Two-photon peak molecular brightness spectra reveal long-wavelength enhancements of multiplexed imaging depth and photostability
Year: 2021
Type: article
Abstract: The broad use of two-photon microscopy has been enabled in part by Ti:Sapphire femtosecond lasers, which offer a wavelength-tunable source of pulsed excitation. Action spectra have thus been primarily reported for the tunable range of Ti:Sapphire lasers (∼700-1000 nm). However, longer wavelengths offer deeper imaging in tissue via reduced scattering and spectral dips in water absorption, and new generations of pulsed lasers offer wider tunable ranges. We present the peak molecular brightness spectra for eight Alexa Fluor dyes between 700-1300 nm as a first-order surrogate for action spectra measured with an unmodified commercial microscope, which reveal overlapping long-wavelength excitation peaks with potential for multiplexed excitation. We demonstrate simultaneous single-wavelength excitation of six spectrally overlapping fluorophores using either short (∼790 nm) or long (∼1090 nm) wavelengths, and that the newly characterized excitation peaks measured past 1000 nm offer improved photostability and enhanced fidelity of linear spectral unmixing at depth compared to shorter wavelengths.
Source: Biomedical Optics Express
DOI: 10.1364/boe.433989
Authors: Ryan T. Lang, Bryan Q. Spring
Institution: Northeastern University
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,913
Awards: Photodynamic Priming of Cancer and Image-guidance for Optimal Immune Response, Multiplexed and dynamically targeted photoimmunotherapy of heterogeneous, chemoresistant micrometastases guided by online in vivo optical imaging of cell-surface biomarkers, Online monitoring and image-guided treatment of chemoresistant micrometastases
OpenAlex ID: W3194602710