SETA BioMedicals

Fluorescence Lifetime Imaging

Fluorescence Lifetime Imaging

Fluorescence Lifetime Imaging Microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent specimen. FLIM is in particular useful as an imaging tool to be used on molecules in living cells. There are a number of parameters that have an influence on the fluorescence lifetime of a probe or label. Such factors are for example ion concentrations, oxygen concentration, interaction of molecules. Unlike fluorescence intensity, fluorescence lifetime is to some extent independent of the fluorophore concentration, photo-bleaching, light scattering and fluctuations of the excitation light intensity. Therefore fluorescence lifetime is a more robust read out parameter and enables less artifact prone measurements of analytes and FRET analysis.

There are two methods of fluorescence lifetime measurements: the time-domain method and the frequency-domain method.

Time-domain FLIM - In the time domain the sample is excited with a short laser pulse with a few hundred picosecond pulse duration and the time between the pulse and the arrival of the photon at the PMT is measured. This process is repeated for a large number of photons and a time-pin histogram is generated which is then fitted with an exponential decay function to give the lifetime of the measured luminescent compound. Fluorescence lifetime images are typically expressed in a pseudo-color according to their lifetimes.

Frequency-domain FLIM – In frequency domain the excitation light is sinusoidally modulated at a specific frequency and the fluorescence lifetime is calculated from the phase shift or demodulation of the emitted light compared to the excitation light. Due to the non-zero lifetime of the fluorescent species, the emitted fluorescence light is phase-shifted and demodulated compared to the excitation light.

FRET Imaging

Since the fluorescence lifetime of a fluorophore depends on both, the radiative and non-radiative rates, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor. FRET can be used to generate lifetime changes that are then measured pixel by pixel to provide the FLIM image.

If you consider using FRET in combination with FLIM in your application we refer you to FRET applications, where we offer a series of FRET pairs with Förster distances from 40 – 80 Angstrom. Should you require a specific FRET pair that is not on the list please contact us. FLT labels with long lifetimes in the range between 8 and 32 ns are listed under FLT labels.

Reactive Labels for FLIM :

Our newly developed Squaraine-rotaxanes are extremely bright, photostable and have several ns lifetimes and are therefore well-suited for FLIM measurements. SeTau 647 is the brightest dye currently on the market for the Kr-ion laser. It combines a extremely high quantum yield of 60% with an extinction coefficient of 200,000 [M-¹.cm-¹]. Some FRET combinations of these dyes with Seta or SQ dyes as acceptors yield FRET pairs with Forster distance up to 80 Angstrom. Please contact us at info@setabiomedicals.com or 217 417 2160 for more information.

Frequency responses of SeTau 665 (K8-4119)
in aqueous buffer solution pH 7.4.

Intensity decays of SeTau 665(K8-4119) in aqueous
buffer solution pH 7.4 in comparison to Cy5 and Alexa 647.

Product	Lifetime [ns]	Pack Size	Product Code
SeTau 647-NHS	3.2	0.5mg	K9-4149-0.5mg
SeTau 647-di-NHS	3.2	0.5mg	K9-4142-0.5mg
SeTau 665-NHS	3.1	0.5mg	K9-4119-0.5mg
SeTau 665-di-NHS	3.1	0.5mg	K9-4113-0.5mg

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Fluorescent Probes for FLIM :

These probes are hydrophic in nature and therefore passively penetrate the cell membranes. Once inside the cell the ethyl ester modified probes are hydrolyzed by esterases thereby forming carboxyl groups that are cell-impermeable. These probes can be structurally modified for targeting of different cellular locations for in vitro and in vivo optical imaging of live and fixed cells. Images stained with some of these probes are stable for hundreds of hours.

Product	Pack Size	Product Code
SeTau 633	1 mg	K9-4145-1mg
SeTau 650	1 mg	K9-4116-1mg
SeTau 656	1 mg	K9-1604-1mg

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Product Specifications:


		Excitation Light Sources								Free Dye						Dye Conjugate
Product Number	Product Name	380	405	436	488	532	594	635	670	Solvent	λ_Ab max. [nm]	ε [M^–1cm^–1]	λ_Em max. [nm]	QY [%]	FLT [ns]	Carrier PB 7.4	λ_Ab max. [nm]	λ_Em max. [nm]	QY [%]	FLT [ns]
K7-545	SeTau-425-NHS		•								425	4200	545	39	26.2	0.0	427	528	16
K7-547	SeTau 405 NHS	•	•							PB 7.4	405	13800	518	80	9.3	BSA, D/P=8	406	518	20	7.6
K7-549	SeTau-404-NHS	•	•							PB 7.4	402	10600	515	52	9.3	BSA, D/P=2	402	503		8.8
K8-1252	Square-635-di-NHS	•						•		PB 7.4	636	216000	648	6	0.26	BSA, D/P=0.5	652	665	20	2.44
K8-1262	Seta-635-di-NHS		•					•		PB 7.4	640	120000	653	5	0.5	IgG, D/P=1	646	658	13
K8-1352	Square-660-NHS		•						•	PB 7.4	658	182000	677	3	0.26	BSA, D/P=1	676	695	13	3.32
K8-1500	Square-655	•	•						•	MeOH	628	177000	654	0.09		1 g/L BSA	652	672	80
K9-4112	SeTau-655-di-NHS							•		PB 7.4	665	200000	673	25	0.9	IgG, D/P=1	657	675	31	1.8
K9-4113	SeTau-665-di-NHS	•							•	PB 7.4	665	140000	715	62	3.1	IgG, D/P=1	664	709	43	3.3
K9-4142	SeTau-647-di-NHS							•		PB 7.4	650	368000	694	65	3.2	IgG, D/P=0.5	649	691	58	3.2
K1-204	SeTau-380-NHS	•								Water	270	23800	480	56	32.5	BSA,	383	468	44	30.6
K4-1082	SeTau-270-NHS	•								Water	272	10800	385	6	10.2	BSA, D/P=1	330	495	5	11.7
K8-1667	Seta-635-NH-di-NHS							•		PB 7.4	640	188000	656	33	1.8	IgG, D/P=1	646	663	21
K8-1300	Square-670-di-COOH	•	•						•	PB 7.4	667	207000	685	7	0.46	6g/L BSA	678	698	28	3.2
K9-4149	Seta-647-NHS							•		PB 7.4	649	200000	695	61	3.2	IgG, D/P=1	648	694	59	3.3
K9-4119	SeTau-665-NHS	•	•						•	PB 7.4	664	160000	712	53	3.1	IgG, D/P=1.2	653	716	50	3.3
K9-4116	SeTau-649-ethylester	•	•					•		CHCl₃	650	309000	703	65		1 g/L BSA	652	672	80
K9-4145	SeTau-633-ethylester						•			CHCl₃	634	88000	683	68
K8-1407	Square-650-pH-NHS							•		PB 7.4	653	135000	671	16	1.17	IgG, D/P=1	662	677	7	1.52