Background/Question/Methods Microbial communities, which are crucial to the cycling of organic matter, can be challenging to study in natural systems. However, quantum dots (nanoscale semiconductors) are a potential approach to quantifying organic nutrient translocation by fungi and plants. Quantum dots (QDs) can be bound to a number of substrates and can be manipulated to fluoresce in almost any color. In this work, we assessed fluorescent quantification techniques to observe the uptake of QD-bound labile (glycine) and recalcitrant (chitosan) organic nitrogen substrates by fungi and plants. We conjugated QDs to the amino groups of the labile amino acid glycine and the recalcitrant polysaccharide chitosan. We injected a slurry of QD-bound glycine and QD-bound chitosan into soil in the field. Additionally, a second slurry of QD controls (unbound quantum dots in solution) was injected in a separate location. We extracted fungal hyphae and plant roots from soil cores taken at each injection point. We used both epi-fluorescent spectroscopy and confocal microscopy techniques to quantify QD-labeled substrates. 

Results/Conclusions Using epi-fluorescent microplate spectroscopy and confocal microscopy, we quantified QD-bound organic nitrogen uptake by fungi on and within plant roots, as well as fungi extracted from the field. Using epi-fluorescent microplate spectroscopy we were able to quickly detect QD fluorescence in multiple bulk samples with detection limits reaching 0.00001 µM QDs. However, slow freeze effects reduced QD fluorescence up to 70%, and background fluorescence added additional variability to the results. Using confocal emission fingerprinting techniques, which use intelligent algorithms to remove autofluorescence, we were able to quantify QD fluorescence separate from background noise. However, this technique requires all parameters of the confocal microscope to remain static, unless calibration curves are used each time a parameter is adjusted (such as increasing laser intensity for a dense sample). Lastly, we used raster image correlation spectroscopy, which correlates the average movement of a fluorescent molecule over time to detect as little as one fluorescent probe per biovolume sample. Since autofluorescence doesn’t correlate, we were able to observe QD fluorescence within fungal hyphae located inside a plant root with a high signal to noise ratio. While some quantitative methods appear more beneficial than others in certain systems, our results highlight the potential to use QD-conjugates to quantify uptake of a variety of organic nutrients in many systems including soil, fungi and plants.