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, and the environment of a fluorescent probe or label. 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.

 

SETA BioMedicals has developed several fluorescent probes and labels that are suitable for lifetime-based imaging applications. Some of these have longer lifetimes up to 31 ns.

			Excitation Light Sources												Characteristics
Product Number (Specs Sheet)	Product Name (Product Info)	Target Group	380	405	436	488	532	594	635	650	680	700	750	780	Medium	λ abs [nm]	ε [M–1 cm–1]	λ em [nm]	QY [%]	FLT [ns]	Buy
K1-204 K1-204	SeTau-380-NHS SeTau-380-NHS	NH2	•	•											BSA, D/P=1	383	23,800	468	44	30.6
K6-207 K6-207	3-DAB 3-DAB			•	•	•									EtOH	460	8,600	665	22	4.0
K7-547 K7-547	SeTau-405-NHS SeTau-405-NHS	NH2	•	•											IgG, D/P=8	405	13,800	518	19	8.4
K7-567 K7-567	SeTau-405-Azide SeTau-405-Azide	triple-CC	•	•											EtOH	391	15,000	498	55	8.5
K8-1342 K8-1342	Seta-670-NHS Seta-670-NHS	NH2	•	•					•	•					BSA, D/P=1.5	681	180,000	696	27	2.43
K8-1352 K8-1352	Square-660-NHS Square-660-NHS	NH2	•	•					•	•					BSA D/P=1.2	676	182,000	695	13	3.32
K8-1407 K8-1407	Square-650-pH-NHS Square-650-pH-NHS	NH2						•	•	•					IgG, D/P=0.8 pH=2.0	662	135,000	677	7	1.52
K8-1663 K8-1663	Seta-633-NHS Seta-633-NHS	NH2						•	•						IgG D/P=1	637	250,000	647	26	1.49
K9-4119 K9-4119	SeTau-665-NHS SeTau-665-NHS	NH2								•	•	•			IgG, D/P=1.5	662		716	46
K9-4142 K9-4142	SeTau-647-di-NHS SeTau-647-di-NHS	NH2							•	•					IgG, D/P=0.5	649	200,000	691	58	3.2
K9-4148 K9-4148	SeTau-647-Maleimide SeTau-647-Maleimide	SH							•	•					PB 7.4	648	200,000	692	45	3.2
K9-4149 K9-4149	SeTau-647-NHS SeTau-647-NHS	NH2							•	•					IgG, D/P=1	648	200,000	694	59	3.3
K9-4150 K9-4150	SeTau-647 SeTau-647								•	•					PB 7.4	647	211,000	693	59	3.1
K7-548 K7-548	SeTau-405-Maleimide SeTau-405-Maleimide	SH	•	•											EtOH	391	15,000	498	32	8.5
K8-1405 K8-1405	Square-650-pH-Carboxy Square-650-pH-Carboxy	NH2						•	•	•					pH 9.0	535	48,000	663	9	0.53
K9-4179 K9-4179	SeTau-680-NHS SeTau-680-NHS	NH2							•	•	•				IgG D/P=1	682		703	47	3.1
K9-3152 K9-3152	SeTau-488-NHS SeTau-488-NHS	NH2				•
K9-4159 K9-4159 new	SeTau-660-NHS SeTau-660-NHS	NH2								•					IgG Conjugate D/P = 1	663	240,000	694	56

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 up to 32 ns are listed under FLT labels.

 

Reactive Labels for FLIM :

Squaraine-rotaxanes are extremely bright, photostable and have several ns lifetimes and are therefore well-suited for FLIM measurements. SeTau-647 (SeTau-647-NHS and SeTau-647-Maleimide) is the brightest dye currently on the market for the Kr-ion laser. It combines an extremely high quantum yield of 60% with an extinction coefficient of 200,000 [M^-1cm^-1]. Some FRET combinations of these dyes with Seta or SQ dyes as acceptors yield FRET pairs with Förster distances up to 80 Angstrom.

Fluorescent Probes for FLIM :

SeTau-633-Ethyl-Ester is hydrophobic in nature and therefore passively penetrates 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 probe 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. 

For more specific information we refer you to B.Smith et al., Angew. Chem. 2007, 119, 5624-5627.

See also Fluorescence Imaging and NIR Fluorescence Imaging.