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.

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

ROSSETTA, ALESSANDRO
2022-04-20

Abstract

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.
FPGA, FLIM, microscopy, TDC, time-tagging, fluorescence, fluorescence lifetime
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/1079516
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