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  • Primer
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Near-infrared II fluorescence imaging

Nature Reviews Methods Primers volume 4, Article number: 23 (2024) Cite this article

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Abstract

Fluorescence imaging in the second near-infrared (NIR-II) window enables deep-tissue imaging with high resolution and improved contrast by taking advantage of the reduced light scattering and tissue autofluorescence in this region of the spectrum. NIR-II fluorescence imaging uses photoluminescent contrast agents — including carbon nanotubes, quantum dots, rare earth-doped nanocrystals, gold nanoclusters, small molecules and their aggregates — and fluorescent proteins, which all exhibit fluorescence in the 1,000–3,000 nm range. After administration of these fluorophores in vivo, live animals can be imaged with specialized detectors and optical instruments, yielding images with contrast and resolution unparalleled by conventional visible and near-infrared fluorescence imaging. This powerful approach enables dynamic imaging of vascular structures and haemodynamics; molecular imaging and image-guided surgery of tumours; and visualization of deep-seated structures, such as the gastrointestinal system. NIR-II fluorescence imaging has revolutionized biomedical imaging over the past 15 years and is poised to make comparable advancements in cardiology, neurobiology and gastroenterology. This Primer describes the principles of NIR-II fluorescence imaging, reviews the most used fluorophores, outlines implementation approaches and discusses specific scientific and clinical applications. Furthermore, the limitations of NIR-II fluorescence imaging are addressed and future opportunities across various scientific domains are explored.

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Fig. 1: Summary of NIR-II fluorescence imaging.
Fig. 2: Representative in vivo NIR-II imaging systems.
Fig. 3: Different NIR-II fluorescent probes and their emission spectral ranges.
Fig. 4: Preparation and administration of NIR-II probes.
Fig. 5: Representative results of NIR-II fluorescence imaging.
Fig. 6: Applications of in vivo NIR-II fluorescence imaging.
Fig. 7: Examples of unexpected outcomes in in vivo NIR-II imaging.

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Acknowledgements

G.H. acknowledges a National Science Foundation (NSF) EAGER Award (2217582) and a Beckman Technology Development Grant. E.L.S. acknowledges the support of a fellowship from the Bio-X Initiative of Stanford University. Z.O. is supported by the Wu Tsai Neurosciences Institute. H.C. acknowledges support from a Stanford Interdisciplinary Graduate Fellowship. C.H.C.K. acknowledges support from the NSF Graduate Research Fellowships Program and from the NeuroTech training programme from the Wu Tsai Neuroscience Institute. D.J. and E.X. acknowledge Grant PID2019-106211RB-I00 (NANONERV) funded by MCIN/AEI/10.13039/501100011033, and the S2022/BMD7403 REMIN-CM project supported by Comunidad Autónoma de Madrid. E.X. is grateful for a Juan de la Cierva Incorporación scholarship (IJC2020-045229-I). The initial draft of Figs. 1, 2, 4 and 7 were prepared using BioRender.

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Authors and Affiliations

  1. Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA

    Elizabeth Lea Schmidt, Zihao Ou, Han Cui, Carl H. C. Keck & Guosong Hong

  2. Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA

    Elizabeth Lea Schmidt, Zihao Ou, Han Cui, Carl H. C. Keck & Guosong Hong

  3. Nanomaterials for Bioimaging Group (nanoBIG), Departamento de Física de Materiales, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain

    Erving Ximendes & Daniel Jaque

  4. Nanomaterials for Bioimaging Group (nanoBIG), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Hospital Ramón y Cajal, Madrid, Spain

    Erving Ximendes & Daniel Jaque

  5. Institute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autónoma de Madrid, Madrid, Spain

    Daniel Jaque

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  1. Elizabeth Lea Schmidt
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Contributions

Introduction (E.L.S., Z.O., H.C., C.H.C.K. and G.H.); Experimentation (E.X., H.C., D.J. and G.H.); Results (E.L.S., Z.O., H.C. and G.H.); Applications (E.X., H.C., D.J. and G.H.); Reproducibility and data deposition (E.L.S., Z.O., H.C., C.H.C.K. and G.H.); Limitations and optimizations (E.L.S., Z.O., H.C., C.H.C.K. and G.H.); Outlook (E.L.S., Z.O. and G.H.); overview of the Primer (all authors).

Corresponding authors

Correspondence to Daniel Jaque or Guosong Hong.

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Nature Reviews Methods Primers thanks Fan Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Brain Image Library: https://www.brainimagelibrary.org/

Distributed Archives for Neurophysiology Data Integration: https://www.dandiarchive.org/

Image Data Resource: http://idr.openmicroscopy.org/about/

The Cancer Imaging Archive: https://www.cancerimagingarchive.net/

Supplementary information

Glossary

Adjuvant

A substance added to vaccines to enhance the body’s immune response to the vaccine’s antigen.

Affibody

Small protein scaffolds derived from the Z domain of staphylococcal protein A, engineered to bind specific target proteins with high specificity and affinity.

Aggregation-induced emission

A phenomenon where a material, often an organic compound, emits light more efficiently when it is aggregated or clustered together than when it is in an isolated, dissolved state.

Autofluorescence

The natural emission of light upon excitation of biological tissues, largely contributed by endogenous chromophores such as NADH (emission ~460 nm) and flavins (500–600 nm), as well as pigmented cellular structures such as lipofuscin (450–650 nm) and reticulin (470–520 nm).

Avalanche photodetectors

Photodiodes that are specifically designed to use the avalanche effect, which involves the multiplication of charge carriers (electrons and holes) due to high applied voltages, to amplify the electrical signals generated by the absorption of photons.

Dichroic mirrors

Optical filters that reflect light below (for short pass) or above (for long pass) a specific cut-off or cut-on wavelength, respectively, while transmitting the rest.

Emission filter

An optical filter, typically positioned in front of the detector, selectively transmits wavelengths corresponding to the emission of a specific fluorophore while blocking other undesired wavelengths, such as those from the excitation light source.

Epifluorescence

The fluorescence observed in an optical microscope or imaging system when the object is illuminated from the side that is being viewed.

Excitation filter

An optical filter, typically positioned in front of the excitation light source, selectively transmits wavelengths suitable for exciting a specific fluorophore while blocking other undesired wavelengths.

High endothelial venules

Specialized post-capillary venous structures found in lymph nodes and Peyer’s patches that facilitate the entry of lymphocytes from the bloodstream into lymphatic tissues.

Human embryonic kidney cells

A cell line derived from human embryonic kidney tissue, known for robust growth and ease of transfection, commonly used in the production of recombinant proteins, viral vectors and in vitro drug toxicity assays.

Indium gallium arsenide

(InGaAs). A compound semiconductor material that is sensitive to infrared light and commonly used in photodetectors for second near-infrared (NIR-II) fluorescence imaging.

Infinity-corrected objective

An optical lens system designed to produce parallel rays between the objective and the eyepiece or camera, typically used in microscopy for clearer imaging and easier integration of additional optical components.

Intralipid

A sterile fat emulsion commonly used in medical settings as a parenteral nutrition supplement and in research as a scattering medium to simulate biological tissues in optical imaging experiments.

Optical density

A measure of how much a substance or an object attenuates the intensity of light that passes through it. Mathematically, optical density is defined as OD = –log10(I/I0), where I is the intensity of light transmitted through the substance and I0 is the intensity of the incident light.

Optical diffuser

A device that scatters light in various directions to produce a uniform illumination.

Optomechanics

Elements including optical tables, breadboards, construction components such as mounts and mechanically integrated optoelectronic devices.

Organic colour centres

Synthetic defects in semiconducting single-walled carbon nanotubes (CNTs) created by covalently bonding organic molecules to the crystal lattice, resulting in quantum emitters that fluoresce in the second near-infrared (NIR-II) spectrum, emitting pure single photons at room temperature.

Overtone absorption

The absorption of light by a molecule at a frequency (or wavelength) that is a multiple of the fundamental frequency of a vibrational mode of that molecule.

Particle image velocimetry

A visual measurement technique used to obtain instantaneous velocity fields by tracking the movement of small particles seeded in a fluid flow.

Peripheral node addressin

A carbohydrate ligand for l-selectin that plays a crucial role in the homing of white blood cells, specifically directing their migration to peripheral lymph nodes during the immune response.

Photodiodes

Semiconductor devices that convert light into an electrical current, the amplitude of which is directly proportional to the light intensity shining on the diode.

Photomultiplier tubes

Electronic devices that detect and greatly amplify weak light signals by converting photons generated by a photocathode into an intensified electrical signal through a series of secondary electron multipliers.

Reactive oxygen species

Chemically reactive molecules that contain oxygen, such as hydrogen peroxide (H2O2), superoxide anion (O2−), hydroxyl radical (•OH) and singlet oxygen (1O2).

Scattering

The deviation of light rays from their original path, a phenomenon exacerbated in animal tissue by the inhomogeneity of refractive indices among components such as water, lipid membranes and subcellular organelles.

Semiconductor diode lasers

Lasers with a semiconductor active medium, akin to a light-emitting diode, but that produce coherent light through stimulated emission from the recombination of electrons and holes.

Signal-to-noise ratio

(SNR). The ratio of fluorescence signal to the background noise, the latter of which comprises the shot noise and dark noise of the photodetector, the read-out noise from the camera electronics as well as autofluorescence and scattering from biological tissues.

Superconducting nanowire single-photon detectors

Ultra-sensitive devices that detect individual photons by measuring the disruption in the bias current, which arises when single photons absorbed by the superconducting nanowire break Cooper pairs.

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Schmidt, E.L., Ou, Z., Ximendes, E. et al. Near-infrared II fluorescence imaging. Nat Rev Methods Primers 4, 23 (2024). https://doi.org/10.1038/s43586-024-00301-x

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