Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications

Abstract

Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultrasound.

Introduction

Many researchers in the biological sciences appreciate the extraordinary contrast and specificity provided by fluorescence microscopy. Extrapolating this imaging paradigm to whole-body animal imaging is enticing. However, the physical realities associated with imaging in live tissue make this a continuously elusive objective, as will be evidenced in this review paper. Nevertheless, the information derived from in vivo fluorescence imaging systems can be regarded as an important complement to microscopy studies performed on cell cultures and tissue slices because it provides information about specific biological processes in fully integrated living systems¹. Fig. 1 illustrates the salient differences between in vitro, ex vivo and in vivo fluorescence from biological applications relating to brain imaging. Though in essence the underlying technological and biological principles appear to be the same, imaging in each of these regimes imposes unique challenges requiring significantly different approaches to system design.

In this review paper, the basic principles of imaging fluorescence in living tissue is described, together with the practical challenges in designing, implementing, and assessing these systems. Methods available to overcome some challenges using advanced imaging system designs are discussed and an appreciation of the importance and challenges relating to modeling light propagation in tissue is provided. Perhaps most important is to realize that there is an intrinsic limit on the biological information that can be extracted from even the most carefully designed in vivo imaging instrument. Understanding these limitations is critical for researchers in the biological sciences wanting to use custom or commercial in vivo systems in the scope of their research. If, at the onset of research planning, the intrinsic limitations do not interfere with investigational endpoints, a choice must be made among several technological offerings. This paper will help to guide these choices for systems currently available commercially and in research laboratories.

The paper is divided into several sections covering the fundamentals of fluorescence imaging through advanced technology topics. Section 2 discusses the intrinsic limitations of whole-body imaging. These limitations relate to the interaction of light with microscopic tissue components as well as with the specificity and sensitivity of the contrast that can currently be generated in living animals. A description is also provided for the different types of imaging technologies that can be used for in vivo imaging emphasizing which biochemical fluorophore properties can be extracted from each. This is followed by a more detailed description of the various hardware components required in whole-body fluorescence imaging, including state-of-the-art illumination and light detection technology. This section concludes with a description of the various imaging geometries that can be used to perform in vivo small animal imaging. Section 3, consists of an extensive survey of the main commercial systems available on the market. This is presented in parallel with a discussion of salient features of in vivo fluorescence imaging in terms of resolution, sensitivity and quantification. Section 4 reviews recent publications where in vivo fluorescence imaging has been used to study certain biological phenomena. Finally, the paper concludes with a Section 5 reviewing the research done in laboratories to improve the capabilities of in vivo fluorescence imaging.