From Eye to Insight

FALCONFCSManual20200730_EN手順
1.

Starting the FCS Wizard

2

page

2.

Adjusting the Corr-ring

3

page

3.

Calibrate Beam Parking

5

page

4.

Setting the reference position

8

page

5.

Measurement

11

page

6.

Save FCS data

16

page

7.

Calibration

20

page

8.

FLCS

26

page

Tips
＊The calibration is a part of the optimization of the FCS measurement conditions.
The calibration routine uses a solution with known parameters which must be inserted in thestand before each calibration.
＊PMT、Standard HyD are not available for FCS＊FCS is available for 0.5 to 100 nM＊Due to the STED-FCS reduce the measurement volume, higher concentration sample isavailable＊ 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

1

FCS Wizard①Click the TCS SP8 in the main menu bar②Select the FCS option

The structure of the FCS wizard is described below.

Overview

The procedure for the steps to be carried out is displayed here

Setup Corr-ring

An optimum adjustment of the correction ring correlates with the best
possible optical resolution of the systems (A basic prerequisite for FCS)
（Ref. page 3）

Setup Imaging

Establish and optimize the parameters for image acquisition.
（Ref. page 4）

Setup FCSDefine and optimize the conditions for the FCS measurement.
（Ref. page 7）

Measurements

Define multiple FCS measuring points in the previous acquired XY, XZ,
XYZ or XZY images (Measurement condition is defined in the Setup
FCS step.) （Ref. page 10）2

Setup Corr-ring
you must always make this adjustment when you change the specimen carrier, even if itcomes from the same batch.
Scan modeXZ-y（Z-Galvo）

Format512x512Zoom

1

Lazer line

488 nmAOBS

Reflection

Detector

PMT＊Super Z Galvano stage
Move the focus using the z-drive of the microscope stand until you see the 2 reflection linesthat appear between the immersion and the glass and the between the glass and thespecimen.
Immersion（water）

Cover slip

MediumFocus on the below. Use a higher zoom （e.g. 4 to 8）
Rotate the correction ring on the objective into a position that provides the signal with thehighest contrast in the reflection between cover slip and medium
Left image: Reflection with poorly set position of the correction ring. The image is out offocus, several secondary peaks are visible
Right: Reflection with correctly set position of the correction ring: The image is sharp, andsecondary peaks are scarcely visible.
＊Without Super Z Galvano: Rotate thecorrection ring on the objective into a position
that provides the signal with highest contrast in
XY image. The Z position move accompany
with the correction ring rotation.
the z position.

3

Then, keep

Move the focus about 10 – 20 m from the center of the image toward to the specimen

The tool on the top edge of the display window enable to show ruler like below image.

4

Calibrate Beam Parking
Before starting an FCS experiment, you must calibrate beam parking in order to ensure thepositioning accuracy of the FCS measuring point. Repeat this procedure about every 3 days.
Use a bleach specimen with fixed fluorescence that is capable of being excited with awavelength in the visible range and can also be photobleached with higher laser intensity.
（e.g. Leica standard sample, Convallaria）

1.

Select the ②Setup Imaging

2.

Focus on the bleach specimen（Press Ctrl + T to show crosshair）
5

3.

In the ⑪Acquisition tab, set the ⑥xyz mode, ⑦scan speed 600 Hz, Bi-directional

4.

Select ⑧laser line、suitable ⑨detector and the detection range

5.

Select Glow(O and U) in the color look up table (LUT)

6.

Switch to the ⑪Workflow tab

7.

Click the ⑫Calibration button, open the upper left dialog. The system automaticallydetermines the following values: Zoom 32 and pixel format 1024x1024.
8.

Activate live mode by clicking the Live button. Configure the gain and offset

9.

At ⑬Define bleach time, you can enter the bleaching duration: the default value is 100

msec.

For thin specimens easy to bleach, a bleaching duration of 500 msec or less isrecommended.
10. Click the ⑭Calibrate button. An image will be acquired automatically. Following thisthe previously selected laser lines are automatically set to 100% AOTF. Following this, one
more image is acquired with the previously configured settings.
11. Now, position the crosshair in the center of the bleach point ⑮
12.

Click the ⑯Set Calibration

The systems saves the calibration data and display it

under X and Y

613. Acquire a reference image
Setup FCS
(Ref. page 19)
Click Setup FCS to open the FCS window
Ref. page 17

7

①Select the detector②Setting the lasers③you can check which notch filter is currently in the beam path（Automatically introduces abarrier filter into the beam path）⑦Prepare the FCS measurement by configuring all required setting under Acquire – Beam
Path Settings.
Set the diameter of the detection pinhole using Pinhole, Airy 1.

8

If you would like to acquire an excitation spectral series at a defined FCS point.

ΛIts purpose is to determine the excitation spectra of fluorochromes. The
wavelength of the excitation laser continuously changes and documents theresulting emission intensity
If you would like to acquire an emission spectral series at a defined FCS point.

λIts purpose is to determine the emission spectra of fluorochromes. The
wavelength of the excitation laser continuously changes and documents theresulting emission intensity
ΛλIf you would like to acquire an excitation/emission spectral series at a defined
FCS point. Its purpose is to determine the excitation spectra of fluorochromes.
The wavelength of the excitation/emission laser continuously changes anddocuments the resulting emission intensity⑥Correlation Assignment： In this process, the first 4 FCS detectors that have been activatedin the beam path setting are each assigned an FCS correlation channel. Thus, correlation
curve is generated for each correlation channel.
FCS（Auto-Correlation）①The dropdown menu represents an FCS correlation channel.
②When click on a dropdown menu, anew dialog opens in which you able to assign anindividual detector or ③multiple detectors simultaneously.
④All activated FCS detectors are assigned to the correlation channel by means of the All
Detectors button
FCCS（Cross-Correlation）
In this dialog, 2 auto correlation channels are selected to calculate the cross correlation
between both.

91. Define an FCS measurement point.
display window.

A crosshair appears in the

Use the mouse drag the crosshair to the position

where you wish to carry out the measurement and click on Add Pointat every point.
2. Click on Run FCS Test to start the measurement.
The measurement results are displayed and evaluated on the submonitor.
After configuring the setting for the FCS measurement, switch to the
Measurement operating step.
Tips- When FCS measurements are taken in solution, the measuring point must always be
defined with the aid of a reference point. The reference that is generated at between coverslip and medium. move the focus on the reflection about 10 to 20 m inwards towards themedium.
・Adjust to the count rate between 10 to 500 kcps (count per second)・Higher cpm value give S/N of Correlation curve
・To avoid the effect of the photobleach, reduce the laser power to 2/3 of maximum cpm
Then, still you can observe the effect of the bleach or triplet, more reduce the laser power
Count per second: The number of photon detected per second is displayed
Count per molecule: The number of photon detected per molecule is displayed

10Measurements
Define the measurement point（Ref. page 16）①Define the FCS measurement point111. Define the FCS measurement point. Drag the crosshair to the measurement positiona Click Add, then the position was shown like ○b
2. ○（Max 200 points）
Also you can define the position of XYZ.
3 . You can save/load defined positions from ○e Save List or ○f Load List.
Remove: Remove current point, Remove All: Remove all defined point
4. You can move the focus point form ○d Move to.
②Time series
Number of Cycles

How many measurement run will be carry out
within an FCS experiments.

Cycle Interval

The time interval for a measurement run.

Minimize

Automatically use smallest possible time interval

Image Before FCS Measurement

An image acquisition is to take place before theactual FCS measurement

Image After Each Repetitions

Acquire image after a certain number of repetitions

Image After Each points

Acquire image after certain number of points

Image After Each Cycles

Acquire image after certain number of runs

Total Number of Measurements

Total number is calculated from the number ofdefined measuring point time series and repeats③Definition of point measurement
Pre-Bleach

You can first photobleaching the specimen at ameasuring point before beginning the FCS
measurement. Pre-bleaching serves to reduce
photobleaching effects during the FCS
measurement

Measurement Duration

Specify the duration of the FCS measurement
1 sec to 10 min

Measurement Until

Specify the number of the photons that must be
detected during the FCS at a particular point

Repetition at Point

How often the FCS measurement will be repeated

Repetition Interval

The time interval for a measurement run.

Minimize

Automatically use smallest possible time interval

Number of Defined Points

Define the measurement points for FCS in the ①

Number of λ-Steps

The number of the steps for spectral series

Base nameEnter the name which the FCS data

12

Run

The Run suffix is automatically appended to thename. Default value is 1.

Comment

The comment add to the module LAS X FLIM/FCS

Cycle
Repetition

Repetition
Point

Acquire
Image

13

Point

Graphical user interface for the evaluation of FCS experiments

13-①Setting for correlation
Clicking the Correlation, open the dialog
Spark Filter
In this dialog, you can remove regions from the intensity
distribution diagram in order to exclude them from thecorrelation calculation. When check box is enabled, bars are
displayed in the diagram, which can be manually dragged withtho mouse to the ares you want to remove. You can change
the width of the bar and both add new bars or remove themfrom the contest menu.

Right click on the diagram, open the contest menu (Next page).

14Add Spark Filter Region

Inserts filters in order to remove regions from the intensity
duration diagram and exclude them from the correlation
calculation

Remove Spark Filter

Delete a previously selected filter

RegionRemove all Spark Filter

Delete all existing filters

Binning Period

Open the Contrate Binning Period dialog where you can set the
support points for the photon counting
In this dialog, you can configure the support positions forphoton counting
Bleaching Correction
Photon bleaching occurs with many fluorochromes and isobserved as a fluorescence decay curve over time.

This decay is also monitored in the case of autocorrelation. You can use this dialog theCorrect Bleaching checkbox, the software detects the region where a fluorescence decay
takes place and corrects it.
Lifetime Filter, Time Gate Filter: Ref. page 25-27
Correlation Assignment： In this process, the first 4 FCS detectors that have been activatedin the beam path setting are each assigned an FCS correlation channel. Thus, correlation
curve is generated for each correlation channel.
FCS（Auto-Correlation）①The dropdown menu represents an FCS correlation channel.
②When click on a dropdown menu, anew dialog opens in which you able to assign anindividual detector or ③multiple detectors simultaneously.
④All activated FCS detectors are assigned to the correlation channel by means of the All
Detectors button
FCCS（Cross-Correlation）
In this dialog, 2 auto correlation channels are selected to calculate the cross correlation
between both.

15

Normalization ：In this dialog, you can enable the normalizationof the correlation function

Off

No standardization of the correction

Normalize at G0

The corrective function is standardized to the value G（tau）=0

Normalize at G（time）

The corrective function is standardized to the value G（tau）=1 s The default value 1 s can be changed in
the Time field

FLIM/FRET/Pattern Fit（Ref. FLIM manual）

Time Gate（Ref. FLIM manual）

Special Settings（Ref. FLIM manual）

Save Results

The current status of all analysis results is stored,
including all settings.

Save Image (Only for FLCS)
1: Higher level directory in the which all data is stored.
2: Frame or image series used as a reference for setting the FCS
measuring point
3: With each click on the Run FCS, a new FCS measurement anda new folder are generated.

The BaseName_Run number

specified in the Run suffix automatically increases by incrementsof 1.
T： Trace
P： Point
R： Run16

13-②After the FCS measurement, you can switch between the following displays
Image

Display the reference frame with the defined FCS measuring points

CountsRepresentation of the counted photons in the FCS volumes over time

13-③Curve fitting setting

FCS Calibration

Open a dialog for the calibration of the FCS calibration volume（Ref. page 16）

Load Calibration

If the results of the current calibration in the table have been
overwritten, you can reload them by clicking this button

Select Fit Model

Open a dialog in which you can select a calculation model forthe curve fitting

Pure Diffusion

Only diffusion contributions are present (no triplet states)

Diffusion with Triplet

There is possibility that molecules from the observation volumetransition to non-fluorescent status (dark states, triplet states)

Triplet Extended 3D

There is possibility that movement restrictions in the 3Dvolume or obstructed diffusion arise

Triplet Extended 2D

There is possibility that movement restrictions arise in amembrane. For STED-FCS

Protonation

If protonation reactions are to be expected

Conformational

For conformational changeTime Range

This specifies the time gate in which the curve fitting is to take
place

17

Some parameters ca either be set to a user-defined value(Value) or adjusted (Fit) or globally(Global) linked. Clicking under Global in the field … of the respective parameter open thefollowing context menu in which you can select for which parameters thesame values are to be used.

Diffusion Components

Enter the number of diffusion components

Fit

Clicking this button calculates the curve fitting listed above forthe curve selected in the navigation table

Fit All

Clicking this button calculates the curve fitting listed above forall curves of the navigation table

You can click the right mouse button to open these context menus that provides functions.

Copy to Clipboard

You can paste the content (parameters and values) into the Excel
table

ExportYou can export the parameter table

Export and show

You can export the parameter table and open the Excel table

LimitsOpen the limit dialog, in which you can configure upper and lower
limits for the respective parameters

Probability Level…

Open the Probability Level dialog, in which you can define the rules
for specifying the accuracy of the respective parameter after the fit（σ1：68.27%、σ2：95.45%、σ3：99.73%）

18

Parameter Plot

Error Correlation plot…

Display the data of a bootstrap analysis for the fit parametersin 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 （σ1：68.27%、σ2：95.45%、σ3：99.73%）

19

FCS Calibration
The calibration is a part of the optimization of the FCS measurement conditions.
The calibration routine uses a solution with known parameters which must be inserted in thestand before each calibration.
(Ref. page 13, FCS Calibration)①The test measurement correlation curve is displayed here

RescaleZoom in and out on the curve using the mouse wheel

Show Legend

Show the legend in the diagram

Show Data Cursor

Show the value for time and intensity for each point

Show Data Cursor

Show the value for time and intensity for each point

Export diagram data…

Export the diagram data to the excel table

Export diagram data and show

Export the diagram data to the excel table and open the
excel table

Copy diagram data clipboard

Store the diagram data in the clipboard

Export ImageStore the diagram as TIFF, JPEG, PNG, BMP, GIF
20

②You can enter the known constant for the calibration here. You can choose between
Diffusion Coefficient, Concentration or Diffusion Coefficient and Concentration③Setting parameters for the calibration
Select Fit Model

Open a dialog in which you can select a calculation model forthe curve fitting

Pure Diffusion

Only diffusion contributions are present (no triplet states)

Diffusion with Triplet

There is possibility that molecules from the observation volumetransition to non-fluorescent status (dark states, triplet states)

Time Range

This specifies the time gate in which the curve fitting is to takeplace

Diffusion Amplitude

Intensity

Diffusion time

The time molecule spend within the observation volume

Triplet TimeTime molecules spend in dark state

Structural Parameterz/w ratio of observation volume

Volume (Concentration)

Calculated volume based on the Concentration

Volume (Diffusion coeff.)

Calculated volume based on the Diffusion coefficient

Average Focal Volume

Average volume of Volume (Concentration) and Volume(Diffusion coefficient)χ2

Fit error or the fit precision for pixel

Fit

By clicking Fit, the calibration with the above listed setting iscompleted④Result of the calibration
Structural Parameter

Ratio of the axial and radial expansion of the focal volume

Effective Volume

Calculated effective observation volume, measured in（fl、10-15 l）

Apply toYou select which channel you would like to assign thecalibration value to.
The results of the calibration are displayed in the parameter
table under Fit Setting
The calibration values are applied for each following
measurement.

21

PicoQuant
Absolute Diffusion Coefficients: Compliation of Reference Data for FCS Calibration22

13-④Representation of the auto-correlation curve or cross-correlation curve in real curve inreal time during FCS tests and the FCS measurement
13-⑤Result table
After the measurement, one row is created for each channel

Right click on the table open the dialog

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, includingcolumns 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 and

The table contents can be exported and show this in Excel

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. page 23)23

13-⑥ For information on how to save the analysis results
13-⑦ Menu for general setting
New Project

Create a new project/experiment

Open ProjectOpen a previously saved experiment in
the .lif, .lei or tif format

BrowseOpen the project browser

Close

Close the selected experiments in the
project directory

Print

Open a printer dialog setting

Print Preview

Show a preview of the printout

Exit

Close the LAS X FLIM/FCS

24

Lifetime Filter： FLCS can separate contribution from background, scattered light.
*For FLCS, the recommended frequency is 40 MHz

No Filter

No filter is applied

Background Removal

Applied a filter that excludes background contribution from thecorrelation calculation

Separation

This option can be used to remedy crosstalk for FCS data records
with a mixture of multiple fluorochromes. In this dialog, you cancreate 2 different photon filters in order to split and correlate theraw data in various channels.
Create Lifetime Filter
Pattern matching25

Create Lifetime Filter
You can create a filter, that based on the fluorescent lifetime of photons, achieves separationin the individual fluorescent components. Filter can be selected for FCS and FCCS.

1. ①Select the channel whose fluorochrome you would like toremove under Channel
2. ②Click Select Fit Model
3. ③Under Time Range, select the time gate around the point
in time of the laser pulse in which the curve is to take place
4.

④ Under Exponential Components, set the number offluorescent lifetime components
5. ⑤Apply the preset parameters. (Ref. page 16)
6. ⑥Click Fit to fit the decay curve. The calculation is carrieda .
out and the result are shown in Fit diagram ○7. ⑦Repeat this step. The software calculation the suitable filters as a result and displayb .
them graphically in the dialog field ○8. Click OK. The dialog window is close26

Create Pattern Matching Filter
To be able to separate the fluorochromes in the FCCS, you can create filters in this dialog
that are based on acquired reference data records with pure fluorochromes.

1. ①Select the channel whose fluorochrome you would liketo remove under channel
2. ②In the patterns dialog, click the Append in order to load
the reference data with a pure fluorochrome (Pattern)
3. The Select Pattern dialog opens
4. The reference data record is displayed in the Patternsadialog and its curve on the right side ○5. ③Click Fit to fit the reference data record curve.

The

calculation is carried out the results are shown in the Fit dialog
as a diagram.
6. Repeat this step accordingly for the suitable filters as ac .
result and display them graphically in the lower dialog field ○7. Click OK. The dialog window is closed

27

Principal of FCS, FCCS, pCF
FCS(A-C), FCCS(D, E), pCF(F, G)

Effect of the molecular size
The larger molecule, theslower diffusion
Effect of the concentration
The higher concentration, thelower correlation

Effect of mixture

Cross-correlated

Non Coross-correlated

1 -> 2 faster than 2 -> 1

Ref: R. Macháň, T. Wohland / FEBS Letters 588 (2014) 3571–3584

28

Statistical filter function

Ref: PicoQuant Application Note, FLCS – Fluorescence Lifetime Correlation Spectroscopy

29

Diffusion coefficient D given byD=𝑅Τ
6𝜋𝜂𝑟𝑁𝐴

Universal gas constant R, the temperature , the particle radius r, viscosity , Avogadro
constant NA30