US 2021/0239955 A1 · NEAR INFRA-RED LIGHT SHEET MICROSCOPY THROUGH SCATTERING TISSUES

Abstract

Provided are methods of near-infrared II light sheet microscopy with excitation and emission up to between about 1320 nm and about 2400 nm, respectively, for optical sectioning through live tissues with improved penetration depth without any invasive surgery. The methods allow for normal and oblique configurations enabled in vivo imaging of live mice through intact tissue, revealing such as abnormal blood flow and T cell motion in tumor microcirculation and mapping out programmed-death ligand 1 and programmed cell death protein 1 (PD-1) in tumors with cellular resolution. 3D imaging through intact mouse heads resolved vascular channels between skull and brain cortex, and monitored recruitment of macrophages/microglia to traumatic brain injury site post injury.

Claims21

Dependent claims

A method of optically sectioning a biological sample, the method comprising the steps:

(a) contacting a biological sample with a fluorescent contrast agent having an excitation wavelength of between about 785 nm to about 2400 nm and a fluorescence emission having a wavelength of between about 800 nm to about 2400 nm;
(b) irradiating the biological sample with at least one excitation light having a wavelength of between about 785 nm to about 2400 nm, wherein the excitation light is configured as a static light sheet and directed through a first plane of the biological sample;
(c) orthogonally detecting an emitted fluorescence having a wavelength of between about 800 nm to about 2400 nm from the irradiated biological sample;
and
(d) generating a first digital image of the fluorescence from the irradiated first plane through the biological sample.

2 more elements

A light sheet microscope comprising along an optical axis:

an illumination objective positioned to direct an excitation light sheet through a plane of a biological sample;
a plurality of achromatic lenses optimized for transmission of light between about 785 nm to about 2400 nm;
a proximal first adjustable mechanical slit;
a cylindrical lens;
a distal second adjustable mechanical slit adjacent to the cylindrical lens and distal to the illumination objective, wherein the slit of said second slit is orientated at right-angles to the slit of the first slit;
a pinhole;
at least one excitation light source in the 600-2400 nm range;
and
at least one removable mirror disposed to direct an excitation light from the at least one light source along the optical axis of the illumination objective, the plurality of achromatic lenses, the proximal first adjustable mechanical slit, the cylindrical lens, the distal second adjustable mechanical slit, and the pinhole;
a detection objective disposed to orthogonally receive fluorescent light emitted from a target irradiated by a light sheet from the illumination objective and to direct said fluorescent light to a detector operably connected to a computer system for generating a digital image of the fluorescent light;
and
at least one emission filter configured to only transmit fluorescent light having a wavelength of between about 785 nm to about 2400 nm.

9 more elements

Dependencies are inferred from the claim text.

Near infra-red light sheet microscopy through scattering tissues

Life of this patent

Near infra-red light sheet microscopy through scattering tissues

Life of this patent

Abstract

Claims21