The BrightEyes-TTM: an open-source time-tagging module for fluorescence lifetime imaging microscopy applications

The aim of this Ph.D. work is to reason and show how an open-source multi-channel and standalone time-tagging device was developed, validated and used in combination with a new generation of single-photon array detectors to pursue super-resolved time-resolved fluorescence lifetime imaging measurements. Within the compound of time-resolved fluorescence laser scanning microscopy (LSM) techniques, fluorescence lifetime imaging microscopy (FLIM) plays a relevant role in the life-sciences field, thanks to its ability of detecting functional changes within the cellular micro-environment. The recent advancements in photon detection technologies, such as the introduction of asynchronous read-out single-photon avalanche diode (SPAD) array detectors, allow to image a fluorescent sample with spatial resolution below the diffraction limit, at the same time, yield the possibility of accessing the single-photon information content allowing for time-resolved FLIM measurements. Thus, super-resolved FLIM experiments can be accomplished using SPAD array detectors in combination with pulsed laser sources and special data acquisition systems (DAQs), capable of handling a multiplicity of inputs and dealing with the single-photons readouts generated by SPAD array detectors. Nowadays, the commercial market lacks a true standalone, multi-channel, single-board, time-tagging and affordable DAQ device specifically designed for super-resolved FLIM experiments. Moreover, in the scientific community, no-efforts have been placed yet in building a device that can compensate such absence. That is why, within this Ph.D. project, an open-source and low-cost device, the so-called BrightEyes-TTM (time tagging module), was developed and validated both for fluorescence lifetime and time-resolved measurements in general. The BrightEyes-TTM belongs to a niche of DAQ devices called time-to-digital converters (TDCs). The field-gate programmable array (FPGA) technology was chosen for implementing the BrightEyes-TTM thanks to its reprogrammability and low cost features. The literature reports several different FPGA-based TDC architectures. Particularly, the differential delay-line TDC architecture turned out to be the most suitable for this Ph.D. project as it offers an optimal trade-off between temporal precision, temporal range, temporal resolution, dead-time, linearity, and FPGA resources, which are all crucial characteristics for a TDC device. The goal of the project of pursuing a cost-effective and further-upgradable open-source time-tagging device was achieved as the BrigthEyes-TTM was developed and assembled using low-cost commercially available electronic development kits, thus allowing for the architecture to be easily reproduced. BrightEyes-TTM was deployed on a FPGA development board which was equipped with a USB 3.0 chip for communicating with a host-processing unit and a multi-input/output custom-built interface card for interconnecting the TTM with the outside world. Licence-free softwares were used for acquiring, reconstructing and analyzing the BrightEyes-TTM time-resolved data. In order to characterize the BrightEyes-TTM performances and, at the same time, validate the developed multi-channel TDC architecture, the TTM was firstly tested on a bench and then integrated into a fluorescent LSM system. Yielding a 30 ps single-shot precision and linearity performances that allows to be employed for actual FLIM measurements, the BrightEyes-TTM, which also proved to acquire data from many channels in parallel, was ultimately used with a SPAD array detector to perform fluorescence imaging and spectroscopy on biological systems. As output of the Ph.D. work, the BrightEyes-TTM was released on GitHub as a fully open-source project with two aims. The principal aim is to give to any microscopy and life science laboratory the possibility to implement and further develop single-photon-based time-resolved microscopy techniques. The second aim is to trigger the interest of the microscopy community, and establish the BrigthEyes-TTM as a new standard for single-photon FLSM and FLIM experiments

High Frequency Digital Frequency Domain Fluorescence Lifetime Imaging System

A system and method is provided for improved fluorescence lifetime measurement. A high frequency digital heterodyning technique is described in which photon counting mode detectors are sampled at a rate slightly slower than a digitally pulsed excitation signal. A digital mixer produces a difference frequency which contains the same information than the high frequency signal. This lower frequency is still suitable for fluorescence lifetime measurements in microscopy. The digital algorithm provides phases and modulations of detected photons binning them into phase steps and time windows. The digital heterodynig algorithm was implemented in a very low cost FPGA (Field Programmable Gate Array)

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids

A MILP methodology to optimize sizing of PV - Wind renewable energy systems

This paper proposes a methodology that is based on mixed-integer linear programming (MILP) to calculate the optimal sizing of a hybrid wind-photovoltaic power plant in an industrial area. The proposed methodology considers the: i) load requirements; ii) physical and geometric constraints for the renewable plants installation; iii) operating and maintenance costs of both wind and PV power plants; and the iv) electric energy absorbed by the public network. The power demand variation associated with the production cycles is considered by using a stochastic simulation tool. To consider both the load and seasonality variability, and to adapt the methodology to the actual operational use of the power plant, the optimization is performed separately for each month of the year. Then, an integrated economic analysis is discussed. The methodology is adopted to analyze an industrial plant in the Rome area used as a train depot and for maintenance purposes. The results, which combine the needs of the plant activity with the availability of renewable energy, enabled the determination of optimal solutions and the relevant savings achievable

Energy storage system for voltage regulation in a 3kV DC Italian railway line

The paper investigates the installation of energy storage systems (ESS) for a 3kV DC railway line located in the North of Italy. The main goals are to compensate voltage drops along the line and to regulate the system's voltage and to recover braking energy of trains. Electromechanical performances of trains and electrical system operation have been simulated by means of a software in order to evaluate the power demand of each train, the TPSSs (Traction Power Substations) power profile and voltage trend along the line, as well as the energy recovery and ESSs installation benefits. Two technological solutions regarding the design of ESS are described and simulated: lithium batteries and supercapacitors storage systems. The first solution is suggested due to the cost savings and the smaller requirements in terms of space occupation and total weight, while the second solution is suggested in case of great power and due to low levels of maintenance The analysis shows the benefits of the implementation of each type of ESS