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The use of fluorescence microscopy to study fate and transport of nanoparticles in the environment can be limited by the presence of confounding background signals such as autofluorescence and scattered light. The unique spin-related luminescence properties of nitrogen vacancy (NV) centers in diamond nanoparticles (NVND) enable new types of imaging modalities such as selective imaging of nanoparticles in the presence of background fluorescence. These techniques make use of the fact that the spin properties, which affect the fluorescence of NV centers, can be modulated using applied magnetic or radio-frequency fields. This work presents the use magnetic fields to modulate the fluorescence of NVND for background-subtracted imaging of nanoparticles ingested by a model organism, C. elegans . With the addition of modest time-modulated magnetic fields from an inexpensive “hobby” electromagnet, the fluorescence of 40 nm NVND can be modulated by 10% in a widefield imaging configuration. Herein, differential magnetic imaging is used to image and to isolate the fluorescence arising from nanodiamond within the gut of the organism C. elegans . This method represents a promising approach to probing the uptake of nanoparticles by organisms and to assessing the movement and interactions of nanoparticles in biological systems.
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This content will become publicly available on December 15, 2025
Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of C. elegans autofluorescence in the cuticle and gut
ABSTRACT Caenorhabditis elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C. elegans autofluorescence has been characterized, it has not been characterized at high resolution using both spectral and fluorescence lifetime-based approaches. We performed high resolution spectral scans of whole, living animals to characterize autofluorescence of adult C. elegans. By scanning animals at 405 nm, 473 nm, 561 nm, and 647 nm excitations, we produced spectral profiles that confirm the brightest autofluorescence has a clear spectral overlap with the emission of green fluorescent protein (GFP). We then used fluorescence lifetime imaging microscopy (FLIM) to further characterize autofluorescence in the cuticle and the gut. Using FLIM, we were able to isolate and quantify dim GFP signal within the sensory cilia of a single pair of neurons that is often obscured by cuticle autofluorescence. In the gut, we found distinct spectral populations of autofluorescence that could be excited by 405 nm and 473 nm lasers. Further, we found lifetime differences between subregions of this autofluorescence when stimulated at 473 nm. Our results suggest that FLIM can be used to differentiate biochemically unique populations of gut autofluorescence without labeling. Further studies involving C. elegans may benefit from combining high resolution spectral and lifetime imaging to isolate fluorescent protein signal that is mixed with background autofluorescence and to perform useful characterization of subcellular structures in a label-free manner.
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- Award ID(s):
- 2146516
- PAR ID:
- 10585212
- Publisher / Repository:
- The Company of Biologists
- Date Published:
- Journal Name:
- Biology Open
- Volume:
- 13
- Issue:
- 12
- ISSN:
- 2046-6390
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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