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1. Fast photostable expansion microscopy using QDots and deconvolution

   https://doi.org/10.1371/journal.pone.0325155  

   Gunawardhana, Loku; Moree, Wilna; Guo, Jiaming; Artur, Camille; Womack, Tasha; Eriksen, Jason L; Mayerich, David (June 2025, PLOS One)

   Pesce, Luca (Ed.)

   Expansion microscopy (ExM) enables sub-diffraction imaging by physically expanding labeled tissue samples. This increases the tissue volume relative to the instrument point spread function (PSF), thereby improving the effective resolution by reported factors of 4 - 20X. However, this volume increase dilutes the fluorescence signal, reducing both signal-to-noise ratio (SNR) and acquisition speed. This paper proposes and validates a method for mitigating these challenges. We overcame the limitations of ExM by developing a fast photo-stable protocol to enable scalable widefield three-dimensional imaging with ExM. We combined widefield imaging with quantum dots (QDots). Widefield imaging provides a significantly faster acquisition of a single field-of-view (FOV). However, the uncontrolled incoherent illumination induces photobleaching. We mitigated this challenge using QDots, which exhibit a long fluorescence lifetime and improved photostability. First, we developed a protocol for QDot labeling. Next, we utilized widefield imaging to obtain 3D image stacks and applied deconvolution, which is feasible due to reduced scattering in ExM samples. We show that increased transparency, which is a side-effect of ExM, enables widefield deconvolution, dramatically reducing the acquisition time for three-dimensional images compared to laser scanning microscopy. The proposed QDot labeling protocol is compatible with ExM and provides enhanced photostability compared to traditional fluorescent dyes. Widefield imaging significantly improves SNR and acquisition speed compared to conventional confocal microscopy. Combining widefield imaging with QDot labeling and deconvolution has the potential to be applied to ExM for faster imaging of large three-dimensional samples with improved SNR. 

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2. Sparse decomposition light-field microscopy for high speed imaging of neuronal activity

   https://doi.org/10.1364/OPTICA.392805  

   Yoon, Young-Gyu; Wang, Zeguan; Pak, Nikita; Park, Demian; Dai, Peilun; Kang, Jeong_Seuk; Suk, Ho-Jun; Symvoulidis, Panagiotis; Guner-Ataman, Burcu; Wang, Kai; et al (October 2020, Optica)

   One of the major challenges in large scale optical imaging of neuronal activity is to simultaneously achieve sufficient temporal and spatial resolution across a large volume. Here, we introduce sparse decomposition light-field microscopy (SDLFM), a computational imaging technique based on light-field microscopy (LFM) that takes algorithmic advantage of the high temporal resolution of LFM and the inherent temporal sparsity of spikes to improve effective spatial resolution and signal-to-noise ratios (SNRs). With increased effective spatial resolution and SNRs, neuronal activity at the single-cell level can be recovered over a large volume. We demonstrate the single-cell imaging capability of SDLFM within vivoimaging of neuronal activity of whole brains of larval zebrafish with estimated lateral and axial resolutions of $\sim <\#comment/>3.5\text{µ<\#comment/>}\mathrm{m}$and $\sim <\#comment/>7.4\text{µ<\#comment/>}\mathrm{m}$, respectively, acquired at volumetric imaging rates up to 50 Hz. We also show that SDLFM increases the quality of neural imaging in adult fruit flies. 

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3. High-contrast, synchronous volumetric imaging with selective volume illumination microscopy

   https://doi.org/10.1038/s42003-020-0787-6  

   Truong, Thai V.; Holland, Daniel B.; Madaan, Sara; Andreev, Andrey; Keomanee-Dizon, Kevin; Troll, Josh V.; Koo, Daniel E. S.; McFall-Ngai, Margaret J.; Fraser, Scott E. (February 2020, Communications Biology)

   Abstract Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology. 

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4. Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture

   https://doi.org/10.1063/5.0101426  

   Keomanee-Dizon, Kevin; Jones, Matt; Luu, Peter; Fraser, Scott_E; Truong, Thai_V (October 2022, Applied Physics Letters)

   Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third. 

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5. Toward implantable devices for angle-sensitive, lens-less, multifluorescent, single-photon lifetime imaging in the brain using Fabry–Perot and absorptive color filters

   https://doi.org/10.1038/s41377-022-00708-9  

   Taal, Adriaan J.; Lee, Changhyuk; Choi, Jaebin; Hellenkamp, Björn; Shepard, Kenneth L. (December 2022, Light: Science & Applications)

   Abstract Implantable image sensors have the potential to revolutionize neuroscience. Due to their small form factor requirements; however, conventional filters and optics cannot be implemented. These limitations obstruct high-resolution imaging of large neural densities. Recent advances in angle-sensitive image sensors and single-photon avalanche diodes have provided a path toward ultrathin lens-less fluorescence imaging, enabling plenoptic sensing by extending sensing capabilities to include photon arrival time and incident angle, thereby providing the opportunity for separability of fluorescence point sources within the context of light-field microscopy (LFM). However, the addition of spectral sensitivity to angle-sensitive LFM reduces imager resolution because each wavelength requires a separate pixel subset. Here, we present a 1024-pixel, 50  µm thick implantable shank-based neural imager with color-filter-grating-based angle-sensitive pixels. This angular-spectral sensitive front end combines a metal–insulator–metal (MIM) Fabry–Perot color filter and diffractive optics to produce the measurement of orthogonal light-field information from two distinct colors within a single photodetector. The result is the ability to add independent color sensing to LFM while doubling the effective pixel density. The implantable imager combines angular-spectral and temporal information to demix and localize multispectral fluorescent targets. In this initial prototype, this is demonstrated with 45 μm diameter fluorescently labeled beads in scattering medium. Fluorescent lifetime imaging is exploited to further aid source separation, in addition to detecting pH through lifetime changes in fluorescent dyes. While these initial fluorescent targets are considerably brighter than fluorescently labeled neurons, further improvements will allow the application of these techniques to in-vivo multifluorescent structural and functional neural imaging. 

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