From Eye to Insight

FALCON FLIM
Manual20200110_EN

FLIM
1. FLIM Calling up

1

Page

2. Calibration of laser and detectors

1

Page

3. Adjust Trigger Level

1

Page

4. Detector setting

5

Page

5. Adjust Photon/Pulse

6

Page

6. Repetition number

6

Page

7. Setting the ROI

9

Page

8. Fit16

Page

9. Crop

10Page

10. FLIM image Fit

27

Page

11. Save image

16, 33Page

12. Phasor plot

35

Page

13. STED

48

Page

Tips
＊Check box for Gating function is not available for FLIM, but the values apply to the FLIM.
＊PMT is not available for FLIM
＊Notch filter supported Pulse laser is required(Notch filter not-supported pulsed laser could available for enough bright sample)＊LAS X FLIM/FCS can be opened in the following LAS X wizard using the FLIM button andis available for FLIM experiment there:- FCS: In the Setup Imaging operating step
- FRET AB, FRET SE: in the Setup operating step in the Workflow or Acquisition tab- Live Data Mode: In the Acquire operating step
- Electrophysiology: In the Acquire operating step
＊Click the「 LAS_X_SingleMoleculeDetection.exe shortcut 」

open the An

evaluation in LAS X FLIM/FCS. LAS X Small is not available for the analysis＊STED 77 nm and WLL pulse timing are synchronized.
WLL pulse picker is not available only when STED 775 nm is ON
STED 775 nm is only available when WLL is 80 MHz

FLIM Calling up

Click the FLIM button. The LAS X FLIM/FCS opens on the second monitor.
In second monitor, the FLIM setting dialog field is displayed.

Calibration of laser and detectors
7 Setting
Click the ○open the FLIM Calibration dialog＊WLL： Automatic/Calibrate*Trigger level is available for background level adjustment (Only SMD-HyD)*Sequential setting is disappear after calibration
1

＊WLL with pulse picker, you can define the repetition rate the dialog.
The dialog open from the buttonin Acquire menu.

Click the red frame, open

the pulldown menu. Then, you can choose the frequency 80, 40, 20 and 10 MHz.
The last used setting frequency is remain＊The dialog open in Hardware Configurator/Laser Config.

Click the ① and ② redbutton, open the pulldown menu. Then you can choose the frequency. The last usedsetting is remain.

①②＊Check the Ref. Line [nm] in red frame is same as Ex laser＊STED 77 nm and WLL pulse timing are synchronized.
WLL pulse picker is not available only when STED 775 nm is ON
STED 775 nm is only available when WLL is 80 MHz

2

＊MP：Manual/NIM2-4（The number depend on the system）Adjust the pulse width with
Trigger level3

F LIM Settings (Main monitor)
Filter Supported WLL-Lines：
All WLL lines supported by a filter system are represented
by a button and are enabled when you click the button.

Line Repetitions

Define the number of scans per line when acquiring a FLIM
image.

The resulting acquisition time for the FLIM image is

automatically displayed under Duration of each image.
Line accumulation for intensity image.
Frame Duration

Define the duration for acquisition of a single FLIM image

Frame Repetitions

Define the number of scans per frame when acquiring a FLIM
image.
Frame average for intensity image

Acquire until max …

Until a certain number of photons have been detected in thephotons/pixel

brightest pixel within a FLIM display window
Frame average for intensity image

Sum of all Channels

All channels

Brightest Channel

Brightest Channel

Dimmest Channel

Darkest Channel

4Specialist Settings ： To increase the speed ofimage acquisition, detector channels for dataacquisition can be combined manually into FLIM
display channels.
If this function is activated, new dialog field (in theFig. left), Manual FLIM Channel Assignment,
opens.
Click the first drop down menu.
The following dialog (in the Fig. left)
opens for assigning the channels.

Single Detector

Assignment of exactly one detection channel.
All activated FLIM detectors are listed in this area. By clicking on an
entry, you can precisely assign one detection channel to selecteddisplay channel.

Multiple Detector

Assignment of several detection channels.
To increase the image-capturing speed, you can assign multiple
detection channels to the selected display channel in this field. Eachtime + is clicked, another drop down menu is added. Then, selecteach desired detection channel from the drop down menus. Clicking
X removes the individual drop down menu again.

All Detectors

Assignment of all active detection channels.
Clicking the button assigns all active detection channels to display
channel.

5A Fast FLIM image is displayed in the sub-monitor for each detection channel. For eachpixel, an averaging of all fluorescence lifetimes and fluorescence intensities that occur in thespecimen is displayed in ta false color image. The contrast for lifetime and intensity is set
automatically, but can be adjusted.
Right click on the image, and choose
Show Data Cursor.
Then, shown this dialog.

If necessary, change the contrast for lifetime and intensity. To
open the dialog for adjusting the contrast, click the control
element next to the channel designation below the FLIM image
display window.

You can change the contrast by using themouse to drag the white margin lines to desired position.
a ：Pixel Intensity Histogram, theUnderneath the image view, ○b ： Lifetime Decay Curve arepile-up control histogram, ○displayed live
Pixel Intensity Histogram：Display the Events over the photon/Laser pulse
Use the pixel intensity histogram to adjust the laser intensity live, so that a maximum of 0.5
photons/laser for HyD and 1.0 photons/laser for SMD-HyD are measured.
The color depends on the false color

6

＊A pulse pile-up is the name of for the case when emitted photons are not taken into accountfor a high count rate due to the dead time of the detector during the counting.
This has the following effects, which ditort the result of the FLIM measurement- The measurement lifetime becomes shorter- A mono-exponential fading on the lifetime (1 component) appear to be double exponential
with a second component a shorter lifetime.
a ：Curve progression with pulse pile-up effect○b ：Ideal curve progression without pulse pile-up effect○＊Required photon number for Fitting
1 component: 100, 2 components: 1000, 3 components:10000

7

FLIM

8-① Setting for analysis
8-② Display window for FLIM image. During FLIM test and FLIM measurements, a fast
FLIM image is already being displayed live. Below the display window, there are controls
for adjusting the contrast of the individual channels.
8-③ Parameter setting are displayed for the fit on the right edge of the screen
8-④ In the diagram area, histogram is displayed in left and the lifetime decay curve isdisplayed on the right
8-⑤ A navigation table is displayed below the diagram in which is a row entry for each image,each ROI and each FLIM display channel.
8-⑥ In this dialog, you can save the FLIM image
8-⑦ In this dialog, you can export FLIM data
Adjust Photon/Pulse

8

18-①
Using ROIs you can limit the region of interest of the fast FLIM image. For this purposes,
various tools are available in this dialog
From the upper left
①Brush②Polygon③Rectangle④Ellipse⑤Circle⑥Individual pixel⑦Selecting a ROI⑧Select all ROIs
⑨Delete selected ROI(s)⑩Undo last operation⑪Restore last operation⑫Automatically form a ROIInvert： Reverse the color display in the FLIM image and shows the image in inverted colors.
Additional function for handling ROIs

New

Creates an entry for ROI. Afterwards, you can draw the ROI.

RenameEnables you to enter a designation for the ROI entry

RemoveDeletes selected ROIs

Reset

Deletes all ROIs

9

From、To

Define a range for the images to be analyzed from the repetitions of thescan. For this purpose, the From and To input fields are available.

Pixel BinningSet the number of pixels compiled during image analysis.

Z/T

The dimension of the current image series is displayedAll

All single images are analyzed

Range

You can define a range for the single images of the series to beanalyzed. For this purpose, the From and To input field are available.

Single

You can select a single image under Index to be used for analysis

Split

Only for Z or T. Each single image od series is represented separately
with a row in the results table and a curve in the lifetime decay or phasor
and can be analyzed indivually

Lifetime BinningSet the number of time frames are compiled in the lifetime decay curve.
This setting only affected the display of the curve (it became smoother).
It is not included in the analysis.

Reflection Filter

If this function is enabled, the values generated by reflections when WLL
are cut from the lifetime curve.

Crop
It is possible to crop lifetime series in time and space and to bin in time as in Z.

10

In this dialog, you can define a time gate, starting the time of theexcitation pulse, in which the analysis of your FLIM image datais carried out. The time gate is applied to each frame.
In this dialog, you can define an intensity range for which theanalysis of your FLIM image data is carry out. The intensity
range is applied to all further analyses and treated as an ROI.
By moving the red lines with the mouse①By moving the red line or entering the ②lower and ③upper value. At the same time, in
the right area of the display window you can see which areas have been selected in theimage.
④Here, you can define the threshold value in the left window are by moving red line① orentering the lower② and upper values③. At the same time, in the right area of the display
window you can see which areas have been selected in the image.
Intensity

All hidden areas not included in the evaluation are displayed red

FastFLIM

All hidden areas not included in the evaluation are displayed gray11

Narrow down the time gate by dragging the red lines with themouse.
The setting is already applied directly to the image display
window (Fast FLIM and Counts) as well as the decay curves.
The raw data is unchanged by this setting.

Standard (High Speed)

For the lifetime decay curve time intervals between two laser
pulses are evaluated only if exactly one photon has been
emitted in them.

For the FLIM image fit, the dead timecorrection is applied.

HyD deadtime is approximately 1.5

nsec.
All Photons

All emitted photons are detected (Raw data)

First Photon

Occasionally, 2 or more photons are emitted after a laser
pulse. In each case, however, only the first emitted photon
is counted.

This ultimately leads to a falsification of theresults known as the pile-up effect.
Manual assign Detectors

To increase the speed of image acquisition, detector
channels for data acquisition can be combined manually into
FLIM display channels.

12

8-②

A
○

1： Reset the zoom for the display window
2： Reduce the zoom for the display window
3： Enter the zoom factor for the display window (%)
4： Increase the zoom for the display window
5： Sets the zoom for the display window to the 100%
B○

During FLIM test and the FLIM measurement, you can choose the following views
Intensity

For each pixel, show the number of the counted photons in gray-scale

Fast FLIM

For each pixel, show an average of the all fluorescence lifetimes andintensities in false color

χ2

For each pixel, show the intensity of the error or the fit precisionFree

You can freely select the parameters for intensity and color using a
dropdown menu in the contrast setting

After the FLIM Image Fit （Ref. page 27）, display options that shown in next page areavailable.

13

Display option for FLIM
Components

For each channel, the special distribution of the intensities is displayed ina separate RGB color

GrayscaleSame as RGB, but the intensities are represented as grayscale

Lifetime

The special distribution of the lifetime for each component is displayed infalse colors (Rainbow colors) and the amplitudes are displayed as anintensity.

Display option for FRET
Efficiency

The average Apparent FRET efficiency is shown in image form as a false
color

FRET

The average FLIM FRET efficiency is shown in image form as a false color

Binding

Proportion of the bound donor molecules to the total number of donor
molecules in % is displayed using a false color image

Distance

The distance of the donor and acceptor are displayed using a false color
image

Display option for Pattern Fit
Components

For each channel, the special distribution of the intensities is displayed ina separate RGB color

GrayscaleSame as RGB, but the intensities are represented as grayscale

C ：Display Options for the Channels○

45①All channels are displayed superimposed in one image②All detection channels are displayed side-by-side③All lifetime channels and detection channel are displayed side-by-side④Displays bars for intensity range and lifetime range for manual setting in left of window⑤Show a lifetime scale below the display window

14

D ：Contrast Setting○

If you disable the checkbox on the button, no image isshown for the corresponding channel.
Clicking the button opens a dialog window that you can use to set the contrast for the current
image display.

Auto Range

Limits the range automatically to optimal
imaging

Full Range

Uses the entire are for displaying theimaging

Narrow down the lifetime scale by dragging the △ by mouse

B Clicking the button opens a dialog window (in the below Fig.) that you can choose○

parameters.

15

8-⑥This dialog allows you to save your analysis results or FLIM images
Save Results…

The current status of all analysis is stored, in the project directory.
The data can be called up again in LAS X FLIM/FCS and further
analyzed.

Save Image…

The colored Fast FLIM or FLIM image is created and stored as anormal intensity image in the .lif format as a 3 channel image (RGB)in the project directory and can be further processed as such. An
evaluation in LAS X FLIM/FCS is no longer possible with this format.
You can enter a designation under Name

Save Pallet Image as RGB
If this function is enabled, the colored Fast-FLIM or FLIM image is created andstored as a normal intensity image in the .lif format as a 3-channel image (RGB) in
the project directory and can be further processed as such.

Auto Range

Scales the image automatically between minimum and maximum sothat the image is displayed properly, but no values are quantified.

Fixed Range

You can select the scaling range for each display Category
separately.

The values are quantifiable.

Per Grey

Scaling by gray-scale value. Here, under Factor or Range, enter a

Level

factor with which the corresponding grayscale value is weighted.
The values are quantifiable.

Range

Scaling by intensity. Here, select a range under Factor or Range byentering values for minimum and maximum.
The values are quantifiable.

Individual Range

Here, you can define the scaling ranges for each component
individually, just like you can for Fixed Range.
The values are quantifiable.
168-③ Fit Settings:

FLIM

Fit

18- ⑤ Clicking this button calculates thecurve fitting with the setting listed above
for all curves a selected in navigation table

Fit All

18- ⑤ Clicking this button calculates thecurve fitting with the settings listed for all
curves of the selected detected channel

FLIM

Open the FLIM image Fit dialog, in which

Image

you can configure setting for the pixel-to-

Fit…

pixel curve fitting of the curves selected in
the navigation table and start the pixel-topixel curve fitting.

Exponential

The number of fluorescent components

Components(To determine it, Ref. Next page)

Select Fit

For FLIM experiments, 2 models are available for calculating the curve

Model…

fittingn-Exponential

The decay curve is fitted taking into the IRF. This also enable using

Reconvolution

the beginning of the decay curve for analysis.

with IRF

statistics of the data and thus enable a correct estimate of relative

This improves theamplitudes from the sum of the exponential decay curves.
n-Exponential

Only the rear area that is not affected by the IRF is used for the analysis.

Tail FitThis attains correct for the lifetimes as the lifetimes are significantly
greater than the IRF width. The disadvantage of this model is that nogood curve fitting can be calculated for components with short curve
fitting. ＊It is not available for the sample that contain lifetime shorter
than IRF (e.g. 500 nsec).

Decay Time

Lifetime (nsec)

Amplitude

Intensity fluorescence

Tail Offset

Background intensity

IRF Background

Background intensity when acquiring IRF (Meaningful if a measured IRF
is used)IRF Shift

Chronological offset of the IRF (Meaningful if a measured IRF is used)

Intensity

The total intensity of the lifetime

Sum AmplitudeTotal of the amplitude17

Mean Photon

Mean photon detected time

Arrival Time
Mean Decay

Mean lifetime (nsec)

Time
χ2

Fit error or the fit precision for pixel

Fit of the Lifetime Decay Curve: Determining the Number of Exponential Components
In order to determine the number and distribution of individual fluorescent components in theimage using the mean lifetime decay curve, the experimentally obtained decay curve must
initially be calibrated with a theoretical exponential curve.
During the FLIM image acquisition,
the Fit dialog shows the decay curveofthefluorescence(Intensity)

or

the

intensity

numberofmeasured photons in the image (allpixels) over time excitation pulse(Time)

a :Average value of the○measured photons over time after the excitation pulse
Select Fit Model and start Exponential Components value 1, click Fit.
The calculation is carried out and the results are shown as follows in the Fit dialog
a , the diagram shows the theoretical exponential
In addition to the measured decay curve ○b and the deviation in the lower range○ccurve with a component○

If the following criteria are met, this indicates that the correct number of components havebeen measured:a and ○
b lie nicely on top of each other- Curves ○c exhibits only minor fluctuations- Curve ○- The FLIM image after the Fit is no noisy- The value for χ2 which indicates the Fit precision, attenuates only slightly (minimum =1)
when the number of the components for the components for the fit is increased
In the above case, all three criteria negate the assumption that these is only a single
exponential component
The Time gate and Intensity Threshold (Ref. page 10) are effective for χ2
18

ExampleIn the following example, the fit has been carried out with 1, 2 and 3 exponential components.
The result allows you to assume that these 3 exponential components here, since thefollowing are true for an Exponential Component value of 3:
a ：The experimental and the theoretical lifetime decay curve lie nicely on top of each other.
○b ：The deviation curve exhibits little fluctuation○c ：The value for χ2 is close to 1○

19

8-③ Fit Settings:

FRET

Fit

18- ⑤ Clicking this button calculates thecurve fitting with the setting listed above
for all curves a selected in navigation table

Fit All

18- ⑤ Clicking this button calculates thecurve fitting with the settings listed for all
curves of the selected detected channel

FLIM

Open the FLIM image Fit dialog, in which

Image

you can configure setting for the pixel-to-

Fit…

pixel curve fitting of the curves selected in
the navigation table and start the pixel-topixel curve fitting.

Förster DistanceThe distance at which the energy transfer efficiency is50%

Unquenched Donor Lifetime

The lifetime of Donor without FRET

Unquenched Donor Amplitude

The intensity of Donor without FRET

Quenched Donor Lifetime

The lifetime of Donor with FRET

Quenched Donor Amplitude

The intensity of Donor with FRET

Tail Offset

Background intensity

IRF Background

Background intensity when acquiring IRF (Meaningful if
a measured IRF is used)

IRF Shift

Chronological offset of the IRF (Meaningful if ameasured IRF is used)

FLIM FRET Efficiency

This is calculated only from the donor molecules in the
pixel at which FRET actually occurs

Apparent FRET Efficiency

The average Apparent FRET efficiency of all molecules

FRET Intensity

Intensity of the interacting donor molecules

Binding

The binding strength between donor and acceptor(Proportion of the bonded donor molecules to the total
number of the donor molecules %)Donor-Acceptor Distance

Average distance between donor and acceptor （nm）

χ2

Fit error or the fit precision for pixel

20

Calculating FLIM FRET efficiency
Create a Fast FLIM images using donor only sample and FRET sample.

In donor only

sample, copy Unquenched Donor Lifetime (Mono-Exponential Donor) or Mean Photon Arrival
Time (Multi-Exponential Donor).

In FRET sample curve fitting, disable the Fit in

Unquenched Donor Lifetime and paste. Then, carry out the curve fitting by clicking on Fit.
Next, carry out pixel-for pixel fit for the image- Mono-Exponential Donor: This calculation model is applied if the fluorescence lifetime ofthe donor has a mono-exponential curve in the absence of the acceptor- Multi-Exponential Donor: This calculation model is applied if the fluorescence lifetime of thedonor has multi-exponential curve in the absence of the acceptor
In the presence of the acceptor, the lifetime of the donor decreases. The FRET efficiency(FRETeff) as a measurement for the molecular bonding is calculated according to the formula
below.

21

8-③ Fit Settings: Pattern Fit

Fit

18- ⑤ Clicking this button calculates thecurve fitting with the setting listed above
for all curves a selected in navigation table

Fit All

18- ⑤ Clicking this button calculates thecurve fitting with the settings listed for all
curves of the selected detected channel

FLIM

Open the FLIM image Fit dialog, in which

Image

you can configure setting for the pixel-to-

Fit…

pixel curve fitting of the curves selected in
the navigation table and start the pixel-topixel curve fitting.

Tail Offset

Background intensity

Amplitude

Intensity of fluorescenceBackground

Background

Shift

Reset the pattern in the fit through a slight time offset

Intensity Sum

Total intensity amount

Intensity Integral

The integrated intensity is the number of photons in the pattern you
have selected in the decay histogram. The number of parameter
depends only the size and brightness of the selected ROI.

Effective Integral

The effective intensity is the intensity if the component in yoursample. It integrates all photons of all pixels for this component.
If you want to know how much of this component is included in yoursamples, the effective intensity specifies the number of photons.

Fractional Intensity

This parameter specifies the proportion/fraction of the photons of
this component. All proportion should add up to 1.

χ2

Fit error or the fit precision for pixel

22

Separating Components with FLIM Pattern Matching

a ,○
b ,○
c : Define reference samples○

You can define reference patterns for the components in 2 ways: By plotting POIs in the
decay diversity map or in the Fast FLIM image.
1. Switch to the Pattern Fit tab
2. In the Patterns dialog field, select a drawing tool and draw a ROI in a range you candefine clearly
In Fast FLIM image: Color differentiation
In the decay diversity map: Definition of the lifetime distribution

3. In the Patterns dialog, for each plotted pattern, click the Add ROI button to add thereference pattern
The entry is created and color allocated to the pattern. At the same time, a lifetime decay
curve for the pixels allocated to the selected ROI in the same color is displayed under Fit(The Orange curve shows the decay curve over the entire image).
4. Repeat step 2 and 3 for each additional ROI that you would like to delineate as acomponent.

23g ： Carry out fit for decay curve○

1. Set the remaining parameter to Fit for adjustment or fix to their own values (Fit) or linkthem globally (Global).
2. Carry out the curve fitting by clicking on Fit in the area of the parameter setting for theselected channels or Fit All for all channels.

After the pixel-for pixel fit for theimage, the resulting images displayedin RGB mode.
d 、○
e 、○
f
、○

24

8-③ Fit Settings: Mean ROI
Only available for time lapse image
1. Turn on the Mean ROI
2. Select FLIM, FRET or Pattern Fit
3. select a drawing tool and draw ROIs

The lifetime changes within the ROIs are recorded.
Below the viewer, the Mean ROI dialog box display the ③average photon arrival time and④the intensities for each ROI. For each ROI there is a curve in the color of the ROI25

8-③ Fit Setting
Fit

If the option is enabled, the value will be determined from thetheoretical curve during curve fitting.

GlobalIf you have selected multiple data records, this gives you ability
to select the same parameter value for all data records.

Right click, display the dialog
Copy to Clipboard

Store the Fit parameters

Export…

Save the Fit parameters as an Excel

Export and show…

Save the Fit parameters and open it as Excel
table

Limits…

Open the limit dialog, in which you can selectconfigure upper and lower limit for therespective parameter.

Probability Level…

You can enter a self-defined vale theaccuracyforallparameters.

The

specification in the Fit Setting is adapted
based on the value.
（σ1：68.27%、σ2：95.45%、σ3：99.73%）
Error Correlation plot…

Display the data of a bootstrap analysis forthe fit parameters in graphic form

Ordinate

Select the parameters for display on the Y-axis

Abscissa

Select the parameters for display on the X-axis

Probability Level

You can enter a self-defined percentage for the confidence interval,
you can also define a multiple of the standard deviation σ.
26

FLIM Image Fit…
Configure the setting for image adaptation for the selected detection channel in this dialog

A Parameter to fit： Calculates the pixel-to-pixel image adaptation automatically.
○

FLIM:
Decay Time

Lifetime

Amplitude

Intensity

Tail offset

Background intensity

IRF Background

Background intensity when acquiring IRF (meaningful if ameasured IRF is used)

IRF Shift

Chronological offset of the IRF (meaningful if a measured IRF is
used)

FRET:
Unquenched Donor Lifetime

Lifetime of the donor fluorescence without FRET

Unquenched Donor Amplitude

Intensity of the donor fluorescence without FRET

Quenched Donor Lifetime

Lifetime of the donor fluorescence with FRET

Quenched Donor Amplitude

Intensity of the donor fluorescence with FRET

Tail Offset

Background intensity

Pattern Fit:
Tail Offset

Background intensity

Amplitude

Intensity of fluorescence

B Histogram： Distribution of pixels over the number of counts○

Threshold

You can define the threshold value the left window area by the red line.
At the same time. in the right area of the display window you can sewhich areas have been selected in the image. If you have selected
Intensity for the view, all hidden areas not included in the evaluation aredisplayed transparently in the Fast FLIM and in red in the Counts.

Pixel Binning

Configure the number of pixels complied for calculating the curve fit If
the intensity is too weak.
27

C In addition to the histogram, a preview image is displayed○

6

7①Reset the zoom for the display window②Reduces the zoom for the display window③Allows you to enter the zoom factor the display window manually and adjust it using slider④Increase the zoom for the display window⑤Sets the zoom for the display window⑥Show the number of counted photons in gray-scale value⑦Shows the distribution of lifetimes in the selected false color coding
D OK
○

28

8-④ Fit
The decay curve of the fluorescence intensities (Intensity) over time after the excitation pulse(Time)

a ：IRF（Instrument Response Function） of the excitation laser pulse○b ：Sum of the fluorescence intensities over time after excitation pulse○

You can select an area by moving the left and right border edge of the diagram using themouse
RescaleZoom in and out on the curve using themouse wheel

Show Legend

Show the legend in the diagram

Show Data Cursor

Show the value for time and intensity foreach point

Intensity logarithmic

Show the intensity value in a logarithmic orlinear viewShow number

Show the total number of the counted

Photons

photons in the selected area

Export diagram

Export the diagram data to the excel table

data…
Export diagram data

Export the diagram data to the excel table

and showand open the excel table

Copy diagram data

Store the diagram data in the clipboard

clipboard
Export ImageStore the diagram as TIFF, JPEG, PNG,
BMP, GIF
29

Histogram

Pixel Intensity

Display the Events over the photon per

HistogramLaser pulse for each pixel

Fast FLIM

Display the Events over the photon

Histogramarrival time for each pixel

Efficiency

Display the Events over the average

HistogramApparent

FRET

efficiency

of

all

molecules for each pixel

Donor-Acceptor

Display the Events over the distances

Distance

between Donor and Acceptor for each

Histogrampixel (The distances are specified in nm
and are reliable a long as the Forster
distance has been defined correctly)

Binding Histogram

Display the Event over the FRET bindingfor each pixel (The bond strength
between donor and acceptor: Proportionof the bound donor molecules to the total
number of donor molecules in %)Decay Diversity

Display the standard deviation of theMapphoton arrival times over the average
lifetime for each pixel

30

Right click on the histogram, display the diagram.

RescaleReset the scaling to the default setting

Show Legend

Show the legend in the diagram

Show Data Cursor

Display the values for time and intensity for each point on the
curve as a tool tip when hovering over them

Intensity logarithmic

Shows the events in a logarithmic view. Otherwise, it is linear.

Show number Photons

Open the Lifetime Histogram dialog, in which you can enter thenumber of data points to be displayed under Number Data point.

Export diagram data…

Export of the result data

Export diagram data

Export of the result data and show the data in an Excel table

and showCopy

diagramdata

Store the result data in the clipboard

clipboard
Export ImageStore the lifetime histogram as an image
TIFF, JPEG, PNG, BMP, GIF

31

8-⑤ Result table
Before the measurement: There are one entry for each FLIM display channel

After the measurement: One row is created for each image, each ROI and FLIM display
channel.

Right click on the table open the window

Select Columns…

Open a new dialog in which you can select which columns(parameters) are shown in the result table

Copy table content

Copies the entire table contents to the clipboard

Copy selected Rows

Copies the content of selected rows to the clipboard

Copy all

Copies the values of all parameters to the clipboard,
including columns not selected under select columns…

Export table content…

The table contents can be exported in Excel or CSV format

Export selected rows …

Selected contents can be exported in Excel or CSV format

Export all…

Entire table contents can be exported in Excel or CSV format

Export table content and

The table contents can be exported and show this in Excel

show…
Export selected rows

Selected contents can be exported and show this in Exceland show…
Export all and show…

Entire table contents can be exported and show this in Excel

Parameter Plot…

You can carry out the data analysis for multiple data records
and graphically display it for selected parameters(Ref. Next page)32

8-⑥This dialog allows you to save your analysis results or FLIM images
Save Results…

The current status of all analysis is stored, in the project directory.
The data can be called up again in LAS X FLIM/FCS and further
analyzed.

Save Image…

The colored Fast FLIM or FLIM image is created and stored as anormal intensity image in the .lif format as a 3 channel image (RGB)in the project directory and can be further processed as such. An
evaluation in LAS X FLIM/FCS is no longer possible with this format.
You can enter a designation under Name

Save Pallet Image as RGB
If this function is enabled, the colored Fast-FLIM or FLIM image is created andstored as a normal intensity image in the .lif format as a 3-channel image (RGB) in
the project directory and can be further processed as such.

Auto Range

Scales the image automatically between minimum and maximum sothat the image is displayed properly, but no values are quantified.

Fixed Range

You can select the scaling range for each display Category
separately.

Per Grey

Scaling by gray-scale value. Here, under Factor or Range, enter a

Level

factor with which the corresponding grayscale value is weighted.
The values are quantifiable.

Range

Scaling by intensity. Here, select a range under Factor or Range byentering values for minimum and maximum. The values arequantifiable.

Individual Range

Here, you can define the scaling ranges for each component
individually, just like you can for Fixed Range. The values arequantifiable.
33

8-⑦
File
New Experiment

Create a new experiment in the project
directory

Open

Open a previously saved experiment andExperiment…

load into the project directory

Import…

Open an image file in the.ptu format forimporting into the project directory

Export…

Open an image file in the .ptu format forexportingExport Raw

Open an image file in the .ptu format for

Data…

exporting

Save

Save the current project or experiment in
the .lif dormat

Save as…

Save the current project or experiment in
the .lif dormat

Close

Close the selected experiments in the
project directory

Close All

Close all opened experiments in the
project directory

Exit

Close the LAS X FLIM/FCS

34

Phasor PlotA Phasor plot is a graphic visualization of the raw data of a FLIM acquisition in a vector
space. Each pixel in a FLIM image is transformed into a point of the phaser diagram.
The position depends on the pixel’s average lifetime. This analysis is fast and returns agraphic display of the measuring curve

Zero lifetime is located at (1, 0) and infinite lifetime at (0, 0); the radius is 0.5. By changing
lifetime from zero to infinity, the phase point moves along a semicircle (universal circle)
from (1, 0) to (0, 0).

Ref: ISS, FLIM Analysis using the Phasor Plots35

𝐼 (t) = 𝐼 (0) ∑ 𝛼 𝑒 /Eq. (1)＊𝐼 (t):The number of the instantly emitted photons at the time t. 𝛼 、𝜏 :The amplitudeand fluorescence decay time of the 𝑖-th component o the mixture, respectively.
Due to the finite response of a system, the measured decay signal is a convolution form of
the intrinsic decay and IRF plus the noise n(t), as shown in Eq. (2).

𝐼(t) = 𝐼𝑅𝐹 ⊗ 𝐼 (t) + 𝑛(𝑡) = IRF ⊗ 𝐼 (0) ∑/𝛼𝑒+ 𝑛(𝑡)

Eq. (2)Each decay trace can be plotted as a single point in the Phasor plot by applying sine andcosine transforms to the measured decay data, as shown by Eq. (3).

This is equivalent

to the real and imaginary components of the Fourier transform of the decay data.

g , (ω) =

∫

, ( )

∫

(

)

s , (ω) =

, ( )

, ( )

∫

∫

(

)

, ( )

Eq. (3)

ω: The laser repetition angular frequency and calculated by multiplying the laser repetition
rate with 2 . By taking Eq. (1) into Eq. (3) and solving the integrals, we can then derive thefollowing relationships between the phasor and the lifetime, given by Eq. (4)

g=∑

𝑠=∑

Eq. (4)＊N is the number of the fluorescent species.
species of the fluorescence lifetime 𝜏

𝑓 is the fractional contribution of the 𝑖-th

to the total intensity.

For a single-lifetime species (N = 1), Eq. (4) is reduced to Eq. (5) and the lifetime can bedirectly determined by the coordinate value of a phasor.

g=

s=

τ=

Eq. (5)For a multiple-lifetime species (N > 1), Eq. (4) can be re-written by Eq. (6).

g=∑g=

(𝑓 𝑔 )s=∑(𝑓 𝑔 )

Eq. (6)s=

Ref: ISS, FLIM Analysis using the Phasor Plots36

A Selection field for the various FLIM methods. Here, you select the Phasor method.
○
B Phasor dialog for settings of the display in the display window.
○
C Display window for the FLIM image. Intensity mode is the default setting for the phasor○

plot method.
D Phasor display window.
○
E Toolbar for the phasor plot.
○
F Data display of the components for the each selected tool for the phasor plot.
○
G Display of the loaded image data records.
○
H Buttons for various save options e.g. you can save the Phasor Plot as an RGB image.
○

37

B
36-○

Harmonic

This parameter indicates the number of the harmonic wave of the Fouriertransformation that the respective lifetime is multiplied by. It corresponds
to the fundamental wave. For very short lifetime, the interesting regions in
the Phasor plot are located in the lower range at a small phase angle. By
increasing this value (to max. 9), you can shift the visualization of theinteresting regions further to the center to a greater phase angle.

ThresholdSpecifying a threshold value filters artifacts out of the Phasor plot. A valueof 5 is present as default.

No Filter

No Filter is applied.

MedianThis is a median filter that smoothes the image while accentuating andcontouring structures in the image. This enhances the visibility of details.
When the checkbox is enabled, you can adjust the filter using the slider orby entering a value. This filter is to be used for analysis purposes.
This filter is to be used, for analysis purpose.

WaveletThis advanced filter use a wavelet transform to reduce noise and preserves
intensity edges in the image. For this, it is necessary to know the noiselevel, which is determined by the detection in counting mode. This filter isused for e.g. optimizing STED acquisitions using a Phasor plot.

Preview

The filter is suitable for the live mode to prepare the experiment. If thischeck box is checked, you can use the slider or enter a value to set the
number of photon from the neighborhood over which the average is taken.

38

D Phasor Diagram36-○

a Universal Circle○
B Circle cursor with phase line (○c ). In the display window, those pixels that are located○within the cursor are displayed in color in the intensity image.
c Phase line.
○
It represents the angle of the phase shift.
D Cluster of lifetimes. They refer to the individual lifetime components.
○

Monoexponential components are located on the semicircle, multiexponential components
below the semicircle.
e Toolbar. Depending on which select, youcan adjust the appearance and perform○

analyses.
Zoom
Open a rectangle using the mouse in order to enlarge a section.
Reset
Resets the zoom to the default setting.
SelectWith this, you can select draw elements (cursor).
Draw Cursor
Draw a second cursor with associated phase line. In the start setting for thephasor method, a cursor is displayed. （Ref. p40）
Draw Ratio Cursor for two Components
With this, you can draw a connecting line between line between two
components. （Ref. p42）
Draw Ratio Cursor for three Components

39With this, you can draw a connecting line between line between threecomponents. （Ref. p43）
Draw Connected Color Coding Line
With this, you can draw a line with a color scaling the phasor plot. （Ref. p41）
Draw Connected Color Coding Lines
With this, you can draw multiple connected line with a color scaling the phasorplot. （Ref. p41）
Draw Connected Color Coding Triangle
With this, you can draw triangle with a color scaling the phasor plot. （Ref. p41）
Draw FRET TrajectoryWith this, you can draw a path for the unquenched donor relative to FRET
efficiency. （Ref. p44）
Draw Cursor
選択後、蛍光寿命の中心をクリック

a You can show and hide the cursor and the phase line foet his component using the○checkbox.
B The associated tool is displayed with its symbol.
○
c Using the color field, you can adjust the color of the cursor and phase line.
○
D By clicking X, you disable the selected tool.
○

Lifetime [ns]The lifetime of component

Radius

The size of cursor

P

If the check mark is set, this data is displayed in the phasor plot.

I

Indicates whether the image is displayed

Name

Designation of the data record in the project directory. For each image ofa data record, a separate row is created.

ChannelDesignation of the acquisition channel.
40Determining and Visualizing the Lifetime using the Phasor Method

1． Select the Phasor method (①). The Phasor diagram (②) is displayed. Two pixel
clusters can be discerned, which points to the presence of two components: a strong one(b) and a weak one (b). In the following, you define the strong components and determine
the associated lifetime.
2． Adapt the setting in the Phasor dialog (②) in such a way that the visualization of the
pixel clusters in the phasor diagram is clearly recognizable.
3． Move the cluster over the first pixel cluster (③) with the mouse button pressed down.
4． Adjust the cursor size to the pixel cluster using the mouse wheel.
5． At the top right edge of the screen (⑥), a field is displayed for the cursor where you canread off the lifetime for the defined component.
Lifetime

Lifetime of the component

RadiusSize of the Cursor

6． In order to define the second component, select the Draw Cursor tool from the toolbar(⑤). A second cursor with phase line is displayed.
7． Repeat steps 3 to 4 dor the second component.

41

Adding Color Coding in the Phasor Diagram

1． Click on the desired tool from the toolbar to select it (①).
2． Draw a line, connecting line or triangle by clicking the starting point (②), holding downmouse button, drawing the line to the next point and releasing the mouse button there (③).
For all further points, repeat the operation (③ and ④).
Depending the selected tools, the lines are plotted with a color gradient from red to blue ().
In addition, a white frame marks the range (⑥) to which the color coding is to be applied.
All pixels outside this range are displayed in white in the intensity image.
3． You can adjust the range by clicking the white line and dragging it to the desired
distance (⑥). In this window, the intensity image is displayed within the defined range
with color coding (⑦).

42Determining the Ration of Two Components in the Phasor Diagram

1． Select the Draw Ration Cursor for two Components tool by clicking it on the toolbar(①).
2． In the Phasor plot, click the center of the area that represents 2 components (②, ③).
3． Finish the graphic by clicking. The connecting line is drawn using the cursor (④)
4． Hold the cursor, hold the mouse button pressed and move the cursor back and forthalong the connecting line and adjust its size (⑤). This component contains two lifetimes
and has its position below the universal circle.
5． In the respective position, the associated data is displayed in the top right area (⑥).
Ratio

Ratio of the two components (②, ③)

Lifetime 1, Lifetime 2

Lifetime of the components (②, ③)

RadiusSize of the cursor (⑤)

43Determining the Ration of Three Components in the Phasor Diagram

1． Select the Draw Ratio Cursor for three Components tool by clicking it on the toolbar(①)
2． In the Phasor plot, click the center of the area that represents 2 components (②, ③,④).
3． Finish the graphic by clicking. The connecting line is drawn using the cursor (⑤).
4． Hold the cursor, hold the mouse button pressed and move the cursor back and forthalong the connecting line and adjust its size (⑥). This component contains two lifetimes
and has its position below the universal circle.
5． In the respective position, the associated data is displayed in the top right area (⑦).
Ratio

Ratio of the three components (②, ③, ④). Total = 1

Lifetime 1, Lifetime 2, Lifetime 3

Lifetime of the components (②, ③, ④)

RadiusSize of the cursor (⑥)

44Drawing a FRET Trajectory in the Phasor Diagram

1． Select the Draw FRET Trajectory tool by clicking it on the toolbar (①).
2． In the Phasor plot, click the center of the region that corresponds to autofluorescence(②)
3． Then, press and hold the mouse button to draw a line the component that corresponds
to the unquenched donor (③)
4． Finalize the graphic by double-clicking. The FRET Trajectory is draw together with this(④).
5． A Cursor for the analysis of the quenched donor (FRET) is displayed. Click the cursor
and move It back and forth along the trajectory with the mouse button pressed (⑤).
6． In the respective position, the associated data is displayed in the top right area (⑥).
Unquenched Lifetime of the unquenched donor

Quenched

Lifetime of the quenched donor (FRET)

Background

Ratio of the background noise

FRET Efficiency

FLIM-FRET efficiency.

45

Images from Phasor Method
画像上で右クリックし、Export Raw Image を選択すると、下記画像が表示されます。

Save Phasor Mask

Save as an image of the Phasor mask that can be used
for further analysis and calculations. The Phasor
mask describe the region of the Phasor plot that
represented in the Fast FLIM image.

Apply Phasor Mask to Image

Applies the Phasor mask to the images. Thisparticularly suitable for STED images where all longer
lifetimes with the excitation ring, are truncated.

Save Phasor GS

Save the values for the g and s coordinates of the
Phasor plot (2 images). The X-axis of the Phasor plot
represents the real component (g) and the Y-axis
represents the imaginary components (s).

46

H
Save Image(Ref. page 33)
Save Result
The current status of all analysis result is stored, including all settings, in the project
directory. The data can be called up again in LAS X and further analyzed.

Save Phasor
You can save the Phasor plot as an RGB image in the project directory. Select Split (Ref.
page 10), then Z Stack or Time series data are saved as video in the format.

47

STED: What Is This ?
The STED method exploits the difference in the lifetimes by applying the component
separation from the Phasor plot to STED images. In STED images, shorter lifetimes aremostly located in the range of the de-excitation donut, whereas the longer lifetimes primarily
occur in the central excitation range. All longer lifetimes that are still present in the range ofthe de-excitation ring (donut) and the image background are filtered out, which increases the
lifetime contrast between the central excitation range and de-excitation ring. This allows for
improving the S/N and the resolution.

48

STED: The User Interface

A ：Selection field for the various STED-visualization method. Here, you select the STED○

method.
B ：Display window for the FLIM image.
○An intensity image is displayed for the STED

method.
C ：Setting range for the various STED method. The STED is default. Time Gate serves○

as a comparison with the conventional methods.
D ：The lifetime Decay Curve of the FLIM image is displayed here.
○
E ：Buttons for various save options, Save Image/Result○49Display Methods: Time gated and STED
The various methods are described for the range FLIM to STED with their adjustment
options and results. You can adjust phasor mask itself under Manual.
The following figure shows the result of the three methods side-by-side. The highest
resolution can be achieved with the STED.

Multi-channel Acquisitions
STED is also possible for multi-channel acquisitions. A button for channel selection isdisplayed for any channel. It is also possible for images without STED to be in one
channel and images with STED to be in another channel.





50

STED: Automatically Optimizing STED Analyses Using the FLIM Phasor Method

Select the STED①, then the STED method② has already been preselected. The lifetime
decay curve of the fast FLIM image is displayed in the area on the right③.

Adjust thesettings ④.

Background

Removes the background using its lifetime properties.

Suppression
Strength

Set the strength of the suppression of the pixels located in the range ofthe de-excitation ring(donut) and in the background. If the value is set
too high, then pixels from the central range will also be suppressed. A
value of 100 is preset.

Denoise

Reduces the noise. A value of 50 is preset.

Time Gate

Define the beginning and end of time gates for the detection here. You
can add/remove time gates by clicking the +/-. For the set time gate, aslider is displayed under the lifetime decay curve and can also be used
for setting the time gate.

Save the Image by clicking the Save Image button⑥.
51

Time gated
You can set a time gate for the detection here. You can improve the resolution by doing this,but with a lower signal strength.

Time Gate ： You can define the beginning and end of the time gate here.

You canadd/remove time gate by clicking the +/-. sing. For the set time gate, a slider is displayed
under curve and can also be used setting the time gate.
Save the Image by clicking the Save Image button.

52

Manual
You have the option here of manually configuring the Phasor mask.

Select the ①STED and ②Manual method. The area on the right shows the Phasor Plotof FLIM image. The line corresponds to the basis for calculating STED.
Configure the desired settings③.
Add black and white color coding to the FLIM image to show the intensities. To do so,select one of the tools from the toolbar④. A black and white LUT is stored for the tools.
Draw a connected line into the desired are of lifetime component⑤.
As the beginning point (Black), select a point with short lifetimes in the border area of the
Phasor (a). Since longer lifetimes also occur in the background, the Phasor shows
curvature to the left. Therefore, set your beginning point in the background area. Thendraw a line along the phasor and, as the end point (White), select a point with long lifetimes
in the border area of the Phasor(b).
The display window will show the STED as an intensity image in grayscale, in other words,
the longer lifetime, the brighter the corresponding pixels⑥.
Save the Image by clicking the Save Image button⑥.

53