FCS

Fluorescence Correlation Spectroscopy (FCS) is a fluorescence microscopy-based technique that provides information on diffusion rate, absolute numbers and changes in particle fluorescence of fluorescently labeled molecules. FCS measures fluorescence intensity fluctuations due to diffusion of fluorescent molecules through the confocal volume element or other measurable changes in the confocal volume element. The fluctuations are correlated over time, yielding quantitative information on molecular mobility and local concentration. Confocal fluorescence microscopy measurements in cells allow studying the interactions and dynamics of biomolecules in their native environment. FCS has therefore become an important tool for various disciplines for following biological events and cellular processes.

SETA BioMedicals offers a number of highly photostable fluorescent labels for use in FCS applications. For a comprehensive list of our proprietary labels we refer you to our Seta dyes.

			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
K8-3335 K8-3335	Seta-555-NHS Seta-555-NHS	NH2					•								PB 7.4	555	155,000	570	7
K8-3345 K8-3345	Seta-555-DBCO Seta-555-DBCO	N3					•								PB 7.4	555	155,000	570	7
K8-3346 K8-3346	Seta-555-Azide Seta-555-Azide	triple-CC					•								PB 7.4	555	155,000	570	7
K8-5035 K8-5035	Seta-650-NHS Seta-650-NHS	NH2							•	•					PB 7.4	651	200,000	671	28
K8-5036 K8-5036	Seta-650-Maleimide Seta-650-Maleimide	SH							•	•					PB 7.4	652	200,000	672	28
K8-5045 K8-5045	Seta-650-DBCO Seta-650-DBCO	N3							•	•					PB 7.4	653	200,000	674	28
K8-5046 K8-5046	Seta-650-Azide Seta-650-Azide	triple-CC							•	•					PB 7.4	651	200,000	671	28
K9-4119 K9-4119	SeTau-665-NHS SeTau-665-NHS	NH2								•	•	•			PB 7.4	664	160,000	712	53	3.1
K9-4142 K9-4142	SeTau-647-di-NHS SeTau-647-di-NHS	NH2							•	•					PB 7.4	650	200,000	694	65	3.2
K9-4145 K9-4145	SeTau-633-Ethyl-Ester SeTau-633-Ethyl-Ester								•	•					CHCl3	634	105,000	683	68
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							•	•					PB 7.4	649	200,000	695	61	3.2
K9-4150 K9-4150	SeTau-647 SeTau-647								•	•					PB 7.4	647	211,000	693	59	3.1
K8-1341 K8-1341	Seta-670-Maleimide Seta-670-Maleimide	SH	•	•					•	•					PB 7.4	667	180,000	688	7

If you consider using FRET in combination with FCS in your application we refer you to FRET Applications, where we offer a series of FRET pairs with Forster distance from 40 – 80 Angstrom. Should you require a specific FRET pair that is not on the list please contact us.

Our newly developed Squaraine-rotaxanes are extremely bright and photostable and well-suited for FCS 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 a quantum yield of ~60% with an extinction coefficient of >200,000 M^-1cm^-1.

SeTau-647-maleimide has been successfully used in the studies of the mechanism of muscle contractions using FCS, specifically fluorescence lifetime correlation spectroscopy (FLCS), lifetime and time-resolved fluorescence polarization methods [26]. The diffussion coefficient was determined to be 2.5 μm²s^-1 and the correlation time was 10.5 ms. Myofibrils were labeled at the myosin light chain 1 position with SeTau-647-maleimide. SeTau dyes have several important advantages over a single isomer of TRITC used in an earlier study. Most importantly, SeTau-647 dyes are excited in the red and thus have reduced autofluorescence. Furthermore, they are well suited for excitation with a 635 nm diode laser, and they have large Stokes shifts (44 nm), a much higher photostability than Cy5 or Alexa 647, have a high extinction coefficient, and a several times longer fluorescent lifetime than Cy5 or Alexa 647. The 3 ns fluorescence lifetime implies a relatively high polarization. SeTau 647 is a unique dye with a high excitation in the red region and a relatively long lifetime, two properties that are usually mutually exclusive [27].

Autocorrelation function of SeTau-647-Maleimide (9.8 nM) in water [27]

An average of 3.6 molecules was observed in the FCS measurement. When the 1/G(0) was plotted vs. the number of SeTau-647 molecules in the detection volume, the extrapolation revealed that the concentration contributed by one molecule of the dye corresponds to app. 75 counts per channel, i.e. the total fluorescence (I_total = I_║ + 2I_┴) from one molecule of SeTau-647 is 225 counts/s. In a typical experiment the signal was 10,000 counts/s, corresponding to about 50 molecules [26].