Illumination sources for small animal in vivo imaging

Illumination sources for small animal in vivo imaging

Noninvasive small animal in vivo imaging is a staple in the pharmaceutical industry for testing a drug’s efficacy and safety, in infectious disease research for monitoring the host’s inflammation and immune response, and in cancer research for detecting a tumor’s progression. It enables scientists to monitor disease processes and progression via methods that are also applicable to human patients.

Typically, researchers tag areas of interest using fluorescent probes or bioluminescence. Fluorescence imaging requires an excitation light source to energize the fluorophores in situ; the resulting emitted light is filtered and detected by a charge-coupled device (CCD). Fluorescence is complementary to other imaging modalities, because fluorescence labeling can be used for other assays, and is a popular standalone technique for in vivo imaging.¹

Getting under the skin of the issue

Traditionally, scientists have performed in vivo imaging using light-emitting diodes (LEDs) in the visible red, green, blue, white (RGBW) range of ~400 to 650 nm. Cellular and tissue imaging in the near-infrared (NIR) spectrum, between 700 and 900 nm, is a newer technique. The NIR range is advantageous for many in vivo imaging applications due to low absorption by biological molecules in this region; although there are instances where RGBW alone, or both illumination sources in combination, may be the better choice.

When to use each illumination source

Photobleaching occurs when a fluorophore photochemically alters such that it permanently loses fluorescence. This is caused by covalent bonds cleaving, or non-specific reactions between the fluorophore and surrounding molecules. The best way to prevent photobleaching is to reduce the light intensity and imaging time to minimize the fluorophore’s light exposure. LED illumination sources can prevent photobleaching.

Autofluorescence may also present a significant problem during in vivo imaging, as many key proteins abundantly expressed within in vivo models (e.g., NADPH, collagen, riboflavin, folic acid) naturally fluoresce. Imaging with NIR lasers helps reduce autofluorescence. Very few endogenous molecules within in vivo models emit fluorescence in the NIR spectrum,² and NIR light scatters less as it exits the body, permitting greater imaging penetrability.³ As such, NIR is useful for looking at deeper internal organs or for performing 3-D reconstructions of non-surface tumors. Longitudinal imaging protocols, such as tissue accumulation studies, benefit from NIR illumination.

NIR-compatible probes are becoming more readily available. Single-walled nanotubes (SWNT), for example, emit in the 1000-1400 nm range and are highly photostable, which allows them to act as NIR tracking probes for longitudinal studies.⁴

Researchers may need to use designer probes that require different illumination sources, making imagers containing both LEDs and NIR lasers particularly useful. This reduces cost, time, and space required for performing a wide range of in vivo imaging applications.

A versatile solution for in vivo imaging

Modern in vivo imagers—with more sensitive detectors and larger repertoires of compatible probes— largely overcome several signal detection and resolution challenges. Instruments such as the UVP iBox Studio are capable of cellular-level spatial resolution without sacrificing whole-body imaging, meaning that researchers can use a single instrument to reveal cellular behaviors in their natural environments and correlate them to systemic or whole-body responses. The iBox Studio in vivo imager is a powerful and affordable imaging system for high resolution small animal fluorescence imaging. The instrument includes a warming plate, keyboard, mouse, and RGBW LEDs. The system is upgradeable for NIR imaging applications, facilitating multiplex experiments or experiments that require designer probes. Small animal anesthesia attachments are also available. In addition to in vivo small animal imaging, the iBox Studio facilitates other types of fluorescence imaging, NIR imaging, multiplex imaging, plant imaging, and colony counting.

References

1. A. Zelmer, T.H. Ward, “Noninvasive fluorescence imaging of small animals,” J Microsc, 252(1):8- 15, 2013.
2. J. Rao et al., “Fluorescence imaging in vivo: recent advances,” Curr Opin Biotechnol, 18(1):17-25, 2007.
3. G. Hong et al., “Near-infrared fluorophores for biomedical imaging,” Nat Biomed Eng, 1:0010, 2017
4. X. Zhang et al., “Near-infrared molecular probes for in vivo imaging,” Curr Protoc Cytom, 60(1):12.27.1-20, 2012.