Time-Resolved Fluorescence Wiki

video_tutorials

Anonymous (anonymous@undisclosed.example.com) — 2014-11-17T16:36:53+00:00

2014/06/18 12:02		current
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	[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]		[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]

-	=== Kurt Thorn: Two Photon Microscopy ===	+	==== Kurt Thorn: Two Photon Microscopy ====
	Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.		Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.

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-	=== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ===	+	==== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ====
	Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.		Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
	The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).		The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).

basics

Anonymous (anonymous@undisclosed.example.com) — 2024-10-11T08:23:47+00:00

2021/09/23 14:59		current
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	{{tag> basics introduction FCS fluorescence microscopy objective resolution NA tutorials}}		{{tag> basics introduction FCS fluorescence microscopy objective resolution NA tutorials}}
-	===== Basics =====	+	====== Basics ======

	* [[video_tutorials\|Fluorescence and Fluorescence Microscopy by Nico Stuurman]]		* [[video_tutorials\|Fluorescence and Fluorescence Microscopy by Nico Stuurman]]

measurement_hardware_instrumentation

Anonymous (anonymous@undisclosed.example.com) — 2018-06-25T07:18:07+00:00

2018/06/25 09:17		current
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	[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]		[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]

-	[[technical docs:Beampath of the Zeiss LSM880]]	+	[[Beampath of the Zeiss LSM880]]

	[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]		[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]

software

Anonymous (anonymous@undisclosed.example.com) — 2025-05-28T07:47:47+00:00

2025/05/28 09:18		current
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	{{tag> software acquisition analysis}}		{{tag> software acquisition analysis}}

-	~~TOC~~

	====== Software ======		====== Software ======

community_news

Anonymous (anonymous@undisclosed.example.com) — 2020-05-04T12:11:21+00:00

2020/04/29 10:35		current
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	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\		* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\
	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\		* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamendal enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\	+	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\
	* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\		* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\
	* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\		* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\

how_to_measure_the_instrument_response_function_irf

Anonymous (anonymous@undisclosed.example.com) — 2023-09-07T22:55:03+00:00

2023/09/08 00:54		current
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	===== Using samples with ultrafast decay =====		===== Using samples with ultrafast decay =====

-	Some detectors (particularly SPADs) have wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission.	+	Some detectors (particularly MPD SPADs) have a wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission wavelength.

	==== General recipe ====		==== General recipe ====

start

Anonymous (anonymous@undisclosed.example.com) — 2021-06-17T14:47:30+00:00

2020/05/04 14:11		current
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-	<WRAP center round box 60%>
-	===== News from the community=====
-	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]]
-	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Exciting fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. \\ \\
-
-
-
-	[[general:community_news\|...see more...]]
-	</WRAP>

applications

Anonymous (anonymous@undisclosed.example.com) — 2026-01-12T10:25:53+00:00

2023/02/23 09:12		current
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	[[howto:antibunching_measurements\|Photon antibunching measurements]]		[[howto:antibunching_measurements\|Photon antibunching measurements]]
		+
		+	[[howto:burst_duration_and_fcs_diffusion_time:burst_duration_and_fcs_diffusion_time\|Burst Duration and FCS Diffusion Time]]


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	[[howto:calculate_ratiometric_single_pair_fret_distributions\|Calculate ratiometric single pair FRET distributions]]		[[howto:calculate_ratiometric_single_pair_fret_distributions\|Calculate ratiometric single pair FRET distributions]]
		+
		+	[[howto:burst_duration_and_fcs_diffusion_time:burst_duration_and_fcs_diffusion_time\|Burst Duration and FCS Diffusion Time]]

faq

Anonymous (anonymous@undisclosed.example.com) — 2025-03-14T09:40:11+00:00

2025/03/14 10:34		current
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	{{topic> faq -draft &nodate&nouser}}		{{topic> faq -draft &nodate&nouser}}
-	[[FAQ:old dongles drivers for FluoFit SymPhoTime 32:old dongles drivers for FluoFit SymPhoTime 32\|Using old dongles for FluoFit and older versions of SymPhoTime (SymPhoTime 32) with Windows 10 or 11]]

interfacing_time_resolved_spectrometer_fluotime_300_microscope_microtime_100

Anonymous (anonymous@undisclosed.example.com) — 2020-05-18T14:19:46+00:00

2020/05/18 16:19		current
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-	{{tag> video FT300 easyTau MT100 FLIM microscopy howto tutorial draft}}	+	{{tag> video FT300 easyTau MT100 FLIM microscopy howto tutorial}}

	====== Interfacing a time-resolved spectrometer (FluoTime 300) with a microscope (MicroTime 100) ======		====== Interfacing a time-resolved spectrometer (FluoTime 300) with a microscope (MicroTime 100) ======

lifetime_component_decomposition_phasor_plot_analysis

Anonymous (anonymous@undisclosed.example.com) — 2025-06-16T07:17:47+00:00

2025/06/16 09:17		current
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	* Sample information:		* Sample information:
-	* Daisy Pollen	+	* Daisy pollen
		+
		+	---

	* Software information: NIS-Elements version 5.42.06 with PicoQuant FLIM-FCS full integration		* Software information: NIS-Elements version 5.42.06 with PicoQuant FLIM-FCS full integration
	{{:lifetime_component_decomposition_in_phasor_plot.mp4?1000\|}}		{{:lifetime_component_decomposition_in_phasor_plot.mp4?1000\|}}
-		+
	* Sample information:		* Sample information:
	* Glial Fibrillary Acidic Protein (GFAP) — labeled with BDP-TMR		* Glial Fibrillary Acidic Protein (GFAP) — labeled with BDP-TMR
	* Mitochondria (via TOM20) — labeled with CY3		* Mitochondria (via TOM20) — labeled with CY3

flim

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:28:34+00:00

2015/11/03 15:28		current
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-	~~TOC~~	+
	{{tag>FLIM}}		{{tag>FLIM}}
		+
		+	~~TOC~~
	====== FLIM ======		====== FLIM ======

align_beam_backreflection

Anonymous (anonymous@undisclosed.example.com) — 2022-08-29T11:17:12+00:00

2022/08/29 13:15		current
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	Adding a drop of water on top of the coverslip reduces the back-scattering intensity by an order of magnitude or so, but the basic shape remains the same. Even if one removes everything extra from the beam path, these fringes remain there, they are universal. Note that all those images above are slightly defocused.		Adding a drop of water on top of the coverslip reduces the back-scattering intensity by an order of magnitude or so, but the basic shape remains the same. Even if one removes everything extra from the beam path, these fringes remain there, they are universal. Note that all those images above are slightly defocused.

-	Such images are used for the daily and fundamental alignment of the MicroTime 200. The detailed procedure is described in the user manual of MicroTime 200. Although the MicroTime is extremely robust we suggest to spend 5-10 minutes every day for verifying the quality of the alignment by inspecting the back-reflection pattern. Have a look how this is used also by watching the tutorial [[howto:exchange_dichroic_mt200\|How to exchange the main dichroic of the MicroTime 200.]]	+	Such images are used for daily and fundamental alignment of the MicroTime 200. A detailed procedure is described in the user manual of MicroTime 200. Although MicroTime is extremely robust, we suggest to spend 5-10 minutes every day for verifying the quality of the alignment by inspecting the back-reflection pattern. Have a look how this is used also by watching the tutorial [[howto:exchange_dichroic_mt200\|How to exchange the main dichroic of the MicroTime 200.]]

	The paper by [[ http://rspa.royalsocietypublishing.org/content/253/1274/358\|B. Richards and E. Wolf: Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system]] shows such 4 lobed patterns for polarized light.		The paper by [[ http://rspa.royalsocietypublishing.org/content/253/1274/358\|B. Richards and E. Wolf: Electromagnetic diffraction in optical systems, II. Structure of the image field in an aplanatic system]] shows such 4 lobed patterns for polarized light.

	Similar figures can be found in this [[https://books.google.cz/books?id=kURdAAAAQBAJ\|classic textbook]].		Similar figures can be found in this [[https://books.google.cz/books?id=kURdAAAAQBAJ\|classic textbook]].

exchange_dichroic_mt200

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:42:54+00:00

2021/06/25 11:19		current
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-		+	{{tag>howto MicroTime200 dichroic microscopy}}
-	{{tag>howto MicroTime200 dichroic microscopy MT200}}	+

	====== How to exchange the main dichroic of the MicroTime 200 ======		====== How to exchange the main dichroic of the MicroTime 200 ======

flim-fret_calculation_for_single_exponential_donors

Anonymous (anonymous@undisclosed.example.com) — 2020-05-06T09:38:57+00:00

2020/05/06 11:19		current
Line 85:		Line 85:

	Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.		Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.
-	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v2.png?400 \|}}	+	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.png?400 \|}}

	* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".		* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".

start

Anonymous (anonymous@undisclosed.example.com) — 2026-02-11T14:19:03+00:00

2024/12/19 16:57		current
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	{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}		{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}

-	===== NovaFLIM / Analysis =====
-
-	{{topic>howto +tutorial +novaflim -draft &nodate&nouser}}

	===== SymPhoTime64 / Analysis =====		===== SymPhoTime64 / Analysis =====

symphotime_tips_and_tricks

Anonymous (anonymous@undisclosed.example.com) — 2020-04-03T14:54:26+00:00

2020/04/03 16:54		current
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-	~~TOC~~

	{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}		{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}
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	====== SymPhoTime 64 Analysis Tips and Tricks ======		====== SymPhoTime 64 Analysis Tips and Tricks ======
		+
		+	~~TOC~~
		+
	===== Summary =====		===== Summary =====

microtime

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:24:05+00:00

2015/11/03 15:23		current
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	* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm		* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm
	* Up to 4 truly parallel detection channels using application-optimized detection with [[glossary:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s		* Up to 4 truly parallel detection channels using application-optimized detection with [[glossary:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s
-	* Time-Correlated-Single Photon Counting ([[glossary:TCSPC]]) and [[glossary:TTTR]] mode for investigation of fast dynamics in FCS and FLIM	+	* Time-Correlated-Single Photon Counting ([[glossary:TCSPC]]) and [[glossary:TTTR]] mode for investigation of fast dynamics in [[glossary:FCS]] and [[glossary:FLIM]]
	* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning		* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning
	* Two optional exit ports for additional hardware, e.g. spectrographs		* Two optional exit ports for additional hardware, e.g. spectrographs

flimfit

Anonymous (anonymous@undisclosed.example.com) — 2014-05-23T09:04:11+00:00

2014/05/23 11:02		current
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-	{{tag> software analysis FLIM }}	+	{{tag> software analysis FLIM open_source}}

	====== FLIMfit ======		====== FLIMfit ======

phasor_plot_structure_separation

Anonymous (anonymous@undisclosed.example.com) — 2025-06-16T08:36:34+00:00

2025/06/16 10:36		current
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	{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}		{{tag> FLIM acquisition Nikon AX Phasor video LSM_upgrade howto}}

-	====== Phasor-Based Structural Separation Using FLIM ======	+	====== Nikon AX: Phasor-Based Structural Separation Using FLIM ======

	Introduction		Introduction

2020/04/29 10:35		current
Line 4:		Line 4:
	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\		* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\
	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\		* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamendal enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\	+	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\
	* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\		* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\
	* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\		* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\