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Abstract Traditional deep fluorescence imaging has primarily focused on red‐shifting imaging wavelengths into the near‐infrared (NIR) windows or implementation of multi‐photon excitation approaches. Here, the advantages of NIR and multiphoton imaging are combined by developing a dual‐infrared two‐photon microscope that enables high‐resolution deep imaging in biological tissues. This study first computationally identifies that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. A NIR two‐photon microscope is constructed next with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single‐walled carbon nanotubes (SWNTs). Spatial imaging resolutions are achieved close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 µm deep into brain slices and through the full 120 µm thickness of aNicotiana benthamianaleaf. NIR‐II two‐photon microscopy can also measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature this study exploits to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. These results suggest dual‐infrared two‐photon microscopy can accomplish in‐tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues.
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Assessing the stability of azopolymer nanotopography during live-cell fluorescence imaging
IntroductionPhotomodifiable azopolymer nanotopographies represent a powerful means of assessing how cells respond to rapid changes in the local microenvironment. However, previous studies have suggested that azopolymers are readily photomodified under typical fluorescence imaging conditions over much of the visible spectrum. Here we assess the stability of azopolymer nanoridges under 1-photon and 2-photon imaging over a broad range of wavelengths. MethodsAzopolymer nanoridges were created via microtransfer molding of master structures that were created using interference lithography. The effects of exposure to a broad range of wavelengths of light polarized parallel to the ridges were assessed on both a spinning-disk confocal microscope and a 2-photon fluorescence microscope. Experiments with liveDictyostelium discoideumcells were also performed using alternating cycles of 514-nm light for photomodification and 561-nm light for fluorescence imaging. Results and DiscussionWe find that for both 1-photon and 2-photon imaging, only a limited range of wavelengths of light leads to photomodification of the azopolymer nanotopography. These results indicate that nondestructive 1-photon and 2-photon fluorescence imaging can be performed over a considerably broader range of wavelengths than would be suggested by previous research.
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- Award ID(s):
- 2014151
- PAR ID:
- 10533923
- Publisher / Repository:
- Frontiers in
- Date Published:
- Journal Name:
- Frontiers in Bioengineering and Biotechnology
- Volume:
- 12
- ISSN:
- 2296-4185
- Subject(s) / Keyword(s):
- live-cell imaging fluorescence microscopy reprogrammable nanotopography azopolymers esotaxis cytoskeletal dynamics 2-photon microscopy
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.3389/fbioe.2024.1409735
