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FALCON Manual

The document provides instructions for using a Leica SP8 FALCON FLIM detector. It describes setup requirements, measurement procedures, fitting methods, and analysis tools for fluorescence lifetime imaging microscopy (FLIM).

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54 views13 pages

FALCON Manual

The document provides instructions for using a Leica SP8 FALCON FLIM detector. It describes setup requirements, measurement procedures, fitting methods, and analysis tools for fluorescence lifetime imaging microscopy (FLIM).

Uploaded by

mangia_11
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Leica SP8 FALCON FLIM detector manual 04.11.

2020

FLIM is only possible using HyD detectors (HyD SMD1 is best) and the pulsed
white light laser, which is emitting from 470 nm to 670 nm.

No FLIM with 405 diode laser possible.

Before you start:

• turn the gated detection at the HyD OFF

• pulse picker can change the frequency of the laser pulses

• for normal fluorophores with lifetimes between 1 and 5 ns we


recommend to use 80 MHz, the laser pulses come every 12.5 ns

• to measure longer lifetimes you may want to change to a lower


frequency, see table:

• for FLIM the detector is always in photon counting mode-no


overexposure possible

• detection for intensity image might also be set to photon counting mode

• make sure in Configuration - Hardware - data transfer mode - Maximum


Integration Time is turned off," Activate X-linearisation" needs to be
unticked in order to do that, please reactivate afterwards
• set detector resolution to 12 bit

FLIM Measurement

• activate FLIM in Aquisition mode


• in FLIM settings choose one of the supported notch filter laser line to
avoid reflection

• activate HyD SMD 1 detector and set detection range to your


fluorophore
• FLIM measurements for 2 channels must be set up sequencially, other
HyD detectors may be used
• press "Live" you will get an instant live FLIM Image in the LasX-FLIM
display
• in the LasX -FLIM display check the Pixel Intensity histogram
• reduce laser power, so that you get less than 1 photon per laser pulse
• capture one image
• hover over the displayed Fast - FLIM Image, with a right mouse click
select "show data curser"
• collected photons per pixel will be displayed in your Fast - FLIM image
• select a meaningful life time range to display (e.g. 0 -5 ns)
• to do a proper 1 component exponential fit about 100 photons/ image
need to be collected
• to do a 2 component fit with the same statistical certainty about 10000
(factor 100 for each component) photons need to be collected
• for 3 component fit about 1 million photons need to be collected for
meaningful statistical data, which is practically not possible (2 channel
FLIM may be considered)
• to collect more photos per pixel, increase line or frame repetitions (10 to
12 are recommended) in FLIM settings (Fig.2) scan speed may be
reduced
• capture an image with the new settings
• Fitting Range: This specifies the time gate around the time point of the
laser pulse in which the curve fitting is to take place.
• Exponential Components: Here, enter the number of fluorescent
lifetime components in the specimen whose lifetime decay was
measured.
• select fit-model and number of exponential components press "fit”
• n-Exponential Reconvolution with IRF In this calculation model, the
decay curve is fitted taking into account the IRF (Instrument Response
Function). This also enables using the beginning of the decay curve for
analysis. This improves the statistics of the data and thus enables a
correct estimate of the relative amplitudes from the sum of the
exponential decay curves.
• n-Exponential Tail Fit In this model, only the rear area that is not
affected by the IRF is used for the analysis. This attains correct results for
the lifetimes, as long as the lifetimes are significantly greater than the
IRF width. The disadvantage of this model is that no good curve fitting
can be calculated for components with short curve fitting.
• try one or more exponential component fit (most dyes show more than
one component decay, exceptions are YFP, CFP, GFPnow)
• the fitted curve should match the measured curve
• the residues should not follow the measured curve
• the Chi square value should be low

• check that you get a reasonable number of counts for the intensity of
each lifetime
• press “FLIM image fit”, exclude background pixels
• press “precise fit” to get 2 or more images that display images with
different life time components
• intensity images are saved automatically in projects
• FLIM images and result table have to be saved manually click on "save
image"or "save result"in the FLIM environment
• for the fast FLIM image right mouse click, then export raw image. We
recommend to save 1 intensity count per grey level and 0.001 ns per
grey level
Using Phasor Plot for FLIM Analysis

Pixel classification and FLIM analysis can be done as well using Phasor plot
(Without fitting).

 Calibration: Opens a dialog in which you can perform a manual phasor calibration,
see Phasor Calibration. This is recommended for high-precision evaluations or
samples with SHG signals, since auto-calibration is not very precise here. In all other
cases, auto-calibration, which by default runs in the background, provides results of
very high accuracy.

 Harmonic: This parameter indicates the number of the harmonic wave of the Fourier
transformation that the respective lifetime is multiplied by. It corresponds to the
fundamental wave. For very short lifetimes, the regions of interest 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 the regions of interest further to the center
to a greater phase angle.

 Threshold: Specifying a threshold value filters artifacts out of the phasor


plot. A value of 5 is preset as default.

 No Filter: No filter is applied.

 Median: This is a median filter that smoothes the image while


accentuating and contouring structures in the image1. This enhances the
visibility of details. When the checkbox is enabled, you can adjust the
filter using the slider or by entering a value. This filter is to be used for
analysis purposes.

 Wavelet: This advanced filter uses a wavelet transform to reduce noise


and preserves intensity edges in the image. For this, it is necessary to
know the noise level, which is determined by the detection in Counting
Mode; refer also to Image Processing Pre-Filter.2 This filter is used, for
example, for optimizing STED acquisitions using a phasor plot.

 Preview: This filter creates different gaussian filters of different widths in


the background, which improves the signal-to-noise ratio even in live
mode. The filter is suitable for the live mode to prepare the experiment.
If this check box is checked, you can use the slider or enter a value to set
the number of photons from the neighborhood over which the average
is taken.

Data Display of the Components

The data of the components for each selected tool is displayed in the upper
right screen area. As an example, data display for three tools is described in
the following. You can find further details in the corresponding instructions.

At the start, the Select tool is selected:


General functions for all tools:

a: You can show and hide the cursor and the phase line for this 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.

Show Image Overlay: If this function is enabled, all components are displayed
superimposed in colors in the fast FLIM image. If the function is disabled, the
fast FLIM image is hidden, and only the pixels of the components remain
visible.

Special data:

 Lifetime: Lifetime of the component

 Radius: Size of Cursor

Data display for the Draw Ratio Cursor for three Components tool:

 Ratio 1, Ratio 2, Ratio 3, etc.: ratio of component 1, 2 and 3.

 Lifetime 1, Lifetime 2, Lifetime 3, etc.: representative lifetime of


component 1, 2 and 3.

Data Display for the Draw FRET Trajectory Tool:


 Background: Ratio of the background noise

 FRET Efficiency: FRET efficiency, see FLIM-FRET. This is calculated only


from the donor molecules in which FRET actually occurs.

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

You can enter a designation under Name.

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