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Fluorescence Lifetime Imaging

Fluorescence Lifetime Imaging Microscopy (FLIM) 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.

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 23800 468 44 30.6
K6-207
K6-207
3-DAB
3-DAB
EtOH 460 8600 665 22 4.0
K7-547
K7-547
SeTau-405-NHS
SeTau-405-NHS
NH2 IgG, D/P=8 405 13800 518 19 8.4
K7-567
K7-567
SeTau-405-Azide
SeTau-405-Azide
triple-CC EtOH 391 15000 498 55 8.5
K8-1342
K8-1342
Seta-670-NHS
Seta-670-NHS
NH2 BSA, D/P=1.5 681 180000 696 27 2.43
K8-1352
K8-1352
Square-660-NHS
Square-660-NHS
NH2 BSA D/P=1.2 676 182000 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 135000 677 7 1.52
K8-1626
K8-1626
Seta-633-di-NHS
Seta-633-di-NHS
NH2 IgG, D/P=1 639 192000 650 33 3
K8-1663
K8-1663
Seta-633-NHS
Seta-633-NHS
NH2 IgG D/P=1 637 250000 647 26 1.49
K9-4119
K9-4119 new
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 200000 691 58 3.2
K9-4148
K9-4148
SeTau-647-Maleimide
SeTau-647-Maleimide
SH PB 7.4 648 200000 692 45 3.2
K9-4149
K9-4149
SeTau-647-NHS
SeTau-647-NHS
NH2 IgG, D/P=1 648 200000 694 59 3.3
K9-4150
K9-4150
SeTau-647
SeTau-647
PB 7.4 647 211000 693 59 3.1
K7-548
K7-548
SeTau-405-Maleimide
SeTau-405-Maleimide
SH EtOH 391 15000 498 32 8.5
K8-1405
K8-1405
Square-650-pH-Carboxy
Square-650-pH-Carboxy
NH2 pH 9.0 535 48000 663 9 0.53
K9-4179
K9-4179 new
SeTau-680-NHS
SeTau-680-NHS
NH2 IgG D/P=1 682 703 47

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.

flim1
flim1

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.

 

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flim1

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.

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Frequency responses of SeTau-647
in aqueous buffer solution pH 7.4
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Intensity decays of SeTau-647 in aqueous
buffer solution pH 7.4 compared to Alexa 647

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

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