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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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Spatially resolved measurements of ballistic and total transmission in microscale tissue samples from 450 nm to 1624 nm
We built a simple and versatile setup to measure tissue ballistic and total transmission with customizable wavelength range, spatial resolution, and sample sizes. We performed ballistic transmission and total transmission measurements of overlying structures from biological samplesex vivo. We obtained spatially resolved transmission maps to reveal transmission heterogeneity from five microscale tissue samples:Danionellaskin, mouse skull bone, mosquito cuticle, wasp cuticle, and rat dura over a wide spectral range from 450 nm to 1624 nm at a spatial resolution of ∼25µm for ballistic transmission measurements and ∼50µm for total transmission measurements. We expect our method can be straightforwardly applied to measuring the transmission of other samples. The measurement results will be valuable for multiphoton microscopy. The total transmission of a sample is important for the collection of multiphoton excited fluorescence and the assessment of laser-induced sample heating. The ballistic transmission determines the excitation power at the focus and hence the fluorescence signal generation. Therefore, knowledge of ballistic transmission, total transmission, and transmission heterogeneity of overlying structures of animals and organs are essential to determine the optimal excitation wavelength and fluorophores for non-invasive multiphoton microscopy.
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- PAR ID:
- 10369525
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2156-7085
- Format(s):
- Medium: X Size: Article No. 438
- Size(s):
- Article No. 438
- Sponsoring Org:
- National Science Foundation
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