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Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study. 

Abstract Image sensors capable of capturing individual photons have made tremendous progress in recent years. However, this technology faces a major limitation. Because they capture scene information at the individual photon level, the raw data is sparse and noisy. Here we propose CASPI: Collaborative Photon Processing for Active Single-Photon Imaging, a technology-agnostic, application-agnostic, and training-free photon processing pipeline for emerging high-resolution single-photon cameras. By collaboratively exploiting both local and non-local correlations in the spatio-temporal photon data cubes, CASPI estimates scene properties reliably even under very challenging lighting conditions. We demonstrate the versatility of CASPI with two applications: LiDAR imaging over a wide range of photon flux levels, from a sub-photon to high ambient regimes, and live-cell autofluorescence FLIM in low photon count regimes. We envision CASPI as a basic building block of general-purpose photon processing units that will be implemented on-chip in future single-photon cameras. 

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.