BrightEyes-TTM, an open-source time-tagging module (TTM) developed at the Italian Institute of Technology (IIT), enables scientists to observe the dynamic processes of molecules inside living cells over time, at a thousandth of a millisecond scale. BrightEyes-TTM can be used to study the variations that occur at the cellular level when a healthy cell becomes diseased. The module is designed to enable implementation of fluorescence single-photon laser-scanning microscopy (SP-LSM) techniques, and works with the single-photon avalanche diode (SPAD) array detectors that are used with SP-LSM. The fluorescent light signals that are emitted by molecules in a cell can be detected by a SPAD array detector. The time between when light is absorbed and fluorescence is emitted by the molecule (i.e., the lifetime) provides information about the molecule’s structure and microenvironment, leading to a more comprehensive view of the function of the molecule inside the cell. The BrightEyes-TTM development involved a multidisciplinary team of engineers, physicians, and biologists. Group picture of the research team at IIT's Molecular Microscopy and Spectroscopy Lab, which is coordinated by Giuseppe Vicidomini. Courtesy of IIT-Istituto Italiano di Tecnologia. The lifetime of the molecule occurs within an extremely short time frame of less than a thousandth of a millisecond. BrightEyes-TTM logs the instant a light particle is emitted from the molecule and captured by the sensor, serving as a “stopwatch” for scientists who wish to view molecular interactions inside cells. The BrightEyes-TTM data acquisition module is designed to exploit the high-throughput, high-resolution, photon-level information provided by SPAD detectors. The module has multiple channels that record at which element of the detector array, and when, with respect to the reference events, a single photon reaches the detector. It has a field-programmable gate array (FPGA) that can tag, in parallel, multiple single-photon events with 30-ps precision and multiple synchronization events with 4-ns precision. The commercially available, low-cost FPGA is equipped with I/O connectors that provide an interface between the FPGA board and the microscope, the SPAD array detector, and the computer. The FPGA-based implementation makes the module easy to update and adapt. The researchers envision a module that can be updated, even remotely, by the IIT team or other groups to keep pace with new SP-LSM techniques and advances in SPAD array detectors. The researchers integrated the BrightEyes-TTM into existing custom SP-LSM architectures, equipped with a 5 × 5 SPAD array detector prototype or a commercial 7 × 7 SPAD array detector. They performed fluorescence-lifetime imaging microscopy (FLISM) on a series of calibration and biological samples, including living cells. As an open-source system, BrightEyes-TTM provides a low-cost way to investigate dynamic processes of molecules. It is now accessible to anyone in the international scientific community who is interested in using the module or adapting it to different sensors. Courtesy of IIT-Istituto Italiano di Tecnologia. The researchers also demonstrated, for the first time, the combination of comprehensive-correlation analysis (CCA) with fluorescence lifetime analysis. The synergy between these two methods could open a path to new fluorescence lifetime fluctuation spectroscopy (FLFS) techniques that provide a more complete picture of the biomolecular processes inside living cells. As proof-of-principle, the researchers correlated the diffusion mode of the green fluorescent protein eGFP with its fluorescence lifetime in live cells. The researchers are using BrightEyes-TTM in an IIT project to investigate the lifetime of proteins and RNA involved in neurodegenerative diseases. BrightEyes-TTM will help the researchers better understand how these two molecular classes interact before and after the pathological processes behind neuronal death. As an open-source system, BrightEyes-TTM is accessible to all in the international scientific community who are interested in using the module or adapting it for use with different sensors. The researchers believe that the potential of SP-LSM will be realized more easily if a broad range of laboratories have access to the module and potentially modify it according to their needs. Sharing the platform with the scientific community could help broaden the use of BrightEyes-TTM to include nonbiological applications, such as quantum microscopy. The research paper provides detailed guidelines, hardware parts lists, and open-source code for the FPGA firmware and operational software for the module. The research was published in Nature Communications (www.doi.org/10.1038/s41467-022-35064-0).
