|Fig. 1: First medical X-ray photograph taken by Wilhelm Conrad Röntgen. (Source: Wikimedia Commons)|
As far as we know, biomedical imaging provides critical information for diagnosing and disease treatment. Nowadays, the physiological information relevant to pathology is attainable on cellular level. Starting from the early X-ray photographs Wilhelm Conrad Röntgen (see Fig. 1), new clinical instruments are constantly being discovered. Examples include computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT). 
Fluorescence imaging is one a new method for mapping vessels and tumor tissues in-vivo and ex-vivo. Near-infrared fluorophores for biomedical imaging have already opened up new fields in fundamental scientific research and clinical practice. Important new knowledge is being about how the human body works through the space-time distribution of signal sources revealed by this method. The importantant limitations of these methods at present are chiefly (1) coarse space-time resolution and (2) lack of in-vitro and in-vivo probes with appropriate functional molecular conjugation.
In-vivo fluorescence-imaging is evades most of these key difficulties. Its many advantages include avoiding hazardous optical radiation and achieving high space-time resolutions by means of real-time wide-field image acquisition. But conventional near-infrared(NIR) fluorescence imaging works in the 700-900 nm NIR window. To reduce photon scattering, light absorption and autofluorescence, it is necessary to work with biocompatible NIR-II (1,000-1,800nm) fluorophores. The newly developed fluorescence imaging approach that extends fluorescence wavelength to the NIR-II window (1000-1700 nm) through these fluorphores has thus received great interest recently.  NIR-II fluorescence imaging in vivo can indeed achive diminished tissue autofluorescence, reduced photon scattering without high levels of light absorption, deep tissue penetration, and high-clarity fluorescence imaging into a living body. 
Unfortunately, strong interactions with water molecules and dominanation by non-radiative decay pathways cuses most NIR-II fluorophores, especially organic molecules, to have a low QY in water phase. This limitation constrains in-vivo NIR-II imaging to reaching real-time tracking at high speeds and frame rates, and deciphering 3D structures of live tissues in a layer-by-layer fashion - by developing Z-resolved imaging techniques in the 1000-1700 nm range.  Up to now only 2D projected epi-fluorescence imaging has been done in the NIR-II window. It is not yet possible to study 3D tissue structures at millimeter depths, partly due to the lack of sufficiently bright fluorophores. 
|Fig. 2: Principle of the confocal microscope. (Source: Wikimedia Commons)|
A confocal microscope is one type of 3D fluorescence microscope in which resolution is increased by rejecting out-of-focus light. This is done by passing light through a pinole with a specific radius, as shown in Fig. 2. The principle of confocal imaging was patented in 1957 by Marvin Minsky.
For obtaining more specific information of the disease, a two-color system has been invented. The two-color system of projection is a name given to a variety of methods of projecting a full-color image using (only) two different single-color projectors. Prof. H. Dai's group at Stanford performed two-color in vivo fluorescence imaging for the first time in the NIR-II window, motivated by the potential of probing of and differentiating multiple components or molecular targets in living biological systems.  Cancer imaging is a key to diagnosis and therapeutic interventions and has been pursued by a wide range of modalities including fluorescence imaging. 
© Hongpeng Gao. The author warrants that the work is the author's own and that Stanford University provided no input other than typesetting and referencing guidelines. The author grants permission to copy, distribute and display this work in unaltered form, with attribution to the author, for noncommercial purposes only. All other rights, including commercial rights, are reserved to the author.
 G. Hong et al., "Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy," Chem. Rev. 115, 10816 (2015).
 G. Hong et al.," Near-Infrared II Fluoresence for Imaging Handlimb Vessel Regeneration With Dynamic Tissue Perfusion Measurement," Circ. Cardiovasc. Imaging 7, 517 (2014)
 G. Hong, A. L. Antaris, and H. Dai, "Near-Infrared Fluorophores for Biomedical Imaging," Nat. Biomed. Eng. 1, 0010 (2017).
 Q. Yang et al., "Rational Design of Molecular Fluorophores for Biological Imaging in the NIR-II Window." Adv. Mater. 29, 1605497 (2017).
 J. T. Robinson et al., "In Vivo Fluorescence Imaging in the Second Near-Infrared Window with Long Circulating Carbon Nanotubes Capable of Ultrahigh Tumor Uptake," J. Am. Chem. Soc. 134, 10664 (2012).