Measurement
	Fluctuation Analyzes Probes Chemical Reactions		How does FCS work? Measurement Calculation Evaluation Result
	Measuring with the ConfoCor 3
	Principle of Fluctuation Analysis
	A fluorescent labeled molecule in solution emits photons as long as it moves through a laser spot. The number of emitted photons per time depends on: number of molecules (concentration) diffussion time (mass of molecules) spot size (instrumental parameter) quantum yield (property of dye)
	ConfoCor 3 uses Confocal Volume Element
	Advanced confocal optics realized in the ConfoCor 3 limits detection to a cylinder approximately 1,5 µm in height. Focus and confocal detection define a measuring volume smaller than ¼ femto liter (1 femto liter=10-15 liter).
	Optical set up used in Fluorescence Correlation Spectroscopy
	The parallel laser light of one or two lasers is directed to the specimen via the primary beam splitter and a high numerical aperture objective that produces a diffraction limited excitation volume. The Stokes-shifted emitted fluorescence light from the sample passes through the primary beam splitter and reaches the detector, an avalanche photodiode, via a pinhole that is needed for confocality. In case of a dual-color experiment, the different emission wavelengths can be separated via a secondary beam splitter before reaching the detectors. The measurement position can be defined by an x,y scanning stage and an objective lens that is movable in z. The scanning mirrors are optional and would also be used for x,y positioning.
	Due to the thermodynamic fluctuationsnumber of molecules will fluctuate in the observed volume element. The measured intensity reflects the varying number of excited molecules.
	molecule emitting photons objective collecting photons detector recording photons
	Number of Pulses represent Intensity
	The number of pulses originating form detected photons, recorded during a specific time interval correspond to a measured light intensity.
	Photons on detector
	Pulses after detector
	Each photon gives a pulse Each photon arriving during the detector's dead time (35 ns) are not recorded Pulses are independent of photon energy (wavelength) The time between two photons / molecule = 10 µs >>> every single photon is counted
	Evaluating Pulses
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