Fluorescence Lifetime (FLT)

FLT Probes and Labels:

			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
K7-545 K7-545	SeTau-425-NHS SeTau-425-NHS	NH2	•	•	•										BSA, D/P=1	427	4,200	528	16	26.2
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
KS-650 KS-650	590–650 nm FLT Standard 590–650 nm FLT Standard								•
K7-544 K7-544	SeTau-425-Maleimide SeTau-425-Maleimide	SH	•	•	•										PB 7.4	425	4,200	545	39	26.2

1. Environment-sensitive lifetime dyes

Selected Square and Seta dyes exhibit shorter fluorescence lifetimes (FLTs) in aqueous media or when labeled to low molecular weight analytes (drugs or small ligands) but increased brightness and up to ten-times longer FLTs when bound to proteins or oligonucleotides. These environment sensitive Square and Seta labels are well-suited as tracers in FLT-based assays, where e.g. the FLT-tracer would be labeled to a small antigen (ligand) displaying a short lifetime but showing a substantial FLT-increase upon binding to a larger protein (antibody or receptor). Square-660-Carboxy is also used as a fluorescent probe that binds to hydrophobic regions in cells. For a general technical note about fluorescence lifetime including additional references see FLT-TN and our product flyer on Fluorescence Lifetime Probes and Lables.

The table below provides data of the expected changes in lifetime for selected FLT dyes. It is important to note that the observed lifetime change of a fluorescently labeled ligand will strongly depend on the nature and size of the biomolecule it binds to:

 

Product	Dye	Dye–BSA Conjugate		Δτ [ns] τ(Dye–conj.) – τ(Dye)
	τmean [ns]	D/P ratio	τmean [ns]
Seta-670-NHS	0.42	1.5	2.43	2.01
Square-660-NHS	0.27	1.2	3.32	3.05
Seta-633-NHS	0.25	0.9	2.81	2.56

 

All of these environment-sensitive FLT labels are excitable with a 633, 635 or 647 nm laser source and Square-660 and Seta-670 can also be excited with a 405 nm laser.

 

a) FLT probes

Probes such as Square-660-Carboxy show large changes in FLT upon binding to either proteins or cell-surfaces. Square-660-Carboxy is water-soluble but with a strong affinity for the hydrophobic regions of biomolecules.

Square-650-pH is a NIR probe that exhibits a pKa around 7 and is therefore useful for measurement of pH changes in the physiologically relevant pH range from pH 6 to pH 8. Importantly both forms (protonated as well as unprotonated) of Square-650-pH are fluorescent and they exhibit different lifetimes which makes this compound useful for lifetime based applications (see Figure below). For more information about these dyes we refer you to their specs sheets and the published literature.

 

pH-Probe	pH	τ [ns]
Square-650-pH	2.0	1.17
	9.0	0.53

b) Hydrophilic FLT labels

These are reactive labels that show changes in FLT upon binding to either proteins, oligonucleotides or cell-surfaces. In general they are labeled to a low-molecular-weight binding partner and interaction with a high-molecular-weight protein or receptor leads to a change in FLT:

 

2. Environment-insensitive lifetime labels

a) Long-lifetime, short-wavelength FLT labels

These proprietary long-lifetime labels with lifetimes up to 32 ns in water are excitable in the UV or blue region. They are useful as polarization probes for the measurement of high MW analytes (small - medium size proteins). The measurement of BSA (MM of 65 K) in a FP assay with SeTau-425-NHS demonstrates that a suitable dynamic range can be achieved even for large antigens with  this long-lifetime polarization probe. Importantly the FLT of these labels does not change upon labeling or binding to proteins or other analytes. Several labels with fluorescent lifetimes of 32, 26 and 9 ns in aqueous environments are available.

 

Relevant Products	Lifetime [ns]
SeTau-380-NHS	32.5
SeTau-405-NHS and SeTau-405-Maleimide	9.1-9.3
SeTau-425-NHS	26.2

 

b) Long-wavelength, squaraine-rotaxane-based FLT labels

SeTau-647-NHS, SeTau-647-Maleimide and SeTau-647-di-NHS are FLT labels with lifetimes that are insensitive to the environment. The average lifetime of these labels is in the order of 3 ns which make them superior lifetime or polarization probes over Cy5 or Alexa dyes (Cy5, Alexa 647: lifetime ~ 1 ns in water). The longer lifetimes make them also more suitable donors in energy-transfer-based assays assuring that even at higher energy transfer rates the lifetime of these labels is not too short to be accurately measured with inexpensive instrumentation.

SeTau-647-maleimide has been successfully used in the studies of the mechanism of muscle contractions using FCS, specifically fluorescence lifetime correlation spectroscopy (FLCS), fluorescence lifetime and time-resolved fluorescence polarizations methods [27].