Short-Wave Infrared Confocal Fluorescence Imaging of Deep Mouse Brain with a Superconducting Nanowire Single-Photon Detector

Abstract

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its noninvasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that the two optimal wavelength windows for high-resolution deep mouse brain imaging are around 1300 and 1700 nm. However, one-photon fluorescence imaging in the wavelength region has been highly challenging due to the poor detection efficiency of currently available detectors. To fully utilize this wavelength advantage, we demonstrated here one-photon confocal fluorescence imaging of deep mouse brains with an excitation wavelength of 1310 nm and an emission wavelength within the 1700 nm window. Fluorescence emission at 1700 nm was detected by a custom-built superconducting nanowire single-photon detector (SNSPD) optimized for detection between 1600 nm and 2000 nm with low detection noise and high detection efficiency. With the PEGylated quantum dots and SNSPD both positioned at the optimal imaging window for deep tissue penetration, we demonstrated in vivo one-photon confocal fluorescence imaging at approximately 1.7 mm below the surface of the mouse brain, through the entire cortical column and into the hippocampus region with a low-cost continuous-wave laser source and low excitation power. We further discussed the significance of the staining inhomogeneity in determining the depth limit of one-photon confocal fluorescence imaging. Our work may motivate the further development of long wavelength fluorescent probes, and inspire innovations in high-efficiency, high-gain, and low-noise long wavelength detectors for biological imaging.

Roadmap on wavefront shaping and deep imaging in complex media

“…Therefore, the depth limit determined by the SBR is similar for 1P confocal and 2P imaging when the excitation wavelength is the same. Both theoretical and experimental studies show that the depth limit for 1P confocal and 2P imaging is between 1.5 and 2 mm, when imaging mouse brain vasculature, in the long wavelength windows of 1300 and 1700 nm [15][16][17][18]. The biggest challenge facing deep tissue long wavelength 1P confocal microscopy is the lack of IR dyes with excitation wavelengths >1200 nm.…”

“…Quantum dots (QDs), including carbon dots, are probably the most promising path so far but making QDs into robust functional indicators may yet prove difficult. Additionally, the recent development of superconducting nanowire detectors (SNDs) is promising for 1P confocal imaging at 1300 and 1700 nm [18]. While still expensive, advancements in materials and manufacturing for SNDs could reduce the cost and make these detectors affordable for biological imaging.…”

Counterion‐Paired Bright Heptamethine Fluorophores with NIR‐II Excitation and Emission Enable Multiplexed Biomedical Imaging

“…The penetration limit of fluorescence imaging is usually described by the effective attenuation length l e (Figure S1), which is derived from the scattering mean‐free path ( l s ) and the absorption length ( l a ) of the tissue (that is, l e =(1/ l a +1/ l s ) −1 ) [2] . As water has a nontrivial contribution to tissue absorption, the theoretical model combining Mie scattering and water absorption thus suggest a “tissue‐transparent” NIR‐II spectral window of 1000–1350 nm and 1500–1700 nm [3] . In 2009, NIR‐II fluorescence imaging was reported initially by using single‐walled carbon nanotubes (SWNTs), [4] and then continuously improved fluorophores combined with various developed imaging methods such as NIR‐II confocal microscopy and light‐sheet microscopy have driven the imaging to achieve unprecedented SNR in deep tissue of living organisms [5] .…”

“…[2] As water has a nontrivial contribution to tissue absorption, the theoretical model combining Mie scattering and water absorption thus suggest a "tissue-transparent" NIR-II spectral window of 1000-1350 nm and 1500-1700 nm. [3] In 2009, NIR-II fluorescence imaging was reported initially by using singlewalled carbon nanotubes (SWNTs), [4] and then continuously improved fluorophores combined with various developed imaging methods such as NIR-II confocal microscopy and light-sheet microscopy have driven the imaging to achieve unprecedented SNR in deep tissue of living organisms. [5] These efforts have recently enabled NIR-II fluorescence multiplexed bioimaging to investigate cell migration, dynamic anatomical and metabolic characteristics in intact animals simultaneously.…”

Optical transitions, exciton radiative decay, and valley coherence in lead chalcogenide quantum dots

“…Lead chalcogenide PbX (X = S, Se, Te) quantum dots (QDs) have found numerous applications in optoelectronics [1][2][3][4][5] and in vivo fluorescence imaging [6][7][8] due to tunability of their fundamental optical transition with the QD size within the near-infrared and mid-infrared ranges. Although lead chalcogenides are N -valley semiconductors with N = 4, the simplest models widely used for their description are restricted to the carrier states in a single valley.…”