This paper introduces the field of metamaterials, details various optical uses of metasurfaces and demonstrates their suitability for imaging with single-photon avalanche diode (SPAD) detector arrays as an integrated optical component. A design for a metasurface-based color filter array (CFA) is presented, the fabrication methodology detailed, and a sample is integrated with a SPAD array. Examples of imaging applications using the integrated assembly are demonstrated, including passive and fluorescence imaging microscopy. The limitations of current metasurface color filtering techniques are highlighted and directions for future advances and applications discussed.
The capability of Single-Photon Avalanche Diodes (SPADs) to detect photons with picosecond timing precision and shotnoise limited performance has given rise to a range of biological and biomedical applications, from Fluorescence Lifetime Imaging Microscopy (FLIM) to Raman Spectroscopy and Positron Emission Tomography (PET). The use of SPAD sensors has also been successfully demonstrated in Single-Molecule Localisation Microscopy. Traditionally implemented as point detectors, recent advances in SPAD technology, such as compact, binary pixels and back-side illuminated, 3D-stacked architectures, have led to 2-D imaging arrays of increasing resolution and fill factor. Combined with high frame rates (in the kFPS range), and negligible read noise, the sensors offer an exciting prospect for capturing fast temporal dynamics in life science cellular imaging. The work in this paper considers the application of SPAD imaging arrays to widefield fluorescence lifetime imaging of high-speed particles in microscopy. We demonstrate, using a time-gated binary SPAD array, that by tracking particles, and spatially re-assigning the underlying photon counts accordingly, lifetime estimates for fast-moving objects are possible. Moreover, we give the first demonstration of FLIM using a SPAD imaging array with on-chip histogramming of photon arrival time, with potential frame rates of several 100FPS. Both FLIM techniques are illustrated using experimental results based on fluorescent microspheres undergoing Brownian motion. The results pave the way towards applications in live-cell microscopy, such as the monitoring of the fluorescence lifetime of highly mobile cell structures, with a view, for example, to study molecular interactions using Förster Resonance Energy Transfer (FRET) measurements.
Single photon avalanche diodes (SPADs) are used in a wide range of applications, from fluorescence lifetime imaging microscopy (FLIM) to time-of-flight (ToF) 3D imaging. SPAD arrays are becoming increasingly established, combining the unique properties of SPADs with widefield camera configurations. Traditionally, the photosensitive area (fill factor) of SPAD arrays has been limited by the in-pixel digital electronics. However, recent designs have demonstrated that by replacing the complex digital pixel logic with simple binary pixels and external frame summation, the fill factor can be increased considerably. A significant advantage of such binary SPAD arrays is the high frame rates offered by the sensors (>100kFPS), which opens up new possibilities for capturing ultra-fast temporal dynamics in, for example, life science cellular imaging. In this work we consider the use of novel binary SPAD arrays in high-speed particle tracking in microscopy. We demonstrate the tracking of fluorescent microspheres undergoing Brownian motion, and in intra-cellular vesicle dynamics, at high frame rates. We thereby show how binary SPAD arrays can offer an important advance in live cell imaging in such fields as intercellular communication, cell trafficking and cell signaling.
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