Applications of IVIS (In Vivo Imaging Systems)

By Vasco Medeiros, BScReviewed by Emily Henderson, B.Sc.

In vivo imaging refers to the analysis and visualization of the body’s internal machinery, performed on a live patient, rather than a tissue sample or some other form of in vitro assay. This form of examination is non-invasive and expounds important cell modes and processes using biomarkers.

Some popular forms of in vivo imaging are near-infrared fluorescent (NIRF) imaging, transfection, transduction, and quantum dots. They have been the driving force behind the exploration of live animal models.

In vivo Imaging through luminescence is a fundamental technique in all of science, fundamental in biochemistry and biology. The two means by which optical data is presented to the user via luminescence, are bioluminescence and fluorescence. The former produces light by fluorescent enzymes (typically luciferase), while the latter emits light via molecular probes.

In vivo screening via fluorescence

Fluorescence, in the world of biology, is a phenomenon that requires a preliminary excitation light source that is exogenous to the animal, emitting excitation photons onto a molecular probe. The probe is encircled in electrons that will enter an elevated excitation state, releasing longer wave photons (light from the probe) as their excitation state relaxes. The light emitted from this probe is then captured by a charge-coupled device (CCD) camera system, allowing researchers to observe whatever is illuminated in vivo.

In the past, immune cell-based therapy has proved challenging under clinical practice because of the confusion that encompasses cellular mechanisms. The development of quantitative fluorescence imaging has addressed this for noninvasive cell tracking. Rinat Meir et al. published a case study underlining this, asserting that T-cells can be labeled with green fluorescent protein for CT imaging, leading to a strong signal intensity when tracking tumor sites.

In vivo screening via bioluminescence

Juxtaposing fluorescent means, bioluminescence does not require a preliminary source of light to excite the sample and will offer a lower signal-noise ratio. The illumination provided by bioluminescence is generated through a chemical reaction originating from the live organism.

A de facto example of Bioluminescence can be found in the case of D-luciferin, embodying the form of a substrate molecular probe. Bioluminescence occurs when the substrate is acted upon by the enzyme firefly luciferase in the presence of cellular ATP and oxygen.

Bioluminescence and fluorescence are the leading practices to view structural components of the brain and are often used to illuminate the progression of malignant cancer cells. The question now remains, how does one obtain a fluorescent probe or moiety, and inject it into a mouse model or clinical patient? This is performed via transfection or transduction.

Transfection incorporates gene transfer into cellular machinery through chemical or physical means, while transduction injects this gene-driven luminescent probe via a viral agent, akin to gene therapy. Some examples of transfection that pertain to these in vivo imaging methods include microinjection, gene gun, and electroporation.

Transduction often uses a viral agent that is in a lysogenic cycle, where phage DNA is introduced into the host genome. This occurs at the site of a target cell population, and the viral genome will replicate. The viral gene being implemented within the host will give rise to bioluminescence, allowing the tissue to be observed

In vivo imaging does not solely pertain to genetic modification, and direct probe injection can be done through other avenues. Some popular examples of non-targeting probes include small organic near-infrared NIR fluorescent dyes or quantum dots.

Fluorescent dyes as non-targeting probes

Some popular small fluorescent dyes include Cy5, Cy7, and indocyanine green. Other dyes such as SiR-NIRs have even been modified through rhodamine scaffolds, allowing for emissions that lay within a suitable range for in vivo imaging. A general feature of these dyes is that they are not designed to be directed towards a specific target. They are neither selective nor specific and are non-discriminatory towards most tissue lines.

Their high quantum efficiency in aqueous media leads to them being resistant to photobleaching, a common problem amongst fluorescence-based assays. These manufactured dyes have minimal tissue absorption, as well as low background noise for serums, proteins, and other biomolecules within the NIR range.

Though most of these dyes are not target-specific, some exceptions can be made in oncology models, due to their enhanced permeability and retention effect (EPR). Tumor masses will retain these dyes for a longer time frame because of their abnormal angiogenesis.

The use of quantum dots in vivo

Quantum dots are another brand of fluorescent probes and are particularly useful within in vivo imaging. These quantum dots are nanocrystals (2-10 nm), made from a semiconductor material coated with a substance that will allow for chemical conjugation of specific ligands. This in turn will produce a probe with specificity, directed towards a particular anagenic site within a given mouse model. These dots have a high emitted: absorbed photon ratio, qualitatively observable by their high brightness.

One auxiliary boon that is granted by quantum dots is their ability to perform multiplexing. Multiplexing is when multiple probes can produce specific emission wavelengths, which can later be collected separately. Multiplexing can occur due to the optimal strokes shift that is provided by the quantum dots, yielding a good separation between the excitation/absorption wavelength, and the emission wavelength.

Further Reading

• All Microscopy Content
• What is Microscopy?
• Microscopy and Artificial Intelligence
• Scanning Electron Microscopy (SEM): An Overview
• Investigating the Intersection between Microscopy and Healthcare