Time-Resolved Fluorescence Wiki - faq

how_can_i_analyze_my_data_using_non-picoquant_software

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:49:47+00:00

2025/01/31 09:01		current
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	====== How can I analyze my data using non-PicoQuant software? ======		====== How can I analyze my data using non-PicoQuant software? ======

-	{{tag> file_format ptu open_source faq}}	+	{{tag> faq file_format ptu open_source analysis software}}

	The PicoQuant raw-data format is freely accessible. PicoQuant has published some demo scripts for reading the different TCSPC/TTTR data formats on Github:		The PicoQuant raw-data format is freely accessible. PicoQuant has published some demo scripts for reading the different TCSPC/TTTR data formats on Github:

laser_intensity_response

Anonymous (anonymous@undisclosed.example.com) — 2025-03-11T08:23:49+00:00

2025/03/11 08:16		current
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	{{tag>FAQ LDH PLS PDL Laser SymPhoTime SPT SEPIA}}		{{tag>FAQ LDH PLS PDL Laser SymPhoTime SPT SEPIA}}

-	===== The intensity response of my laser is not linear =====	+	====== The Intensity Response of my Laser is not linear ======
	or		or
-	===== My laser does not turn on with low power =====	+	====== My Laser does not turn on with low Power ======

-	==== Background ====	+	===== Background =====

	In virtually all laser scanning microscopes from other manufacturers, the laser intensity is regulated via an AOTF. The AOTF is calibrated in a way that the intensity of the laser increases linearly with a linear setting in the LSM software. This can create confusion when customers are used to other systems and then work with PQ-hardware.		In virtually all laser scanning microscopes from other manufacturers, the laser intensity is regulated via an AOTF. The AOTF is calibrated in a way that the intensity of the laser increases linearly with a linear setting in the LSM software. This can create confusion when customers are used to other systems and then work with PQ-hardware.
	Worst case they think their head is damaged.		Worst case they think their head is damaged.

-	==== Answer ====	+	===== Answer =====

	In the SymPhoTime and SEPIA II software, not the laser intensity is regulated, but the current driving the laser. Any diode has a different intensity response.		In the SymPhoTime and SEPIA II software, not the laser intensity is regulated, but the current driving the laser. Any diode has a different intensity response.
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	As the pulse shape changes with the settings on the laser driver or the software, we recommend to use optical attenuation to regulate the intensity, for PQ-produced systems either available on the Laser Coupling Unit or - if it's a directly coupled laser - at the attenuation screw in front of the laser.		As the pulse shape changes with the settings on the laser driver or the software, we recommend to use optical attenuation to regulate the intensity, for PQ-produced systems either available on the Laser Coupling Unit or - if it's a directly coupled laser - at the attenuation screw in front of the laser.

-	==== Additional info ====	+	===== Additional info =====

	In order to avoid confusion, set the intensity to 100% as a default in the software/on the driver and attenuate only optically. This setting is usually good enough, unless the sample lifetime is extremely short.		In order to avoid confusion, set the intensity to 100% as a default in the software/on the driver and attenuate only optically. This setting is usually good enough, unless the sample lifetime is extremely short.

novaflim_calculated_irf

Anonymous (anonymous@undisclosed.example.com) — 2025-03-11T08:19:29+00:00

2025/02/18 13:31		current
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	====== NovaFLIM Calculated IRF ======		====== NovaFLIM Calculated IRF ======
		+	{{tag> faq software novaflim IRF}}
		+

	In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.		In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.
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	* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)		* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)

-	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.	+	The IRF is determined only during the initial TCSPC fit, which means it may vary depending on the different exponential components. During the pixelwise fitting in FLIM, this IRF is applied without any modifications. Consequently, all image points are fitted using the same fixed IRF established during the initial fit.
		+
		+	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.

novaflim_license

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:50:52+00:00

2025/02/14 15:39		current
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-	====== NovaFLIm Licenses ======	+	====== NovaFLIM Licenses ======
		+	{{tag> faq software NovAFLIM license}}

	For NovaFLIM, we use a software license instead of a hardware dongle to make license management more efficient. Compared to a hardware dongle, this approach eliminates the time and costs associated with handling and shipping dongles, as well as the risk of losing or removing them.		For NovaFLIM, we use a software license instead of a hardware dongle to make license management more efficient. Compared to a hardware dongle, this approach eliminates the time and costs associated with handling and shipping dongles, as well as the risk of losing or removing them.

start

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T09:43:52+00:00

2025/02/14 09:36		current
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	==== Why do I not get the same count rates on both SPAD detectors? ====		==== Why do I not get the same count rates on both SPAD detectors? ====

-	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).	+	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).

-	If you are using either a proper (see below) 50/50 cube beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:	+	If you are using either a proper (see below) 50/50 beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:

	1. Misalignment of the Lens in Front of the SPAD:		1. Misalignment of the Lens in Front of the SPAD:
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	- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).		- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).
-	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).	+	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).
	- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.		- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.
	- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.		- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.
-	- If the detectors are the same type, and the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked.	+	* If the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked and maximized.
		+	* Follow the "Everyday Alignment" Procedure from the MicroTime 200 manual to correctly align the lens. It might be that also "Fundamental Alignment" is necessary.
	- Introduce the beamsplitter and measure the combined count rate from both detectors.		- Introduce the beamsplitter and measure the combined count rate from both detectors.
		+	* The sum of the signal on both detectors should be the same as from the previous measurement (i.e. 100% going to Detector 1 or 2).
		+	* If it is not (again, allow for 10% error), check the alignment.

-	If the sum	+	If you have followed these instructions, and the light is not split 50/50, then this is likely a property of the beamsplitter. Make sure that is it the correct kind of beamsplitter (see above), and/or contact us for advice.
-		+
-		+
-	By following these steps, you can determine if the discrepancy in count rates is due to misalignment or the characteristics of the beam-splitter.	+
	===== SymPhoTime =====		===== SymPhoTime =====

the_dark_counts_of_my_spads_increased._when_do_i_need_to_take_action

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:46:14+00:00

2025/01/31 08:57		current
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	====== The Dark Counts of my SPADs increased. When do I need to take action? ======		====== The Dark Counts of my SPADs increased. When do I need to take action? ======
-	{{tags> detector faq SPAD dcps dark_counts}}	+	{{tag> detectors faq SPAD dcps dark_counts}}

	SPAD detectors (Excelitas SPCMs or MPD PDMs) are relatively robust devices, but they can exhibit ageing, which shows mostly in the level of dark counts. Dark counts are the count rate on the detector when no light at all can get to the active area of the detector.		SPAD detectors (Excelitas SPCMs or MPD PDMs) are relatively robust devices, but they can exhibit ageing, which shows mostly in the level of dark counts. Dark counts are the count rate on the detector when no light at all can get to the active area of the detector.

why_do_i_not_get_the_same_count_rates_on_both_spad_detectors

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:47:18+00:00

2025/02/14 15:42		current
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	====== Why do I not get the same count rates on both SPAD detectors? ======		====== Why do I not get the same count rates on both SPAD detectors? ======
		+	{{tag> detectors faq SPAD MT200}}

	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).		In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).