Fluorescence imaging in vivo: recent advances

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In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.

Introduction

In vivo fluorescence imaging resembles fluorescence microscopy in that both use a low-light camera and appropriate filters to collect fluorescence emission light from samples, but differs in that it works at a macroscopic level. The objects for imaging are whole-body small animals instead of cells in culture dishes or on slides. This extension into the in vivo setting allows visualization of biology in its intact and native physiological state, but it is a technically challenging process for at least two reasons. First, thick, opaque animal tissue absorbs and scatters photons and generates strong autofluorescence, all of which obscure signal collection and quantification [1]. Second, the complicated in vivo environment puts additional demands on the contrast agent or imaging probe: it has to be biologically stable once distributed in an organism, preferentially accumulate at the intended target site, and produce imaging contrast specific to the target.

Significant advances in mathematical models for describing photon propagation in tissues and in the available instrumentation for illumination and detection have been made in the past to improve the capacity of quantitative fluorescence imaging in tissue, and have recently been reviewed [2, 3••]. This short review is not intended to be a comprehensive discussion of all aspects of in vivo fluorescence imaging, but instead focuses on the latest progress in imaging probe chemistry and reporter gene techniques.

Section snippets

Imaging probe chemistry

Molecules that absorb in the near-infrared (NIR) region, 700–1000 nm, can be efficiently used to visualize and investigate in vivo molecular targets because most tissues generate little NIR fluorescence [4, 5]. The most common organic NIR fluorophores are polymethines (Figure 1a). Among them, pentamethine and heptamethine cyanines comprising benzoxazole, benzothaizole, indolyl, 2-quinoline or 4-quinoline have been found to be the most useful [6]. Their physical properties, biodistribution,

Small organic fluorophores

Chemists continue to develop new NIR fluorochromes with improved fluorescence quantum yield, high chemical and photostability, low aggregation tendencies and low cytotoxicity. For example, Dehaen and colleagues used palladium-catalyzed coupling reactions to synthesize several BODIPY derivatives with emission ranging from green to near-infrared [22]. Zhao and Carreria reported conformationally restricted Aza-BOBIPY dyes with improved physical properties and chemical and photo stability [23].

Polyvalency to improve probe affinity

Tight and specific binding of the probe to the target is always the key to successful imaging. As many imaging targets are located outside of the cell surface, the principle of polyvalency can be applied to improve the binding affinity of the probe [34]. Two recent studies have reported the design of oligomeric RGD peptides for imaging the αvβ3 integrin receptor in xenografted tumors in vivo. Cheng et al. [35] synthesized monomeric, dimeric and tetrameric cyclic RGD units and conjugated them to

Red and near-infrared fluorescent proteins

Reporter technologies employ reporter genes, and have been an indispensable tool to study gene expression and regulation. There are several reporter genes for in vivo imaging [45]. Among them, fluorescent proteins (FPs) remain highly popular because they are genetically encoded and easy to use with no need for systematic delivery of the imaging probe. FP-based in vivo imaging in cancer research allows direct visualization of primary tumor growth, invasion, metastatic seeding and colonization,

High-resolution in vivo fluorescence imaging

Unlike fluorescence microscopy, in vivo fluorescence imaging detects bulk signals from thousands of cells with much lower resolution. By contrast, fluorescence microendoscopy uses small size optical probes (typically 0.25–1 mm in diameter) that are minimally invasively inserted into solid tissues to achieve high-resolution imaging deep within tissues [57]. Combined with fluorescent probes, this technology could allow the interrogation of the biology at the single-cell and single-molecule level

Conclusions

In vivo fluorescence imaging visualizes the fluorescent emissions from fluorophores in whole-body living small animals. With the increasing availability of fluorescent probes and reporters, it is gaining momentum as an important translational tool between basic research and clinical application. Although traditional small-molecule NIR dyes continue to be used, the development of fluorescent organic, biological, and inorganic nanoparticles for in vivo fluorescence imaging offers powerful tools

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We are grateful to Luke Higgins for initial work on this article and to Edwards Graves for critical reading of the manuscript. This work is supported by grants from the Burroughs Wellcome Fund, the Department of Defense Breast Cancer Research Program Concept Award, the National Cancer Institute Centers of Cancer Nanotechnology Excellence (1U54CA119367-01), and the National Institute of Biomedical Imaging and Bioengineering (EB3803).

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