RESUMO
Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors.
RESUMO
We describe a novel method of generating monodisperse subfemtoliter aqueous droplets on demand by means of piezoelectric injection. Droplets with volumes down to 200 aL are generated by this technique. The droplets are injected into a low refractive index perfluorocarbon so that they can be optically trapped. We demonstrate the use of optical tweezers to manipulate and mix droplets. For example, using optical tweezers we bring two droplets, one containing a calcium sensitive dye and the other calcium chloride, into contact. The droplets coalesce with a resulting reaction time of about 1 ms. The monodispersity, manipulability, repeatability, small size, and fast mixing afforded by this system offer many opportunities for nanochemistry and observation of chemical reactions on a molecule-by-molecule basis.