Building images photon by photon to increase the information content provided by microscopes

The world of laser scanning microscopy is evolving rapidly, thanks to the advent of fast and compact detector arrays. These arrays replace the single element detector typical of traditional confocal laser scanning microscopes, enabling new and unique features.

While conventional detectors only provide the intensity value of the collected light, a pixelated detector also allows the spatial distribution of the incident light to be recorded, thereby creating a small image of the illuminated area for each scan point.

The additional spatial information provided by detector arrays enables a super-resolution technique known as image scanning microscopy (ISM). ISM computationally constructs a single image from a set of raw multidimensional data produced by a microscope. The final image is constructed with better signal-to-noise ratio (SNR), better optical section and better spatial resolution than those of a traditional confocal microscope.

In detail, the lateral resolution of the ISM image can exceed the Abbe limit by up to a factor of two. These advantages are, however, obtained by exploiting only spatial information; Modern fluorescence bioimaging can be further enriched by time-resolved acquisition, which provides access to structural and functional information encoded in fluorescence dynamics (e.g., fluorescence lifetime).

Recently, researchers at the Italian Institute of Technology (IIT) Genoa developed a compact and efficient ISM microscope equipped with a single-photon avalanche diode array (SPAD) detector capable of providing high-level structural and functional imaging. resolution in a single architecture. The research is published in Advanced Photonics.

The reported SPAD array detector includes 25 independent diodes arranged in a square grid. The small size and asynchronous readout enable rapid detection of incident fluorescence photons. The data acquisition scheme, based on the digital frequency domain (DFD) method, is a heterodyne sampling technique that allows the construction of the fluorescence decay histogram with a temporal resolution of up to 400 ps , suitable for most fluorescence imaging applications.

The technique is simple enough to allow the histogram to be calculated on the same Field-Programmable-Gate-Array (FPGA) board used to control the microscope and record the detected signal, thereby simplifying the microscope architecture.

Using the unique spatial and temporal information provided by the SPAD array detector, the authors demonstrated the combination of fluorescence lifetime (FL) measurements with ISM (FLISM). In addition to conventional ISM advantages, the improved SNR of FLISM images allows for more robust estimation of fluorescence lifetime.

The report highlights the versatility of the microscope in combining ISM and time-resolved measurements with stimulated emission microscopy (STED), using the separation by lifetime tuning (SPLIT) technique. The result is an image with improved lateral resolution and contrast obtained without changing the acquisition pattern. Additionally, time-resolved measurements enable imaging of multiple species with a single detector for increased structural specificity.

The system can distinguish different dyes with their fluorescence lifetime values, thanks to the phasor representation of fluorescence dynamics. Even using dyes with similar lifetime values ​​and overlapping excitation spectra, it can distinguish between different fluorophores using the pulsed interleaving excitation technique.

Indeed, by alternating laser excitation pulses of different colors, the spectral information is effectively encoded in the temporal dimension. Thanks to the excellent temporal resolution of the proposed microscope, the contribution of the two fluorescent dyes can then be separated to avoid crosstalk.

According to Giuseppe Vicidomini, principal investigator at IIT’s Molecular Microscopy and Spectroscopy Laboratory and corresponding author, “the results of this work suggest that the future of laser scanning microscopy is closely linked to SPAD array detectors, capable of enrich the microscopy dataset with additional data. spatial and temporal information without the need to modify the optical architecture of a confocal microscope.

The work demonstrates that SPAD array detectors combined with a tailor-made acquisition system make photon-resolved ISM easily accessible and usable.