Gly: glycerin
IMM: Universal objective for water, glycerin, Oil immersion. (Require setting of the appropriate correction
circle to use each IMM)

Notre）If the immersion liquid is attached to dry lens, clean the dry lens certainly. Otherwise it causes thedecrease of image quality.

34

３．Microscope observation method ( by your eyes.)

３－１． Transmitted method（Including DIC）①①②

On the Touch Screen use theconfigure the contrast method.

tab to②
Select the contrast method
Transmitted method list.
BF： Brightfield Transmitted Light
DIC： Differential Interference Contrastfrom③③ Touch the TL-Shutter key on the Touch
Screen to open or close the Shutter (active
illumination axis on or off). Alternatively, Shutter
control button on the left side of the stand canalso be used.

④The brightness is adjusted by the knob on the left side of the stand.

③

Shutter Control④⑤⑤ On the Touch Screen use theconfirm the current intensity.

35

tab to３－２．Operating the fluorescence① On the Touch Screen use thetab toconfigure the contrast method.
②Select FLUO
③ Select the desired cube using thecorresponding key from FLUO-Filtercubes.
※Set of Filter cubes are depending on thesystem. Please refer the Fig1.1about the specof each FLUO filter cubes.

②

①

④
③

④ Touch the IL-Shutter key on the Touch
Screen to open or close the Shutter (active
illumination axis on or off). Alternatively, Shutter
control button on the left side of the stand can also be used.
⑤The brightness is adjusted by the knob on the left side of the stand.

④ Shutter Control⑤

Fig.1 FLUO Filter Cubes
FilterTypical fluorophore
DAPI
DAPI
FITC
FITC (Band Pass）
FITC LPFITC (Long Pass）
RHOD
Rhodamine

Excitation
UV
Blue
Blue
Green

Excitation filter
BP350/50
BP480/40
BP470/40
BP515-560⑥

Dichroic
400
505
510
580

Fluorescent filter
BP 460/50
BP527/30
LP515
LP590

⑥ On the Touch Screen use thetab toconfirm the current FIM (Fluorescence
Intensity Manager). The brightness isadjusted in 5 defined increments.

36

Leica Microsystems K.K.
Tokyo
1-29-9 Takadanobaba,Tokyo, 169-0075 Japan
Phone: +81-3-6758-5630
Fax: +81-3-5155-4333
Osaka
10F 5-4-9 Toyosaki, Kitaku, Osaka City, Osaka 531-0072 Japan
Phone: +81-6-6374-9771
Fax: +81-6-6374-9772
Nagoya2F 2-15-20 Nishiki, Nakaku, Nagoya City, Aichi, 460-0003 Japan
Phone: +81-52-222-3939
Fax: +81-52-222-3784
Fukuoka
12F, 8-30 Tenyamachi, hakataku, Fukuoka City, Fukuoka 812-0025 Japan
Phone: +81-92-282-9771
Fax: +81-92-282-9772

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