Image scanning microscopy (ISM), a superresolution technique enabled by the advent of fast and compact detector arrays, provides signal-to-noise ratio (SNR), optical sectioning, and spatial resolution better than that of a traditional confocal microscope. The lateral resolution of the ISM image can surpass Abbe’s limit by up to a factor of two. However, these advantages are achieved by exploiting only spatial information; modern fluorescence bioimaging can be further enriched by time-resolved acquisition, which enables access to structural and functional information encoded into the fluorescence dynamics (e.g., fluorescence lifetime). Researchers at the Italian Institute of Technology (IIT) in Genoa developed a compact and effective ISM microscope equipped with a single photon avalanche diode (SPAD) array detector capable of providing high-resolution structural and functional imaging in a single architecture. The reported SPAD array detector consists of 25 independent diodes arranged in a square grid. The small size and the asynchronous read-out enable fast detection of impinging fluorescence photons. The data acquisition module records essential spatial and temporal features of photons. Based on the digital frequency domain principle, the module reduces data transfer and storage load without compromising performance. A newly developed compact ISM microscope equipped with a single photon avalanche diode (SPAD) array detector provides high-resolution structural and functional imaging. Courtesy of A. Zunino/IIT. The acquisition module uses a heterodyne scheme to record the photon arrival time with a sampling period down to 400 picoseconds (ps), which meets the needs of most fluorescence imaging applications. Instead of transferring the arrival time of each detection event to the computer, the module uses a fluorescence decay histogram that is built directly on a field-programmable-gate-array (FPGA) board. Computation of the histogram is done on the same FPGA board that is used to control the microscope and record the detected signal, which simplifies the architecture of the microscope. The researchers integrated the data acquisition module and control system into a single-photon laser scanning microscope (SP-LSM) architecture equipped with a commercial SPAD array detector. They validated the digital frequency domain module embedded in the microscope control and data acquisition sections by implementing different fluorescence lifetime ISM (FLISM)-based imaging techniques. When researchers demonstrated the combination of fluorescence lifetime (FL) measurements with ISM, the SNR of the FLISM images improved, enabling a more robust fluorescence lifetime estimation. The researchers combined the new microscopy platform with fluorescence lifetime phasor analysis, enabling superresolution imaging of multiple fluorophores without spectral emission separation. Dyes were distinguished by leveraging different fluorescence lifetimes. Even when dyes had similar lifetime values and overlapping excitation spectra, the new ISM platform was able to distinguish the different fluorophores by using a pulsed-interleaving, multiwavelength excitation scheme. The photon arrival time in FLISM with respect to the fluorophore excitation event enabled the mapping of the fluorescence lifetime distribution in the sample at superresolution. This information could help scientists understand the properties of the biomolecular environment, decipher biomolecule structural changes, and implement multispecies imaging. The researchers also combined time-resolved stimulated emission depletion (STED) microscopy with ISM and time-resolved measurements, using the separation by lifetime tuning (SPLIT) technique. This resulted in an image with enhanced lateral resolution and contrast, obtained without modifying the data acquisition scheme. Time-resolved measurements also enable multispecies imaging with a single detector for increased structural specificity. The results achieved by the IIT research team demonstrate that, with the addition of a SPAD array detector with a tailored data acquisition module, a conventional microscope can be transformed into an information-rich imaging system that provides high-resolution structural and functional imaging in a single architecture. “The results of this work suggest that the future of laser scanning microscopy is tightly connected to SPAD array detectors, capable of enriching the microscopy dataset with additional spatial and temporal information without the need to change the optical architecture of a confocal microscope,” said IIT senior researcher Giuseppe Vicidomini. The research was published in Advanced Photonics (www.doi.org/10.1117/1.AP.6.1.016003).
