Witness the Future of Microscopic Imaging: SPAD's Quantum Leap in Resolution

Thanks to the emergence of swift and compact detector arrays, Image Scanning Microscopy (ISM), a modern super resolution technique, provides a Signal-to-Noise Ratio (SNR), optical sectioning and spatial resolution superior to its traditional florescence confocal counterpart. In particular, ISM lateral resolution can exceed Abbe’s limit by a factor of two, thus significantly advancing the state-of-the-art in contemporary fluorescence bioimaging.

Witness the Future of Microscopic Imaging: SPAD's Quantum Leap in Resolution

However, these benefits arise from the exploitation of spatial information only. By making the leap to time-resolved acquisition one can envision an even richer realm of modern fluorescence bioimaging. Ultimately, this will not only provide structural information, but also elucidate functionality embedded in the fluorescence dynamics, which is now commonly exploited by modern bioimaging techniques. An example of such functionality is the fluorescence lifetime.

Researchers at the Italian Institute of Technology (IIT) in Genoa have ingeniously crafted a compact and efficient ISM microscope, featuring a single-photon avalanche diode (SPAD) array detector. This innovation allows for high-resolution structural and functional imaging within a unified architecture.

The disclosed SPAD array detector comprises 25 independent diodes arranged in a square grid. Its diminutive size and asynchronous read-out facilitate swift detection of impinging fluorescence photons. The data acquisition module captures crucial spatial and temporal features, employing the digital frequency domain principle to reduce data transfer and storage load while maintaining optimal performance.

Witness the Future of Microscopic Imaging: SPAD's Quantum Leap in Resolution
The acquisition module uses a heterodyne scheme, which allows the record photon arrival times, with a sampling period as short as 400 picoseconds (ps) -- short enough for most fluorescence imaging applications. The module doesn't send the arrival time for each of those detection events to the computer; rather it decodes the fluorescence decay histogram right on the the useful field of view projection of the microscope's focal plane. The result? Not only is the microscope's architecture greatly simplified, but so too is control and data acquisition.

"Many scientists will find that an MSI in Turbiscan available will help them to answer the scientific questions that they are currently investigating more quickly and easily, and will additionally allow them to pose new questions about the pahsegropmy and stability of their systems," Rouilly said.

The team integrated a data acquisition module and control system into a Single-Photon Laser Scanning Microscope (SP-LSM) architecture that has a commercial SPAD array detector. For validation, they implemented a variety of fluorescence lifetime ISM (FLISM)-based imaging techniques on the digital frequency domain module embedded in the microscope control and data acquisition sections. Combining FL measurements with ISM has demonstrated an improved SNR of FLISM images and more robust fluorescence lifetime estimation.

At the end of the day, this new microscopy platform and the workflow IF/IFF phasor analysis make it possible to achieve super-resolution imaging of multiple fluorophores without spectral emission separation. Dyes with distinct fluorescence lifetimes can be parsed using a pulsed-interleaving, multiwavelength excitation scheme even when their values are similar and their excitation spectra are overlapping.

While IMR not only finds the fractional intensities of different lifetimes, and additionally maps their positions, reports their coordination in space by way of measuring the proximity of prompt and delayed photons to one another. As in SMD it gives away more than the number of lifetime components, can you perform a multicomponent fit to an IMR data-set and (with caution) use a simple color-coded super resolution STED (IN-STOP-STED) image to show what is happening?

The answer was yes - and it gives a sub-wavelength resolution detailed view of the structural changes that bound the complex with one attire of fluorescent molecules following another. By combining time-resolved measurements and ISM with the time-resolved stimulated emission detection (STED) feature, implemented by separating phosphorescent from strongly scattering and prompt from delayed photons, using the separation by lifetime tuning (SPLIT) technique in a joint measurement, the researchers were able to achieve an almost two fold enhancement in lateral resolution and contrast without any changes to data acquisition scheme, to elucidate the structural changes of the polymers that manifest the molecular dress of individual biomacromolecular compounds. Moreover, unlike existing super-resolution fluorescence microscopy techniques, they showed that their approach can visualize the polymers that are significantly different but both below the imaging resolution. And unlike others, like fluorescence lifetime imaging (FLIM), the measurements have no need to be performed in separate schemes and are multichannel super-imaged - for example, a single detector provides environmental sensitivity with enhanced structural specificity for multispecies imaging.

The findings by the IIT research team show that the marriage of a SPAD array detector and a dedicated data acquisition module can transform a conventional microscope into a rich-information imaging system, able to provide high-resolution structural and functional imaging within a single, compact architecture.

“The implications of this study hint to a future in which laser scanning microscopy and SPAD array detectors could seamlessly coexist, enhancing microscopy datasets with additional spatial and temporal information without the need for changes to the optical architecture of a confocal microscope,” added IIT senior researcher Giuseppe Vicidomini.

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