Parallel FCS and FcCS

Parallel FCS and FcCS

Gösch M., Blom H., Serov A., Rochas A., Besse P.A., Rigler R., Lasser T.

The detection of single molecules using confocal spectroscopy in the field of biology, chemistry and medicine is of major interest and tremendous improvements have been made in the last decade. The underlying principle, however, a pinhole and a single excitation volume element have remained the same as well as the technical limitations.

We present approaches to address these technological hurdles and to design and implement a confocal system with parallel detection capability [1] that increases the throughput and speeds up analyses time. We measure in multiple spots FCS and FCCS on experimental set-ups containing diffractive optical excitation, CMOS (complementary metal oxide semiconductor)-detector arrays and a combination of these.

A step forward towards a highly integrated parallel confocal fluorescent detection set-up has been taken, by introducing CMOS-detector arrays in conjunction with diffractive optical elements (DOEs). A combination of a 2 x 2 DOE and a 2 x 2 CMOS-detector array is utilized in order to see if parallel detection of single molecule is feasible. Autocorrelation curves on Tetramethylrhodamine (TMR) are taken in multiple spot FCS measurements. The recorded curve for both, the CMOS-SPAD array and a conventional SPCM are compared. The CMOS-SPAD shows a much better performance from the afterpulsing point of view, but heavy losses for the count rate per molecule are apparent due to the low quantum yield. However, equal concentration values are obtained [2]. This detection scheme potentially improves and speeds up the analysis of biological and chemical samples because on-chip processing, data analysis, and a high level of integration could easily be realized.

Additionally, two individual DOEs were used to build a parallel 2 x 2 cross correlation set-up. This technology has been developed for the determination of the gene expression profile by a novel analytical method based on FCCS analysis. Here, the long analysis time and the constantly present low concentration of the sample motivated the implementation of such a set-up. Additionally, other analytical methods such as high-throughput screening (HTS), could profit from this method decreasing their analysis time in future.

[1] Blom H., Gösch M., Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units, Current Pharmaceutical Biotechnology 5:231-241 (2004)
[2] Gösch M., Serov A., Rochas A., et al., Parallel Single Molecule Detection with a fully integrated Single Photon 2 x 2 CMOS Detector Array, Journal of Biomedical Optics 9#5 (2004)