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--- 2ffcs [2013/08/05 13:33] – 192.168.0.50
+++ 2ffcs [2026/08/18 20:49] (current) – removed - external edit (Unknown date) 127.0.0.1
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-====== 2FFCS ======
-===== Intro =====
-FFCS (sometimes also written as 2fFCS) is an abbreviation **2 F**ocus **F**luorescence **C**orrelation **S**pectroscopy. Dual focus FCS might also be used.
-Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.
-===== Experimental Setup =====
-==== Pinhole Selection ====
-The conjugated pinhole size should be chosen slightly larger than the excitation spot diameter. The conjugated pinhole size is the pinhole diameter divided by the magnification of the objective.
-e.g:
-    *60x 1.2N.A. Objective, 640nm excitation
-    *excitation FWHM = 350 nm (typical for 4x out-coupler!, the 10x out-coupler will have a larger excitation spot)
-Usually the size of the confocal volume or the diameter of the excitation spot is given as its FWHM. However the airy disc diameter is bigger and the 1/e2 diameter must be used.
-=== Single Focus ===
-<math>
-ln(2)=\frac{FWHM^2}{w_0^2} </math>
-<math> w_0=0.849 \cdot FWHM </math>
-The diameter of the excitation spot therefore is:
-<math> d_{exc}=2 \cdot w_0 = 594 nm </math>
-Together with the 60x magnification of the objective the airy disc at the pinhole location is
-<math> d_{PH}=60\cdot 594nm = 35.6\mu m </math>
-This equals 1 airy unit (AU)
-Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.
-PQ usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.
-=== Two Foci ===
-The distance between the foci in two-focus FCS is determined by the Nomarski prism. Usually the distance is around 400 nm. Therefore the 2 foci (separated by 400 nm) will occupy:
-<math> d_{exc, 2fFCS} = 2 \cdot w_0 + 400 nm = 994 nm </math>
-Together with the 60x magnification of the objective both airy discs together, at the the pinhole location, have a diameter
-<math> d_{PH, 2fFCS}=60\cdot 994nm =59.6\mu m </math>
-== Adjusting the Pinhole ==
-    -use a suitable dye solution sample to get an approx. count-rate of 105 cps. e.g. 10-9M aqueous Atto655
-    -make sure both lasers have the same power
-    -using the time trace oscilloscope optimize the pinhole position **with** Nomarski Prism and **both** lasers.
-    -switch to TCSPC oscilloscope
-    -select linear scale
-    -optimize with horizontal knob, this knob affects both lasers equally
-    -optimize with vertical knob until both lasers show same intensity. When turning, you should see the decreasing of one laser while the other stays at the same level for a bit then, also the second decreases.
-    -if you can't get them both to their **respective** maximum value at the same time, the pinhole is too small. (This means that you are either cutting one or the other or both)