Time-Resolved Fluorescence Wiki

calculate_ratiometric_fret-images - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-21T03:39:02+00:00

2014/02/03 13:54		current
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-	====== Calculate_ratiometric_FRET_Images_with_the_FRET-Image_Script_Step_by_step_v1.3 ======
-	<sub>{{Calculate_ratiometric_FRET_Images_with_the_FRET-Image_Script_Step_by_step_v1.3.odt\|Original file}}</sub>
-
-	{{calculate_ratiometric_fret-images_Image_0.png?128}}Tutorial
-
-	Calculate Ratiometric FRET-Images with the FRET-Image-Script
-
-	Summary
-
-	This tutorial shows step-by-step, how the "FRET Image" script of SymPhoTime 64 can be used to calculate pixel-by-pixel the average FRET efficiency in an image containing several cells transfected with Cerulean/YFP constructs. To calculate the average FRET efficiency, a ratiometric approach based on the intensities in the donor and the acceptor channel is used.
-
-	{{?482}}
-
-	Step-by-Step Tutorial
-
-	- Start SymPhoTime 64 software.
-	- Open the "Samples" workspace via "File\open Workspace" from the main menu.
-	{{?482}}
-
-	- //Response:// The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-	{{calculate_ratiometric_fret-images_Image_1.png?185}}
-
-	* Highlight the file "CENP-labelled _cells_for_FRET.ptu" by a single mouse click.
-	{{calculate_ratiometric_fret-images_Image_2.png?184}}
-
-	- Select the "Analysis" tab and in there, open the drop down menu "Imaging".
-	{{calculate_ratiometric_fret-images_Image_3.png?348}}
-
-	{{?482}}
-
-	- Start the "FRET Image" script by clicking on "Start".
-	{{calculate_ratiometric_fret-images_Image_4.png?123}}
-
-	* //Response:// The FRET image script is applied to the file "CENP-labelled _cells_for_FRET.ptu". Thereby, a new Window opens:
-	{{calculate_ratiometric_fret-images_Image_5.png?642}}
-
-	{{?482}}
-
-	* Set the pixel "Binning" to 2 points.
-	* As data channels, check only Channel 1 and 2, as in these channels the relevant photons are recorded.
-	{{calculate_ratiometric_fret-images_Image_6.png?271}}
-
-	* Press "Calculate".
-	* //Response://
-	* Automatically, the two active detector channels are assigned to the donor and acceptor channel. By default, detector channel 2 is considered the donor channel and detector channel 1 is assigned as acceptor channel.
-	{{?482}}
-
-	* A false color image is calculated, in which the intensity of detector 1 and 2 are plotted in green and red, respectively.
-	{{calculate_ratiometric_fret-images_Image_7.png?475}}
-
-	* Invert the color assignment to display the acceptor channel in red and the donor channel in green. Therefore, click on the bar next to the image to open the display options and invert the channel selection.
-	{{calculate_ratiometric_fret-images_Image_8.png?303}}
-
-	{{calculate_ratiometric_fret-images_Image_9.png?304}}
-
-	* //Response://The image is adapted, the acceptor is now plotted in red.
-	{{?482}}
-
-	- Set the Min. Photons per FRET event to "700" Cnts (counts).
-	- Click on "more" to open the additional options for entering the correction parameters.
-	{{calculate_ratiometric_fret-images_Image_10.png?212}}
-
-	{{calculate_ratiometric_fret-images_Image_11.png?244}}
-
-	* As parameters, enter "70" for "Gamma" and "7" for "Cross Talk".
-	{{?482}}
-
-	* Click "Calculate FRET".
-	{{calculate_ratiometric_fret-images_Image_12.png?276}}
-
-	* //Response://
-	* The FRET image is calculated, and the corresponding histograms are calculated. In the FRET image the FRET efficiency is only calculated for pixels with intensities higher than the threshold.
-	* The FRET efficiency color scale is automatically set from 0 to 100% FRET efficiency.
-	* The FRET efficiency histogram and the Distances histogram are shown.
-	{{calculate_ratiometric_fret-images_Image_13.png?642}}
-
-	* It can be seen that a few fluorescent dots on the lower right are over the set intensity threshold. As these signals are not in an area of interest, place the mouse cursor over the FRET image, activate the context menu with a right mouse click and select the "Rectangle ROI" tool.
-
-
-	{{calculate_ratiometric_fret-images_Image_14.png?301}}
-
-	- Now it is possible to remove the unwanted area by constantly pressing the "CRTL" button and the left mouse button, draw a rectangle over these areas and release mouse and keyboard. Do this repetitively until all unwanted signals are removed.
-	{{calculate_ratiometric_fret-images_Image_15.png?642}}
-
-	- Press "Calculate FRET" again to update the histograms.
-	- //Response://The FRET efficiency and distance histograms are updated. This can be seen clearly at the small peak around 20% FRET efficiency which disappears now.
-	{{calculate_ratiometric_fret-images_Image_16.png?642}}
-
-	- Store the result by clicking on "Save Result.
-	{{calculate_ratiometric_fret-images_Image_17.png?302}}
-
-	- //Response:// A result file is generated and linked to the raw data file.
-	{{calculate_ratiometric_fret-images_Image_18.png?150}}
-
-	- The image analysis is now finished. There are several possibilities to continue:
-	- To adapt the histogram plots, change the plot parameters on the right of the histogram to the desired values and click "Recalculate". To save these plots, click "Save Results" again.
-	- To export certain analyses, place the mouse cursor over the area of interest, activate the context menu with a right mouse click and choose the export function of interest to export the data.
-	- In case one wants to analyze many images with the same FRET pair, it is possible to save the starting values for the fits by clicking on "Save Defaults" and store the starting values as a User Profile by entering the main menu ("Settings\Save User Profile as"). The software will need to restart to adapt the changes.
-	- If you don't want to do anything more, just close the result window.
-

check_overlap_of_different_color_confocal_volumes - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-19T11:36:26+00:00

2013/08/06 16:51		current
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-	====== Check Overlap of different color confocal volumes ======

-	Because both lasers are fed through the same fiber, there cannot be any optical beam displacement. According to my records, your system has the latest apochromatic fiber outcoupler, therefore there is practically nothing you can improve: lasers mounted in FCU are always well overlapped. This is a simple consequence of the clever [[MicroTime 200]] design.
-
-	However, in theory a confocal volume overlap of different colors will never be perfect. The reason is that the degree of chromatic correction of the objective has its own limits. The best results are obtained by the latest UPLSApo (UltraPlan Super Apochromat) objectives.
-
-
-	With a [[PIE]] capable setup, you can evaluate the overlap by imaging a single, sub-resolution, multicolor particle, for example a 100 nm diameter TetraSpeck bead, using both lasers simultaneously.
-
-	You need an appropriate dual band major dichroic and PIE excitation with the Sepia. In this case there is no need for detection color splitting. One detector is sufficient. Use a single long pass filter, target and image a single bead. Of course, you have to set the laser intensities so that both laser excitations contribute with approximately half of the final detected sum count rate.
-
-	In [[SymPhoTime]], calculate two images: one for each PIE time window, that is, one for each excitation laser. (It is easy with the SPT, use the graphical tool for setting the time-gates.) By comparing the two images, you should see no significant displacement.

residuals - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-19T03:28:39+00:00

2013/08/09 09:35		current
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-	This page is redirected to [[:glossary:residuals]].	+

t3-mode - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-19T03:17:00+00:00

2013/08/05 12:57		current
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-	====== T3-mode ======
-	Jump to: navigation, search

-	A special measurement mode of the [[PicoHarp 300]].
-
-	In T3 Mode input channel 0 is dedicated to a sync signal. As far as the experimental setup is concerned, this is similar to histogramming mode. The main objective is to allow high sync rates (up to 84 MHz) which could not be handled in T2 Mode. Accommodating the high sync rates in T3 Mode is achieved as follows: First, the sync divider is employed as in histogramming mode. This reduces the sync rate so that the channel dead time is no longer a problem. The remaining problem is now that even with a divider of 8, the sync rate is still too high for collecting all individual sync events like ordinary T2 Mode events. Considering that sync events are not of primary interest, the solution is to record them only if they arrive in the context of a photon event on channel 1. This is the same as the original [[TTTR]] mode concept as it was first conceived for the TimeHarp system. The event record is then composed of two timing figures: 1) The start-stop timing difference between the photon event and the last sync event, and 2) the arrival time of the event pair on the overall experiment time scale (the time tag). The latter was originally obtained from an independent asynchronous clock. This made it difficult to combine the start-stop timing with the time tag or to know the sync period the event belonged to. For the PicoHarp's T3 Mode a smarter approach was chosen: The time tag is now obtained by simply counting sync pulses. From the T3 Mode event records it is therefore possible to precisely determine which sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows to reconstruct the arrival time of the photon with respect to the overall experiment time.
-
-	T3 Mode measurements always implicitly provide routing information if a router is used. Routing information is represented by four bits in each TTTR record, that allow up to four routing channels to be distinguished. However, these bits are only meaningful if a router was actually used for the measurement. In case of the PHR 40x routers, only two routing bits are used. Throughput constraints and crosstalk considerations apply the same way as in interactive routed measurements.
-
-	Each T3 Mode event record consists of 32 bits. There are 4 bits for the channel number, 12 bits for the start-stop time and 16 bits for the sync counter. If the counter overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered. The 12 bits for the start-stop time difference cover a time span of 4096*R where R is the chosen resolution. At the highest possible resolution of 4 ps this results in a span of 16 ns. If the time difference between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in histogramming mode, where the number of bins is also finite. However, by choosing a suitable sync rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment scenarios. R can be chosen in doubling steps between 4 ps and 512 ps.
-
-	Dead time in T3 Mode is the same as in the other modes (95 ns typ.) Since there is only one photon channel, only autocorrelations can be calculated (unless a router is used). This can be done at the resolution R but of course only starting from lag times larger than the deadtime.
-
-	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in th dead time anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate is fully available for input channel 1. For all practically relevant fluorescence detection applications this is more than sufficient.
-
-	For maximum throughput, T3 Mode data streams are normally written directly to disk. The current PicoHarp software does not provide any immediate data visualization during a T3 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The PicoHarp software installation CD contains demo programs showing how T3 Mode files can be read by custom software. The implementation of custom measurement programs requires the PicoHarp programming library, which is available as a separate option.

lifetime_fitting_using_the_tcpsc_fitting_script - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-19T01:27:42+00:00

2014/02/14 11:16		current
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-	====== Lifetime Fitting Using the TCPSC Fitting Script ======
-
-
-	===== Summary =====
-
-
-	This tutorial shows step-by-step, how to fit the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.
-
-	===== Step-by-Step Tutorial =====
-
-
-	Select a file and start the script
-
-	* Start [[products:SymPhoTime64\|SymPhoTime 64]] software.
-	* Open the "Samples" workspace via "File\open Workspace" from the main menu.
-
-	Note: The "Samples" workspace is delivered with the SymPhoTime 64 and on the CD-ROM and contains example data to show the function of the SymPhoTime data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
-
-	Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_1.png }}
-
-	* Highlight the file ''ATTO655_diff_FLCS-pattern.ptu'' by a single mouse click.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_2.png }}
-
-	* Select the "Analysis" tab and in there, open the drop down menu "TCSPC".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_3.png }}
-
-	Note: The drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}
-
-	* Start the "TCSPC Fitting" script by clicking on "Start".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_4.png }}
-
-	Response: The TCSPC Fitting script is applied to the file ''ATTO655_diff_FLCS-pattern.ptu''. Thereby, a new Window opens:
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_5.png?600 }}
-
-	Note: The window contains two different regions:\\
-	- Left: Fitting and analysis options.
-	- Center/right: TCPSC fitting graph showing the TCSPC histogram of the measured sample.
-
-	* In the decay form on the left, select n-exponential reconvolution as fitting model.
-	Response:
-	* In the TCSPC window, the IRF is displayed in bright red and the data fitting limits are moved to the border of the TCSPC window.
-	* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}
-
-	Note: The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\
-	For explanation about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.
-
-	* Click: "Initial Fit" (marked in orange).
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_7.png?600 }}
-
-
-
-	Response:
-	* In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= raw data fit values) are displayed.
-	* Fit values appear in the fitting table.
-
-	Note:
-	Usually, a decent fit is characterized by the following criteria:\\
-	The fitted curve overlays well with the decay curve. In the residual window, the values spread randomly around 0.\\
-	The χ²-value approaches 1.\\
-	The calculated fitting values are reasonable.\\
-	Usually the fitting model with least parameters is selected.\\
-	=> In this example, the fit is already sufficient.
-
-
-	* Store the result file by selecting: "Save Results".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_8.png }}
-
-	Response: A result file (''TCSPC_Fitting.pqres'') is stored under the raw data file (''ATTO655_diff_FLCS-pattern.ptu'').
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_9.png }}
-
-	* Later, clicking on the result file reopens the file in the same way as it was stored.
-	* Now all necessary steps are done. There are several possibilities how to continue:
-
-	===== Export the Fitting Values =====
-
-	* To transfer the fitting data into another program, place your mouse cursor over the fitting table into a grey region, use a right mouse click to open the context menu and select "Copy" or simple use "<CRTL>" + "C" to copy the fitting table into the clipboard.
-	* Open Notepad or Excel or any other program you want to copy the data into, and past the data there using "<CRTL>" + "V".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_10.png?236 }}
-	⇓
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_11.png?358 }}
-
-	===== Export the Complete Decay =====
-
-	* If the complete decay should be exported to plot the graph in another program, place the cursor over the decay graph, use the right mouse click to open the context menu and select one of the ASCII export options, in this case "selected cell".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_12.png?412 }}
-
-	Response: A window opens and asks for a file name to store the exported result file. Select e.g. the name ''decay.dat''.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_13.png?355 }}
-
-	Note: The .dat file contains the TCSPC curve, the estimated instrument response function (IRF) and the fitted curve.
-
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_14.png?600 }}
-
-	===== Process Several Files and Calculate Averages =====
-
-	* If several raw data files are marked in the workspace and the TCSPC fitting script is then applied, the decays of all loaded files are displayed in the script. For illustration, mark the two files ''Cy5_diff_IRF+FLCS-pattern.ptu'' and ''ATTO655_diff_FLCS-pattern.ptu'' with one mouse click each and start the TCSPC fitting script.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_15.png }}
-
-	Response: The TCSPC fitting window is opened and the TCSPC histograms of both files are loaded. The TCSPC histogram from the file "ATTO655_diff_FLCS-pattern.ptu is marked in green, indicating that it is the active file. Under decay data all files are listed, the active file is always highlighted in green.
-
-	Note: The file ''Cy5_diff_IRF+FLCS-pattern.ptu'' contains a lifetime measurement of the dye Cy5 in water. In the TCSPC histogram it can be clearly seen that the lifetime of this dye is significantly shorter than the lifetime of ATTO655.
-
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_16.png?600 }}
-
-	* Click on "Initial Fit" to perform a monoexponential tailfit of the ATTO655 dataset.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_17.png?600 }}
-
-	* If both dyes can be fitted with the same model, click "Fit All".
-	Response: Both data sets are fitted with a single exponential tailfit model. The values of the last dataset are displayed in the fitting table.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_18.png?268 }}
-
-	* To toggle between the fitted values of the different data sets and check the fits, click onto the different datasets to activate them.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_19.png }}
-
-	* To export all fitting values for all fits, activate the "Paramater Plot".
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_20.png }}
-
-	Response: The parameter plot is shown. The parameters to be displayed are plotted as points in a graph, with the first point on the left belonging to the first data set, etc.
-
-	Note: The parameter plot is in this case not very illuminating, as only 2 datasets are present. It's full potential can be generated, if the same dye is measured several times, because in this case it graphically shows the deviation of the fitting values. It also calculates an average for each fitting value over the data sets. In our example, it is of course meaningless, as two different dyes were fitted.
-
-	* Place the mouse over the graph, activate the context menu with a right mouse click and select the "Export ASCII (All cells)" option. This allows to store the fitting values of all fitted data sets at once e.g. to load them into Excel or a similar program for further processing.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_21.png?600 }}
-
-	Response: A window opens and asks for a file name and a folder to store the data, e.g. as ''fitting values.dat''.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_22.png }}
-
-	* Store the file. You can open the data e.g. with the notepad function of the PC to have a look at the data structure. Every data set is stored as a line and the fitting values are arranged as columns. The order of the data sets corresponds to the order of the data sets in the SymPhoTime table.
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_23.png?600 }}
-
-	* Don't forget to save the data in SymPhoTime by clicking on "Save results". This generates an analysis result file (''.pqres''), which in this case is storedalong with both corresponding raw data files (''.ptu'').
-	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_24.png }}
-
-

flim - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T22:23:30+00:00

2013/08/09 09:31		current
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-	This page is redirected to [[:glossary:flim]].	+

least_squares - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T21:33:54+00:00

2013/08/09 09:32		current
Line 1:		Line 1:
-	This page is redirected to [[:glossary:least_squares]].	+

roi_fitting_using_the_flim_script - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T20:16:43+00:00

2014/02/11 14:04		current
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-	====== ROI fitting using the FLIM script ======
-
-
-	===== Summary =====
-
-
-	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In detail, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.
-
-	==== Step-by-Step Tutorial ====
-
-
-	=== Select a file and start the script ===
-
-	* Start [[products:SymPhoTime64\|SymPhoTime 64]] software.
-	* Open the "Samples" workspace via "File\open Workspace" from the main menu.
-
-	Note: The "Samples" workspace is delivered with the SymPhoTime 64 and on the CD-ROM drive and contains example data to show the function of the SymPhoTime data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
-
-	Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-	{{ roi_fitting_using_the_flim_script_Image_1.png }}
-
-	* Highlight the file ''DaisyPollen_cells_FLIM.ptu'' by a single mouse click.
-	{{ roi_fitting_using_the_flim_script_Image_2.png }}
-
-	* Select the "Analysis"-tab and in there, open the drop down menu "Imaging".
-	{{ roi_fitting_using_the_flim_script_Image_3.png?348 }}
-
-	Note: The drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}
-
-	* Start the [[glossary:flim\|FLIM]] script by clicking on "Start".
-	{{ roi_fitting_using_the_flim_script_Image_4.png }}
-
-	Response: The FLIM script is applied to the file ''DaisyPollen_cells_FLIM.ptu''. Thereby, a new Window opens:
-	{{ roi_fitting_using_the_flim_script_Image_5.png?600 }}
-
-	Note: The window contains five different regions:\\
-	- Upper left: imaging analysis options.\\
-	- Upper center: [[glossary:fast_flim\|Fast FLIM]] image, displayed in false color scale. The brightness encodes the intensity while the color encodes the average "Fast FLIM" lifetime, i.e. the mean arrival times of the photons after the laser pulse. When not defined otherwise, intensity and color scale stretch from minimum to maximum. As the mean arrival time of the background photons in the areas where no fluorescence is detected, is randomly spread over the TCSPC window, the mean photon arrival time of the dark background is usually very long (up to 1/2 of the TCSPC window), which makes the color scale loaded by default sometimes unsuited for displaying the lifetime contrast in the actual sample. In this case, the scale has to be adapted.\\
-	- Upper right: Fluorescence Lifetime Histogram. Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot.\\
-	- Lower left: Lifetime fitting parameters. Here lifetime fitting models can be defined. Fitting is then applied to the lifetime graph(s) on the lower center (TCSPC histogram).\\
-	- Lower center/left: By default, this set shows the TCSPC histogram of all photons in the image in green (= dataset 0) and the TCSPC histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the images TCSPC histogram.
-
-	* To enhance the lifetime contrast within the daisy pollen, adapt the color scale from 0.7 to 2 ns by typing these values as limits for the Fast Lifetime color scale.
-
-	Note: The default lifetime scale stretches from -0.5 to 41.6 ns. The physically impossible value of -0.5 ns is explained by the fact, that SymPhoTime places the 0 value at the rising edge of the decay in the TCSPC window. As a mean arrival time is calculated, it is possible that in dark regions only one or two photons caused dark count noise are detected which are randomly detected before the laser pulse in the TCSPC window.\\
-	Reasonable limits can either be found by dragging the limit bars in the color scale "Fast Lifetime" histogram or by considering the peaks of the "Lifetime Histogram". Entering fixed values has the advantage that different images can be compared later.
-
-	{{ roi_fitting_using_the_flim_script_Image_6.png?600 }}
-
-	Response: The color scale in the image adapts and lifetime contrasts in the pollen become visible.
-
-	=== Select 3 Regions of Interest (ROI) ===
-
-
-	* Keep the mouse pointer over the image and open the context menu by a right mouse click.
-	{{ roi_fitting_using_the_flim_script_Image_7.png?349 }}
-
-	* Select a ROI tool in the context menu, e.g. the "FreeROI" tool.
-
-	Note: The different ROI tools are used in the following way:\\
-	Paint ROI: highlights the pixels the mouse cursor is guided over while the left mouse button is activated. The faster the cursor is moved the broader is the area of pixels highlighted around the mouse.\\
-	Free ROI: moving while keeping the left mouse button activated marks the area within the drawn shape.\\
-	Rectangle ROI: to draw rectangular ROIs.\\
-	Ellipse ROI: to draw elliptic ROIs.\\
-	Magic Wand ROI: when activated, clicking activates all neighboring pixels with a similar Fast Lifetime. Especially useful for marking spotted structures.
-
-	* Draw a ROI in the blue rim of the pollen by maintaining the left mouse button activated while circling the selected part of the image as shown:
-	{{ roi_fitting_using_the_flim_script_Image_8.png?600 }}
-
-	Response: \\
-	* Everything except the circled area is displayed only in grey scale.
-	* In the lower part of the window, a new decay appears in grey, which contains the photons from the selected pixels. This new decay is labeled "ROI 0".
-	{{ roi_fitting_using_the_flim_script_Image_9.png?600 }}
-
-	* On the upper left, open the drop down menu "Region of Interest" and click on "New".
-	{{ roi_fitting_using_the_flim_script_Image_10.png }}
-
-	Response: \\
-	* In the "Region of Interest" drop down, the ROI number is set to "1".
-	* The whole image is displayed in color scale again.
-	* In the decay legend, ROI 1 is added.
-	* As ROI 1 contains still all photons of the image and equals the "Overall decay", no new curve is displayed.
-	* Place the cursor over the image, select in the context menu (right mouse click) the "Rectangle ROI" and mark a rectangular ROI in the green central part of the pollen.
-	{{ roi_fitting_using_the_flim_script_Image_11.png }}
-
-	Response: \\
-	* Everything except the marked area is displayed only in grey scale.
-	* In the lower part of the window, a new decay appears in grey, which contains the photons from the selected pixels. This new decay corresponds to "ROI 1".
-	* Mark a third ROI. Therefore, on the upper left, open the drop down menu "Region of Interest" and click on "New".
-	Response: \\
-	* In the "Region of Interest" drop down, the ROI number is set to "2".
-	* The whole image is displayed in color scale again.
-	* In the decay legend, ROI 2 is added.
-	* Place the cursor over the image, select in the context menu (right mouse click) the "Magic Wand ROI" and click on a red region in the pollen.
-	{{ roi_fitting_using_the_flim_script_Image_12.png }}
-
-	Response: \\
-	* Everything except the marked area is displayed only in grey scale.
-	* In the lower part of the window, a new decay appears in grey, which contains the photons from the selected pixels. This new decay corresponds to "ROI 2".
-
-	Note: The new decay wich stems from the last and smaller ROI contains considerably less photons. Ideally, decays selected for ROI fitting should contain ~10000 – 50000 photons in the peak.
-
-	* As the decay contains much less photons than the other ROIs, we add other regions to the same ROI. In order to recognize the red regions, go to the context menu and select "Invert ROI":
-	{{ roi_fitting_using_the_flim_script_Image_13.png }}
-
-	Response:\\
-	* Everything except the originally selected pixels for ROI 2 are displayed in color scale.
-	* Keep the <CRTL>-button pressed while clicking on the other red dots in the image. With every selection, the corresponding red dot is greyed out.
-	{{ roi_fitting_using_the_flim_script_Image_14.png }}
-
-	* Again, select "Invert ROI" in the context menu.
-	{{ roi_fitting_using_the_flim_script_Image_15.png }}
-
-	Response: Now only the red dots of the pollen image are highlighted, everything else is greyed out. The decay corresponding to ROI 2 contains now the photons from all marked dots.
-
-	Note:\\
-	To review the different regions, go to the drop down menu "Regions of Interest" and change the number between 0 and 2. The image will display the corresponding image with the selected ROI.\\
-	To export the images with a marked ROI, keep the cursor over the image with the selected ROI, open the context menu with a right mouse click and select one of the export options:\\
-	Bitmap exports the image as a bmp file. If the scale bar is displayed, the image including the scale bar is exported.\\
-	Bitmap with color scale exports the image as a bmp file including the intensity and color scale next to the image.\\
-	1x1 bitmap exports the image as a bmp file with one pixel of the image being exported as one pixel in the image file. In the other bitmap export options, the image is exported as displayed.\\
-	ASCII exports the image as ASCII array using a text file format. Thereby one array encodes the intensity values, while a second array encodes the Fast Lifetime values. After removal of the header and the unwanted array, individual arrays can be imported in graphic programs, e.g. ImageJ as a text image. These images contain the full count resolution of the raw image.
-
-	=== Perform a Lifetime Fit on the Selected Regions (ROIs) ===
-
-	* In the decay form on the lower left, select n-exponential reconvolution as fitting model.
-	{{ roi_fitting_using_the_flim_script_Image_16.png?600 }}
-
-	Response:\\
-	* In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.
-	* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.
-	{{ roi_fitting_using_the_flim_script_Image_17.png?600 }}
-
-	Note: The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\
-	For explanation about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.
-
-	* Under "Decay" select "ROI 0".
-	Response:\\
-	* The TCSPC curve of ROI 0 is highlighted in green.
-	* Click: "Initial Fit" (marked in orange).
-	Response: \\
-	* In the TCSPC window, the fit of ROI 0 is displayed as a black line. Below, the residuals (= raw data fit values) are displayed.
-	* Fit values appear in the fitting table.
-	{{ roi_fitting_using_the_flim_script_Image_18.png?600 }}
-
-	Note: Usually, a decent fit is characterized by the following criteria:\\
-	The fitted curve overlays well with the decay curve. In the residual window, the values spread randomly around 0.\\
-	The χ²-value approaches 1.\\
-	The calculated fitting values are reasonable.\\
-	Usually the fitting model with least parameters is selected.\\
-	In this example it is obvious that the applied single exponential reconvolution fit is not suited to fit the decay curve.
-
-	* Increase the "Model Parameters" to n = 2.
-	* Again, click on "Initial fit":
-	Response:\\
-	* The fit quality increases, but still the fit quality is not sufficient.
-	{{ roi_fitting_using_the_flim_script_Image_19.png?600 }}
-
-	* Increase the "Model Parameters" to n = 3.
-	* Again, click on "Initial fit".
-	Response:\\
-	* Now a good fit result is achieved.
-	{{ roi_fitting_using_the_flim_script_Image_20.png?600 }}
-
-	* To check whether this is really the best possible fit, increase the "Model Parameters" to n = 4.
-	* Again, click on "Initial fit".
-	Response:\\
-	* The fit becomes even slightly better, but looking at the fitting parameters, we find an amplitude A4 with a negative value "-40". This is not expected, and not physically logical. Therefore, this decay is best described by a 3-exponential model.
-	{{ roi_fitting_using_the_flim_script_Image_21.png?600 }}
-
-
-	* Decrease the "Model Parameters" to n = 3.
-	* Again, click on "Initial fit".
-	Response:\\
-	* Now a good fit is achieved.
-	{{ roi_fitting_using_the_flim_script_Image_22.png?600 }}
-
-	* To compare the fit with other ROIs, we select: "Fit all".
-	{{ roi_fitting_using_the_flim_script_Image_23.png?600 }}
-
-	Response: \\
-	* Every decay is fitted with a 3-exponential fit.
-	* As the last fit is ROI 2, this curve is shown in green at the final window.
-	* To compare the fitting data, either toggle between the graphs by selecting as "Decay" ROI 0, ROI 1 or ROI 2 or click on "Parameter Plot" (right next to the TCSPC graph).
-
-	Response:\\
-	* Instead of the TCPSC curve, the fitted parameters appear in a graph.
-	* Select the parameter you would like to display (e.g. T_av_Int, the intensity-weighted average lifetime, which - neglecting background photons – roughly corresponds to the Fast Lifetime).
-	{{ roi_fitting_using_the_flim_script_Image_24.png?600 }}
-
-	Note: On the x-axis, the dataset name is plotted. The order is the same as in the decay menu:\\
-	1 = Overall decay, generated from all photons of the image.\\
-	2 = Pixel decay, including the photons from the last selected pixel.\\
-	3 = ROI 0\\
-	4 = ROI 1\\
-	5 = ROI 2\\
-
-	* Keep the cursor over the graph, and after a right mouse click, select from the context menu the "Export ASCII (All Cells)" option.
-	{{ roi_fitting_using_the_flim_script_Image_25.png?600 }}
-
-	Respond: A window opens, asking for a file name to store the fitting values. The data are stored as a .dat file.
-	{{ roi_fitting_using_the_flim_script_Image_26.png?600 }}
-
-	* The .dat file contains the fitting values as table and can be opened e.g. using the Notepad program. They can also be imported into Excel or similar programs.
-	{{ roi_fitting_using_the_flim_script_Image_27.png?600 }}
-
-	* Store the result file by selecting: "Save Results".
-	{{ roi_fitting_using_the_flim_script_Image_28.png?241 }}
-
-	Response:\\
-	* A result file (''FLIM.pqres'') is stored under the raw data file (''DaisyPollen_FLIM.ptu'').
-	{{ roi_fitting_using_the_flim_script_Image_29.png }}
-
-	* Later, clicking on the result file reopens the file in the same way as it was stored.
-
-	Note:\\
-	This tutorial explains only a subset of the functions of the FLIM script. Also check the script explaining pixel fitting (Lifetime-fitting Using the FLIM-analysis).
-
-

tcspc - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T19:42:52+00:00

2013/08/09 09:36		current
Line 1:		Line 1:
-	This page is redirected to [[:glossary:tcspc]].	+

2ffcs - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T18:49:39+00:00

2013/08/05 13:33		current
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-	====== 2FFCS ======
-
-	===== Intro =====
-
-	2FFCS (sometimes also written as 2fFCS) is an abbreviation 2 Focus Fluorescence Correlation Spectroscopy. Dual focus FCS might also be used.
-
-	Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.
-
-	===== Experimental Setup =====
-	==== Pinhole Selection ====
-
-	The conjugated pinhole size should be chosen slightly larger than the excitation spot diameter. The conjugated pinhole size is the pinhole diameter divided by the magnification of the objective.
-
-	e.g:
-
-	*60x 1.2N.A. Objective, 640nm excitation
-	*excitation FWHM = 350 nm (typical for 4x out-coupler!, the 10x out-coupler will have a larger excitation spot)
-
-	Usually the size of the confocal volume or the diameter of the excitation spot is given as its FWHM. However the airy disc diameter is bigger and the 1/e2 diameter must be used.
-
-	=== Single Focus ===
-
-	<math>
-
-	2ln(2)=\frac{FWHM^2}{w_0^2} </math>
-
-	<math> w_0=0.849 \cdot FWHM </math>
-
-
-	The diameter of the excitation spot therefore is:
-
-	<math> d_{exc}=2 \cdot w_0 = 594 nm </math>
-
-	Together with the 60x magnification of the objective the airy disc at the pinhole location is
-
-	<math> d_{PH}=60\cdot 594nm = 35.6\mu m </math>
-
-	This equals 1 airy unit (AU)
-
-
-	Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.
-
-	PQ usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.
-
-	=== Two Foci ===
-
-	The distance between the foci in two-focus FCS is determined by the Nomarski prism. Usually the distance is around 400 nm. Therefore the 2 foci (separated by 400 nm) will occupy:
-
-	<math> d_{exc, 2fFCS} = 2 \cdot w_0 + 400 nm = 994 nm </math>
-
-	Together with the 60x magnification of the objective both airy discs together, at the the pinhole location, have a diameter
-
-	<math> d_{PH, 2fFCS}=60\cdot 994nm =59.6\mu m </math>
-
-	== Adjusting the Pinhole ==
-
-	-use a suitable dye solution sample to get an approx. count-rate of 105 cps. e.g. 10-9M aqueous Atto655
-	-make sure both lasers have the same power
-	-using the time trace oscilloscope optimize the pinhole position with Nomarski Prism and both lasers.
-	-switch to TCSPC oscilloscope
-	-select linear scale
-	-optimize with horizontal knob, this knob affects both lasers equally
-	-optimize with vertical knob until both lasers show same intensity. When turning, you should see the decreasing of one laser while the other stays at the same level for a bit then, also the second decreases.
-	-if you can't get them both to their respective maximum value at the same time, the pinhole is too small. (This means that you are either cutting one or the other or both)

frap - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T18:29:54+00:00

2013/08/06 16:15		current
Line 1:		Line 1:
-	This page is redirected to [[:glossary:frap]].	+

fret - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T16:09:40+00:00

2013/08/06 14:58		current
Line 1:		Line 1:
-	====== FRET ======

-	FRET stands for Förster Resonance Energy Transfer
-
-	Non-radiative energy transfer from a donor fluorophore (which is usually directly excited) to an acceptor fluorophore (for which direct excitation usually should be avoided).

determination_of_the_focal_width_with_the_focal - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T14:47:46+00:00

2014/02/14 10:31		current
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-	====== Determination of the Focal Width with the Focal Width Analysis ======
-
-
-	===== Summary =====
-
-
-	This tutorial shows step-by-step, how to determine the Point Spread Function (PSF) of a microscopic system using the Focal Width Script. It requires the image of a subresolution bead, in which the center is marked in the image. The script applies then a Gaussian fit of the bead and calculates the Full Width Half Maximum (FWHM) in both scanning directions.
-
-	===== Step-by-Step Tutorial =====
-
-	==== Select a file and start the script ====
-
-	* Start [[products:SymPhoTime64\|SymPhoTime 64]] software.
-	* Open the "Samples" workspace via "File\open Workspace" from the main menu.
-
-	Note: The "Samples" workspace is delivered with the SymPhoTime 64 and on the DVD-ROM and contains example data to show the function of the SymPhoTime 64 data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial, e.g. to a folder "C:\SPT_User".
-
-	Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-	{{ determination_of_the_focal_width_with_the_focal_Image_1.png }}
-
-	* Highlight the file ''TS-Bead_immo_xy-scan.ptu'' by a single mouse click.
-
-	Note: This file contains the image of a TetraSpeck Bead with 100 nm diameter. As this is smaller than the optical resolution, beads of this size can be used to measure the focal width in a microscope system.
-
-	{{ determination_of_the_focal_width_with_the_focal_Image_2.png }}
-
-	* Select the "Analysis" tab and in there, open the drop down menu "Alignment".
-	{{ determination_of_the_focal_width_with_the_focal_Image_3.png }}
-
-	Note: The drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}
-
-	* Start the "Focal Width" script by clicking on "Start".
-	{{ determination_of_the_focal_width_with_the_focal_Image_4.png }}
-
-	Response: The "Focal Width" script is applied to the file ''TS-Bead_immo_xy-scan_DualFocus.ptu''. Thereby, a new Window opens:
-	{{ determination_of_the_focal_width_with_the_focal_Image_5.png?600 }}
-
-	Note: The window contains two different regions:
-
-	- Left: Image plotting analysis options.
-	- Center/right: Image, plotted in an intensity grey scale. To adjust brightness and contrast, click on the vertically aligned arrows on the right.
-
-	Optional: The following parameters can be adapted:
-	* Pixel binning by changing the "Binning", e.g. to twopoints.
-	* Select the second excitation peak by clicking on the white area of the "Set Time Gate" graph.
-
-	Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.\\
-	If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the MicroTime 200), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.
-
-	{{ determination_of_the_focal_width_with_the_focal_Image_6.png }}
-
-	* When finished, click "Calculate" to update the image.
-	* Click into the intensity center of the bead.
-	{{ determination_of_the_focal_width_with_the_focal_Image_7.png?600 }}
-
-	* The image fitting is already done and you can click on "Save Result" to store the data. This generates an analysis results (''.pqres'')-file, which is assigned to the corresponding raw data (''.ptu'') file.
-	{{ determination_of_the_focal_width_with_the_focal_Image_8.png }}
-
-	* Optional: Apply the same script to the file ''TS-bead_immo_xz-scan.ptu'' to get an idea about the ratio of the focal width in xy vs. z.
-

calculate_and_fit_fcs_traces_with_the_fcs_script - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T14:43:51+00:00

2014/02/13 14:21		current
Line 1:		Line 1:
-	====== Calculate and Fit FCS Traces with the FCS Script ======

-	===== Summary =====
-
-
-	This tutorial shows step-by-step, how an autocorrelation curve of a fluorophore dissolved in water is calculated and fitted using the FCS script.
-
-	==== Background Information ====
-
-	Note: ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm.\\
-	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.\\
-	Note that the diffusion coefficients of all dyes are temperature dependent.\\
-	FCS measurements are usually point measurements acquired with very sensitive detectors (typically SPAD or Hybrid PMT detectors).\\
-	FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.\\
-
-	==== Step-by-Step Tutorial ====
-
-	=== Select a file and start the script ===
-
-	* Start [[products:SymPhoTime64\|SymPhoTime 64]] software.
-	* Open the "Samples" workspace via "File\open Workspace" from the main menu.
-	* The "Samples" workspace is delivered with the SymPhoTime 64 on aDVD-ROM and contains example data to show the function of the SymPhoTime data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
-
-	Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_1.png }}
-
-	* Highlight the file ''ATTO4888_485nm_pulsed.ptu'' by a single mouse click.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_2.png }}
-
-	* Select the "Analysis"-tab and in there, open the dropdown menu "FCS".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_3.png }}
-
-	Note: The drop-down menus can be opened and closed by clicking on the grey button on the left side of the header of the drop down menue: {{grey_button.png}}
-
-	* Start the FCS-script by clicking on "Start".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_4.png }}
-
-	Response: The FCS-script is applied to the file ''ATTO488_485nm_pulsed.ptu''. Thereby, a new Window opens:
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_5.png?600 }}
-
-	Note: The window contains three different regions:\\
-	- Left: analysis and display options. For explanation of the different parameters, place the mouse cursor over this part of the window and press <F1> to open the corresponding help page.\\
-	- Upper center/right: the intensity time trace. The display option can be changed using the "Trace Settings" of the analysis options. The large window shows the inset of the complete trace above marked in green. The photon counting histogram on the right displays the frequencies of the different intensity values. Usually, this trace is used to check, whether the signal is stable during the measurement, which is necessary for FCS. Also, large intensity spikes stemming from aggregation of the fluorescent sample can be identified in this graph. In measurements inside cells, often a slight decrease at the beginning ot the trace is noticed. In this case, this region can be excluded from calculating the FCS trace by adjusting the upper vertical red limit bars in the small upper trace.\\
-	- Lower center/right: the FCS trace window. As first the FCS correlation criteria have to be defined and the curve needs to be calculated, this graph does not contain any trace at this stage.
-
-	* In this example, all light is recorded with detector 1, therefore we leave the FCS options as shown and press "Calculate" in order to calculate the autocorrelation function.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_6.png }}
-
-	Response:\\
-	* The FCS curve is calculated and plotted.
-	* The first decreasing part is due to detector afterpulsing and an unwanted artifact from the detector. If cw light is used for exciting the dye, this artifact cannot be removed in an autocorrelation, and the corresponding part of the curve which is influenced by this artifact needs to be excluded from curve fitting.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}
-
-	Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly Hybrid PMTs) also have so low afterpulsing that the problem does not occur.
-
-	* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.
-
-	=== Background Removal for Pulsed Excitation ===
-
-	* If the sample has been excited with a pulsed laser, there's another way to remove afterpulsing:
-	* Open the "FLCS Background Correction" dropdown panel and check the "Enable" box.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_8.png }}
-	⇓
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_9.png }}
-
-	* This button calculates a simple FLCS filter on the data: the fluorescence decay versus the background in the TCPSC window. As the detector afterpulsing is not correlated with the laser pulse on the time - range of interest, photons caused by detector afterpulsing behave similar as normal background photons.
-
-	Note: If you want to check what is calculated with the FLCS background correction, click on "more" to open the graphs of the TCSPC curve and the calculated filters.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_10.png }}
-
-	* Click on "Calculate" again.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_6.png }}
-
-	* It is not necessary to calculate first a FCS curve without the photon filter. You can also directly activate the FLCS background correction and calculate the FCS curve.
-
-	Response: The FCS curve is re-calculated. As a result, the steep decrease at the beginning of the curve is now missing and an artifact free curve is obtained.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_12.png?600 }}
-
-	* If you do not want to fit the data: Save the calculated curve by clicking on "Save Result".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_13.png }}
-
-	Response: A result file is generated and linked to the raw data file.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_14.png }}
-
-	=== Fitting FCS curves using the FCS Fitting script ===
-
-	Note: In order to provide correct results, the confocal volume VEff and the structural
-	parameter k have to be calculated first. Check the tutorial "Calibration of the
-	confocal volume for FCS using the FCS Calibration Script" for a description
-	about how to calibrate the confocal volume.\\
-	If no calibration values are stored, the effective volume VEff is set to 1 fl and the
-	structural parameter k is set to 5. While a structural parameter value of 5 is
-	usually reasonable, the confocal volume depends on the wavelength and can
-	differ significantly.\\
-	Check your system regularly for correct alignment using a reference dye as
-	ATTO488.\\
-	In this tutorial, we use the default values to keep the description easy. If your
-	system is already calibrated, the results (especially the concentration C and the
-	diffusion constant D) will depend on your calibration.
-
-	* If you do want to fit the data: Transfer the curve to the FCS fitting script by clicking "Transfer to Fit".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_15.png }}
-
-	Response:\\
-	* The FCS curve is automatically stored. Therefore, it is not necessary to store the data before transferring them to the FCS fitting script.
-
-	Note: It is also possible to open a stored FCS curve directly with the FCS fitting script.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_16.png }}
-
-	* The FCS Fitting window pops up.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_17.png?600 }}
-
-	Note: This window consists of different sections:\\
-	Left: fitting panel to select a fitting model and see the fitting parameter. If you
-	need information about how the fitting models are defined and what is the
-	meaning of the parameters, click on the "Help" button next to the fitting model.
-	This opens the corresponding help window.\\
-	Center/right: here the FCS curve is plotted. Below the graph the residuals are
-	plotted. As we have not performed a fit yet, this area is still empty.\\
-
-	* Select the correct Fitting Model. We start with the default model "Pure Diffusion".
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_18.png }}
-
-	Note: There are several models available. Click on the "Help" button to see the
-	corresponding fitting equations:\\
-	Pure Diffusion: Use this model to analyze diffusion of a dye in a solvent (i.e.
-	water). It is assumed that the dye does not show any fluctuation between
-	various states with different photophysics at a timescale short or equal to the
-	diffusion time through the confocal volume. Examples for these dyes are
-	ATTO655 or ATTO425. Also dyes with a moderate triplet contribution can be
-	fitted with this model if the excitation intensity is so low that the triplet population
-	can be neglected. See: S.R. Aragón, R. Pecora, J. Chem. Phys. 1976, 64,
-	1791-1803; R. Rigler et al., Eur. Biophys. J. 1993, 22, 169-175.\\
-	Triplet: Use this model if the dye you use shows some contributions caused by
-	transitions into the triplet state or similar photophysics, while otherwise freely
-	diffusing in a solvent. See: J. Widengren et al, J. Fluoresc. 1994, 4, 255-258.\\
-	Triplet Extended (3D): Use this model if your dye is observed in conditions
-	where either the average count rate is not completely stable and/or the dye is
-	diffusing in all three space directions through a crowded environment as a
-	polymer or a cell. For cell measurements, this is usually the model of choice.
-	See also M. Wachsmuth et al., J. Mol. Biol. 2000, 298, 677-89.\\
-	Triplet Extended (2D): Use this model if diffusion of a dye within an artificial or
-	cell membrane is observed. This model is made for dyes moving within a plane,
-	e.g. modified lipids in a membrane. If the diffusion is not anomalous, set the
-	anomaly parameter to 1 and keep it fixed. See e.g. Korlach et al., PNAS 1999,
-	96, 8461-8466 or L. Wawrezinieck et al., 2005, Biophys. J. 89, 4029-4042.\\
-	Conformational: Use this model if your dye is undergoing conformational
-	changes when diffusing through the confocal volume, which are characterized
-	by stretched exponential kinetics. See e.g. S. Wennmalm et al, PNAS 1997, 94,
-	10641-10646.\\
-	Protonation: Use this model if analyzing a diffusion of a sample with an internal
-	+ external acid/base equilibrium which has an effect on the photophysical
-	properties of the dye. This model was used to characterize GFP. See e.g.
-	U.Haupts et al., PNAS 1998, 95, 13573-15578.
-
-	* Press "Initial Fit" to calculate the fitting curve.
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_19.png }}
-
-	Response:\\
-	* The curve is fitted and plotted in the main window.
-	* The residuals (= difference between fitted curve and raw data) are plotted below the curve.
-	* The fitting parameters are displayed in the parameter panel.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_20.png?600 }}
-
-	Note: It can be clearly seen that at short correlation time ranges, the curve does not fit
-	the data well. This is an indicator that the sample undergoes some
-	photophysical fluctuations in the μs – time range, usually triplet fluctuations.
-	Therefore, we have to change the fitting model.
-
-	* Select the "Triplet" model.
-	Response:\\
-	* The parameters in the parameter panel are adapted.
-	* Press "Initial Fit".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_21.png }}
-
-
-	Response: \\
-	* The curve is fitted and plotted in the main window.
-	* The residuals (= difference between fitted curve and raw data) are plotted below the curve.
-	* The fitting parameters are displayed in the parameter panel.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_22.png?600 }}
-
-	Note: This time, the curve fits the data significantly better.\\
-	A diffusion time of a few tens of μs is typical for a dye in water, as well as triplet
-	time in the range of a few microseconds.\\
-	The fitted diffusion coefficient D is significantly higher than the real value of
-	400μm²/s. This is due to the incorrect calibration. If the diffusion coefficient is
-	known, the size of the confocal volume can be decreased and the curve refitted,
-	until the fitted diffusion coefficient is realistic.\\
-	Please look at the corresponding tutorial to learn how to calibrate the confocal
-	volume.
-
-	* Save the fit by clicking "Save Results".
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_23.png }}
-
-	Response: A result file is generated and linked to the raw data. We can now close the fitting window.
-
-	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_24.png }}

mcp - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T12:31:04+00:00

2013/08/05 13:45		current
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-	====== MCP ======

-	Microchannel Plate PMT (MCP)
-
-	A microchannel plate PMT is a sensitive photon detector. It consists of an array of glass capillaries (10-25 μm inner diameter) that are coated on the inside with a electron-emissive material. The capillaries are biased at a high voltage. Like in the PMT, an electron that strikes the inside wall of one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates a gain of 103 to 106 and produces a current pulse at the output. The timing jitter of MCPs is sufficiently small to perform time-resolved photon counting on a sub-nanosecond- scale, usually outperforming PMTs. Good but also expensive MCPs can achieve timing uncertainties as low as 20ps. Microchannel plates are also used as an intensifier for low-intensity light detection with array detectors.

tttr - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T12:28:03+00:00

2013/08/05 13:02		current
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-	====== TTTR ======
-
-	===== What is TTTR? =====
-
-	TTTR stands for Time-Tagged Time-Resolved. The basic concept of this measurement mode is to record every individual photon of a [[TCSPC]] experiment. Both the arrival time as well as the TCSPC timing information (i.e. the time from the preceding excitation pulse or to the following excitation pulse in reverse start stop experiments) are recorded.
-
-	===== TTTR at PQ =====
-
-	The [[TimeHarp 200]] (also the [[TimeHarp 100]]) and the [[PicoHarp 300]] support TTTR mode measurements. With the the [[PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[TimeHarp 200]] / [[TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.
-
-	While recording of TTTR mode files is possible with the [[TimeHarp 200]] or [[PicoHarp 300]] software, analysis is supported mainly by the SymPhoTime software package.
-
-	Due to its capability of including external TTL pulses as markers in the stream of photon events TTTR mode is suitable for [[FLIM]] measurements. The markers are used to synchronise the photon events with a scanning process allowing to sort the photons into image pixels. Again, this is mainly supported by the SymPhoTime software. Only in combination with the [[SCX 200 FLIM]] measurements based on TTTR mode can be performed directly from the [[TimeHarp 200]] software.
-
-

bifl - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T11:46:12+00:00

2013/08/06 14:38		current
Line 1:		Line 1:
-	====== BIFL ======
-
-	===== What is "BIFL"? =====
-
-	BIFL stands for Burst Integrated Fluorescence Lifetime: The photon events of a complete burst are accumulated into a [[TCSPC]] histogram which is used for lifetime analysis methods afterwards. Usually a burst is defined as a time interval for which the intensity of a time trace or [[MCS]] measurement is above a chosen threshold. Evaluation of all bursts present in an [[MCS]] trace, for example, results in a lifetime histogram.
-
-
-	===== BIFL at PQ =====
-
-	BIFL is supported by the [[SymPhoTime]] software, alongside with a variety of burst related techniques.
-
-

irf - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T11:42:03+00:00

2013/08/06 15:55		current
Line 1:		Line 1:
-	This page is redirected to [[:glossary:irf]].	+

pattern_matching - removed - external edit (Unknown date)

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T07:39:19+00:00

2014/02/13 14:13		current
Line 1:		Line 1:
-	====== Pattern Matching ======

-	===== Summary =====
-
-
-	Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.
-
-	==== Open an Image ====
-
-	The following steps describe how to perform a Pattern Matching Analysis on raw data. This allows for separating different dyes or regions of different lifetime/decay-behavior in a sample.
-
-	* Copy the sample workspace from the DVD to a local disc drive and open it.\\
-	Response: The sample workspace contains several examples of measurements and analyzes. The files of the sample workspace are displayed in the [[helpkey:SC_WorkspaceWin\|workspace panel]] on the left side of the [[helpkey:SC_MTMainScript\|main window]].
-
-	{{ pattern_matching_Image_1.png }}
-
-	* Click on the file ''FRET_GFP and mRFP.ptu''.\\
-	Response:\\
-	* The file is highlighted in the workspace tree view.
-
-	* Open the //Imaging// drop down window of the //Analysis// working mode and press the //Start// button of the //Pattern Matching// box.
-	{{ pattern_matching_Image_2.png }}
-
-	Response: The software calculates a //Decay Diversity Map//, a simple intensity image and an overall decay from the raw data file.
-
-	==== Select Patterns ====
-
-	Note: Once an image is generated, the next step is to select the patterns which are to be used for decomposing the image.
-
-	* Variate the settings of the Rainbow-image via the sliders for intensity and lifetime to enhance the contrast between the different regions.
-	{{ pattern_matching_Image_3.png?600 }}
-
-	Response: The image now clearly shows two populations of different lifetimes. This means, that probably two patterns are sufficient to fit the image.
-
-	Note: Have a look at the //Decay Diversity Map//. This new feature tells you more about the different populations in the picture. The //Decay Diversity Map// is a 2D histogram. The x-axis shows a histogram of the average lifetimes of the pixels in the image. The y-axis shows a histogram of the Delta-Tau values of each pixel in the image.\\
-	This quantity relates to the deviation of the fluorescence decay from a mono-exponential decay. While a small value depicts a mono-exponential decay, a bigger Delta-Tau value indicates a multi-exponential one.\\
-
-	{{ pattern_matching_Image_4.png?600 }}
-
-	Note: In the example, two peaks in the average lifetime (x-axis) can already be seen.
-
-	* Zoom into the //Decay Diversity Map// to see the two different populations and their different lifetime-properties.
-	{{ pattern_matching_Image_5.png?600 }}
-
-
-
-	Response: The zoomed region is now displayed in the same resolution as before.
-	{{ pattern_matching_Image_6.png?600 }}
-
-	* Click the //Optimize View// button to enhance the resolution.\\
-
-	Response: More detailed contour lines are displayed. In our example the two populations and their different properties are depicted. The first population has a shorter lifetime and a slightly bigger Delta-Tau value compared to the second population. This indicates the first one to be the [[glossary:fret\|FRET]] population, as the lifetime is shorter and the decay "less" mono-exponential. The bigger deviation from mono-exponentiality results from the fact, that the molecules in question are composed of a mixture of one population performing FRET and an additional population of Donor-only or non-FRET molecules. The second therefore is the non-FRET population without any FRET performing molecules.
-	{{ pattern_matching_Image_7.png?600 }}
-
-	* In the right-click menu select the magic wand ROI (region-of-interest).
-
-	{{ pattern_matching_Image_8.png?600 }}
-
-	* Select the innermost contour line by clicking on it.
-	{{ pattern_matching_Image_9.png?600 }}
-
-	Response:\\
-	* The selected ROI is highlighted in the //Decay Diversity Map//. Also the location of the selected pixels in the intensity image is depicted.
-	* Click //Add ROI// in the //Patterns// box.
-	{{ pattern_matching_Image_10.png }}
-
-	Response: The selected ROI appears as pattern in the box and its overall decay is displayed in the //Patterns// plot.
-	{{ pattern_matching_Image_11.png?600 }}
-
-	* Rename the pattern by clicking on it. Change the name to "//FRET pattern//".
-	{{ pattern_matching_Image_12.png }}
-
-	* Now repeat steps 5-8 to add the second population as "//non-FRET pattern//" to the list of patterns.
-	{{ pattern_matching_Image_13.png?600 }}
-
-	{{ pattern_matching_Image_10.png }}
-
-	Response: Both decay-patterns appear in the //Pattern// box and in the Patterns diagram.
-	{{ pattern_matching_Image_15.png?600 }}
-
-	Note: Another option to acquire the patterns would be to select the different populations in the intensity image also using the magic wand ROI. Several non-connected regions can be selected by pressing the Shift-key while clicking. The procedure resembles the one described above.\\
-	Patterns can also be imported from other measurements. For more details, see Additional Information at the end of this step-by-step manual.\\
-	Patterns can be saved by simply clicking the save button in the Pattern box. This allows for analyzing them with the TCSPC Fitting feature and calculating the values of the average FRET efficiency and binding (not shown).
-
-	==== Perform a Pattern Fit ====
-
-	Note: The desired patterns are already selected (in the example the FRET and the non-FRET pattern) and can now be used for fitting.\\
-	It is recommended to perform an initial fit before starting the Pattern Fit. This is because some parameters of the initial fit will be used in the Pattern Fit as start parameters and also because an initial fit is a good indicator, whether the defined patterns suffice to describe the image.\\
-	The initial fit {{initial_fit.png}} feature decomposes the overall decay (average over all pixels) into the user-defined patterns. This is done in terms of a linear combination. In general, for i Patterns it finds the corresponding amplitude αi:
-
-	//Decay<sub>overall</sub> = Σ α<sub>i</sub> ( Pattern<sub>i</sub> - Bkgr<sub>i</sub> )//
-
-	In addition, a shift parameter can shift the decay in time.\\
-	If the residuals of the initial fit show no trend or outliers, it is safe to assume that the selected patterns suffice to describe the image and a Pattern Fit can be started.
-
-	{{ pattern_matching_Image_16.png }}
-
-	Note: The Pattern Fit {{pattern_fit.png}} algorithm is similar to the initial fitting one, but instead of decomposing the overall decay, it performs the decomposition on each pixel. The linear decompositions of the particular pixels give different αi's for every pixel.\\
-	After the fitting process a false color RGB image is displayed which shows the amplitudes αi of the first three patterns in three different colors for each pixel.
-
-	* Since the patterns are taken from the same measurement as the overall decay, it is justified to first set the //Limits of the Shift// values to 0, as there shouldn't be any shifts. (Unselecting the Fit-boxes doesn't prevent the plot-procedure from fitting the Shift values by pressing “Inital Fit”).
-
-	{{ pattern_matching_Image_17.png }}
-
-	* Clicking //Initial Fit// will perform a fit for the overall decay (all photons of the image) by mixing the different patterns. As standard a Monte-Carlo search algorithm will be followed by a MLE optimization procedure.
-
-	{{ pattern_matching_Image_18.png }}
-
-	Response: Both decay-patterns, the overall decay and the resulting curve are displayed in the //Fit// diagram. The residual plot on the bottom should show no trends or big deviations in comparison to its noise level. This result shows that the image can be readily described by two different decay patterns belonging to two different species, the FRET and non-FRET population. The result would be even better by adding a background pattern (see Additional Information at the end of this step-by-step manual). From this result we can learn that the FRET pattern is composed of FRET molecules with only one specific FRET efficiency. Molecules with a different efficiency would result in deviations of the residual-curve and the need of a third pattern.
-
-	{{ pattern_matching_Image_19.png?600 }}
-
-	* Start fitting the image by pressing //Pattern Fit// in the //Pattern Matching// box. This process may take several minutes.
-
-	{{ pattern_matching_Image_20.png }}
-
-	Response: The intensity image will be replaced by a pattern-fitted image. In this image the decay of each pixel is fitted by mixing the patterns. The pixel color is dominated by the most prominent pattern in the pixel-decay. In the //Decay Diversity Map// the location for each pattern is depicted in the same color as in the image.
-
-	{{ pattern_matching_Image_21.png?600 }}
-
-	* Pressing //Pattern Distribution// shows black and white intensity images for the patterns, visualizing the most prominent regions of each pattern.
-	{{ pattern_matching_Image_22.png?600 }}
-
-	=== Additional Information ===
-
-	Note: There is a lot of possibilities to tailor the Pattern Matching Analysis to your needs and the given sample. Here are some examples using the same demonstration sample as above.\\
-	If decay measurements of the different components are available, one can also import patterns from different measurements.
-
-	* To import a non-FRET pattern in our example: Click //Import// in the //Patterns// box.
-
-	{{ pattern_matching_Image_23.png }}
-
-	* In the pop-up window, select the reference-measurement (in the example select //''non-FRET sample.ptu''//) and confirm by clicking //OK//.
-
-	{{ pattern_matching_Image_24.png }}
-
-	Note: This will import the overall decay of the reference measurement. Make sure the measurement contains only the desired component. To make a better selection of the pattern to be imported, open the external measurement (in the example "non-FRET sample.ptu") in another Pattern Matching Analysis and follow the step-by-step instructions to extract the desired non-FRET pattern.
-
-	* After selecting this pattern, click Save in the Patterns box.
-
-	{{ pattern_matching_Image_25.png }}
-
-	* Close the Analysis and import the saved pattern in the main analysis as described above, but instead of "non-FRET sample.ptu" select the created pattern (''Pat_non-FRET sample.pres'').
-
-	{{ pattern_matching_Image_26.png }}
-
-	Response: The imported pattern appears in the //Pattern// box and its overall decay is displayed in the //Patterns// plot.
-
-	Note: The fit of the overall decay can be improved by using a background pattern besides the patterns for the different regions.
-
-	* Extract the two patterns as described above and add them to the list of patterns.
-
-	{{ pattern_matching_Image_27.png }}
-
-	* Select the //Free ROI// tool in the right-click menu in the intensity image.
-
-	{{ pattern_matching_Image_28.png }}
-
-	* Mark a region in the black part of the image to extract a background pattern.
-
-	{{ pattern_matching_Image_29.png }}
-
-	Response:\\
-	* The selected ROI is highlighted in the intensity image (although hardly visible at standard settings). Also the location of the selected pixels in the //Decay Diversity Map// is depicted.
-	* Click //Add ROI// in the Patterns box.
-
-	{{ pattern_matching_Image_30.png }}
-
-	Response:\\
-	* The selected ROI appears as pattern in the box and its overall decay is displayed in the //Patterns// plot.
-	* Rename the new pattern by clicking on it. Change the name to "Background pattern".
-	* Limit the Shift values to 0 and perform an initial fit as described in the step-by-step instructions, section Perform a Pattern Fit.
-
-	{{ pattern_matching_Image_31.png?600 }}
-
-	Response:\\
-	* All pattern decays, the overall decay and the resulting fit are displayed in the //Fit// window. The residuals are better than before, as they show no trends or waves at all, but only noise.
-	* Start fitting the image by pressing //Pattern Fit// in the //Pattern Matching// box. This process may take several minutes.
-
-	{{ pattern_matching_Image_20.png }}
-
-	Response: The intensity image will be replaced by a pattern-fitted image. In this image the decay of each pixel is fitted by mixing the patterns. The pixel color is dominated by the most prominent pattern in the pixel-decay. In the //Decay Diversity Map// the location for each pattern is depicted in the same color as in the image.
-
-	{{ pattern_matching_Image_33.png?600 }}
-
-	* Pressing //Pattern Distribution// shows black and white intensity images of each pattern showing the most prominent regions of each pattern. The Background is now displayed in the third image.
-
-	{{ pattern_matching_Image_34.png?600 }}

dokuwiki.svg - created - external edit

Anonymous (anonymous@undisclosed.example.com) — 2026-08-18T06:49:26+00:00

	current