A technique for the study of fluorescent molecules in transparent fluid media, fluorescence-correlation spectroscopy (FCS) evaluates the correlated fluctuations of laser-induced photons emitted from the molecules as they pass through a focal spot, allowing study of inter- and intramolecular dynamics. Because these dynamics can occur in real time and over large spatial areas, spatially resolved excitation and detection strategies at multiple spots with fast data-acquisition rates are needed, particularly for cellular applications. Although two-and four-spot FCS using either individual avalanche photodiodes (APDs) or a 2 x 2 CMOS array detector has been demonstrated, researchers at Dresden University of Technology (Dresden, Germany) have paved the way for wider-view multispot FCS detection using an electron-multiplying charge-coupled device (EMCCD).1
To show the merits of EMCCD detection compared to multispot APD and other detection schemes, the researchers first used an FCS setup that allows them to use two different detectors to prove that the EMCCD noise and detection sensitivity were comparable to those for APD detection by. The setup consisted of an inverted microscope with a 60x water-immersion objective and a 488 nm laser beam that illuminated the sample. A microscope switch alternated between the EMCCD detector-an iXon DV860 from Andor (Belfast, Northern Ireland) with 128 x 128-pixel area-and the SPCM-AQR-13 APD detector from PerkinElmer Optoelectronics (Fremont, CA). The input to the APD was via a 50-µm-core-diameter multimode fiber.
With the APD signal fed into a digital correlator displaying instant count rate and correlation function, and the EMCCD signal evaluated with a home-built algorithm to compute and fit correlation curves, sample solutions of quantum dots and standard Alexa dye were analyzed. Using a vertical-shift-speed setting of 0.3 µs per line (yielding a frame transfer time of 38.4 µs) and with full electron-multiplying gain for the EMCCD, the researchers compared fitted correlation curves for APD and EMCCD detection and found that for a 2 x 2-pixel detection area on the EMCCD (which has a slightly larger focal volume compared to the one obtained by the circular multimode fiber aperture), the signal-to-noise ratio deduced from the signal trace was comparable to the one obtained from APD detection.
To improve analysis of the dye molecules with fast diffusion times of say 60 µs-a challenge for CCD detectors because of the slow readout and frame-transfer time-the researchers adopted use of a “fast kinetic mode” that allowed continuous acquisition with exposure times down to 1 µs. Even in this faster mode, analysis of a sample showed that deduced particle numbers and diffusion times were in good agreement with APD detection when taking the CCD signal from 2 x 2 pixels.
Proof of principle
Next, to show that a single EMCCD detector could be used as a wider-view replacement for APD multiple-spot detection (usually requiring multiple APD detectors), the researchers performed a simple proof-of-principle measurement using two separate excitation volumes and parallel detection along the horizontal axis of the CCD. The laser beam was split and reunited by two pellicle beamsplitters so that the reflected beam entered the objective back aperture at a slight angle. The two focal volumes were separated by 2 µm within the sample or 120 µm (5 pixels) in the image plane, respectively. To compare with standard APD detection, two separate measurements were obtained by moving the fiber to the two detection positions. The resulting autocorrelation curves showed good agreement between the EMCCD and APD detection techniques, but with the EMCCD having the advantage of much greater flexibility with regard to the illumination pattern (see figure).
Compared to APD detection, EMCCD detection offers the advantage of the free choice of distance between multiple detection volumes and the ability to compute data from multiple volumes in a single measurement. “The possibility of simultaneous multispot FCS analysis is crucial for intracellular measurements with large sample inhomogeneities, and for spectrally resolved FCS,” notes researcher Petra Schwille. “The laser spot array can easily be produced by diffractive optical elements, but the multispot detection has so far been problematic. One solution for the future is certainly the use of APD arrays, but we believe that with the rapid improvement of both sensitivity and speed, EMCCD cameras will soon be the detectors of choice for spatially resolved FCS.”
REFERENCE
1. M. Burkhardt and P. Schwille, Optics Express 14(12) 5013 (June 12, 2006).
