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.
The development of a very-compact DNA sequencer instrument based on Single Photon Avalanche Diode (SPAD) for
microchip electrophoresis is here reported. The planar epitaxial SPAD combines the typical advantages of microelectronic
devices with high sensitivity. We present a miniaturized system based on a custom array of SPAD, purposely designed
to be compatible with Amersham Biosciences commercial markers. This system is the first example of very compact,
ultra-sensitive, portable and low cost DNA sequencer. It may represent a breakthrough in DNA sequencing system and
open the way to the development of a new category of portable low-cost apparatus.
One of the main drawbacks of Single Photon Avalanche Diode arrays is the optical crosstalk between adjacent detectors.
This phenomenon represents a fundamental limit to the density of arrays, since the crosstalk increases with reducing the
distance between adjacent devices. In the past, crosstalk was mainly ascribed to the light propagating from one detector
to another through a direct optical path. Accordingly, deep trenches coated with metal were introduced as optical
isolation barriers between pixels. This solution, however, was unable to completely prevent the crosstalk. In this paper
we present experimental evidence that a significant contribution to crosstalk comes from photons reflected internally at
the bottom of the chip. These photons can bypass trenches making them ineffective. We also propose an optical model
suitable to predict the dependence of crosstalk on the position within the array.
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.