Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques
to access a variety of molecular properties such as size, brightness, conformation, and binding constants. However, this
is limited to low concentrations, which results in long acquisition times in order to achieve good statistical accuracy.
Data can be acquired more quickly by using parallelization. We present a new approach using a multispot excitation and
detection geometry made possible by the combination of three powerful new technologies: (i) a liquid crystal spatial
light modulator to produce multiple diffraction-limited excitation spots; (ii) a multipixel detector array matching the
excitation pattern and (iii) a low-cost reconfigurable multichannel counting board. We demonstrate the capabilities of
this technique by reporting FCS measurements of various calibrated samples as well as single-molecule burst
Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in
all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume
(typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit
of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the
advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data
acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation
and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate
the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence
measurements obtained with different novel multipixel single-photon counting detectors.
In the last years Time-Correlated Single-Photon Counting (TCSPC) has increasingly been used in many different
scientific applications (e.g.: single molecule spectroscopy, fluorescence lifetime imaging, diffuse optical tomography).
Many of these applications are calling for new requests on the development of instrumentation that operates at higher
and higher conversion rates and that is able to resolve optical signals not only in the time domain, but also in wavelength,
polarization and position. To exploit the potential of parallel analysis over multiple acquisition channels, a new
generation of TCSPC devices is needed that is characterized by low size and costs. The core block of TCSPC
instrumentation is the time-interval measurement section, which can be implemented with a Time-to-Amplitude
Converter (TAC); the converter can be integrated on a single chip in order to reduce the overall size and cost of the
system. This paper presents a monolithic TAC that has been designed to achieve the high resolution, good differential
linearity and fast counting rate required in modern applications. The TAC here described is built on a commercial
0.35 μm CMOS technology, and is characterized by resolution better than 60 ps, differential nonlinearity limited to
0.5% rms and short dead-time of 80 ns. The low area occupation (1.4x1.8 mm) and minimal need for external
components allow the realization of very compact instruments with multiple acquisition channels operating
simultaneously at very high count rates.
We present a multichannel photon counting module that exploits a monolithic array of single-photon avalanche diodes
(SPADs). The detector array consists of eight 50μm diameter SPADs featuring low dark counting rate and high photon
detection efficiency (50% at 550nm); inter-pixel crosstalk probability is as low as 2•10<sup>-3</sup>. The use of highly integrated
active quenching circuits makes it possible to design a very compact read-out circuit, yet providing eight fully
independent counting channels and gating capability. The detection module maintains the same physical dimensions of
commercially available single element modules and can be used as a plug-in replacement to add multichannel
capabilities to existing measurement setups. Full characterization of module performance is here presented.