In addition to their fascinating optical properties, the elemental and electronic structures of GNP are crucial attributes that make them powerful nanoprobes. GNP are electron-dense and thus act as an efficient electron contrast agent for electron microcopy (EM) imaging. GNP can be easily detected in biological compartments and their detailed shape and precise location can be confirmed at the spatial resolution of electron microscopes (lower than the dimensions of the used GNP themselves). As mentioned in the previous section, the old use of immunogold is an example of utilizing GNP as a contrast agent for EM imaging, which enabled a rich understanding of cellular structures and components. Monoclonal antibodies labeled with GNP have been used to detect RNA polymerase II inside the nucleus of living HeLa cells with localization accuracy in the 10 nm scale ^[4]. Similar to the early work on labeling monoclonal antibodies, GNP have recently been explored as an electron microscopy contrast agent to label polymeric nanocarriers. The rationale is to encapsulate GNP inside the matrix of polymeric nanocarriers and to allow the later visualization using EM imaging modalities. This approach exhibits various advantages: (1) the encapsulated smaller GNP can be embedded in the interior matrix of the polymeric nanocarriers and not on their surface, and thus it is not expected to alter the surface characteristics of the labeled nanocarriers; (2) only a low number of encapsulated GNP are required to track and visualize labeled polymeric nanocarriers; (3) the availability of rich knowledge and various procedures to prepare GNP with tunable size and shape as well as surface chemistry facilitates an efficient encapsulation into polymeric nanocarriers; (4) the well documented chemical stability of GNP is a clear advantage for the long term studies in tracking/visualization of the vehicle; (5) the ability to tune the size and shape of GNP (spheres, rods, cubes etc.) may allow multiplexed EM detection of polymeric nanocarriers labeled with different gold nanostructures; (6) the biocompatibility of GNP is a clear advantage compared to many other inorganic nanoprobes such as quantum dots or silver nanoparticles.

It has been reported on the efficient encapsulation of spherical GNP (15 nm) into poly(lactic-co-glycolic acid) (PLGA) nanocarriers (200 nm) to visualize the latter inside the cytoplasmic compartments of HeLa cells, with spatial details that cannot be revealed by conventional alternative probes and imaging tools such as fluorescence microscopy ^[5]. Luque-Michel labeled PLGA nanocarriers with small GNP probes that enabled the localization of PLGA nanocarriers inside culture cells using the high backscattered electron capability of the encapsulated gold nanoprobes ^[6]. Abstiens et al. ^[7] proposed two labeling modalities for dual tracking and visualization of PLGA carriers: (1) encapsulation of fluorescent dyes into the core; and (2) assembly of 2.2 nm GNP at the surface to enable detection using fluorescence and electron microscopies. The two modalities complemented each other as the former provided a wider field of view with much lower spatial resolution and the latter provided high spatial resolution at the expense of a lower area available for imaging per analysis (i.e., a narrower field of view).

In addition to the capability of visualizing GNP using electron microscopy, the dense electronic nature of gold atoms enables significant X-ray attenuation. Computed tomography (CT) imaging, which relies on X-ray attenuation, is one of the most used imaging platforms in clinical practice due to its availability, short scan time, cost-effectiveness and high spatial resolutions. Since tissues and biological fluids have low attenuation capability, contrasting agents (typically iodine-based media) are required for resolved CT imaging in clinical practice. However, it was found that GNP could provide more efficient X-ray attenuation (5.16 for GNP compared to 1.94 cm²/g for iodine, at 100 keV) and they thus hold true promise in CT imaging ^[8][9][10]. Based on this property, targeted gold nanoprobes have been employed to localize cancer using a standard clinical CT scan ^[11][12]. Kim et al. showed that labeling stem cells (SC) with gold nanoprobes has enabled the in vivo imaging of SC using CT without notable adverse effects to the labeled cells with a detection limit of as low as 2 × 10⁴ cells/mL in vivo ^[13]. In vitro studies confirmed the capability of GNP to act as a strong contrasting agent for CT imaging of encapsulating PLGA nanocarriers.

Inductively coupled plasma mass spectrometry (ICP-MS) is known to be one of the most powerful analytical tools for the analysis of trace metals. Thus, GNP can be quantified in complex biological samples using ICP-MS with high sensitivity and selectivity and low interference and background levels (LOQ 15 pg/mL) ^[14]. In fact, it has been shown that ICP-MS is able to monitor the uptake of few GNP into a single cell ^[15]. Wang et al. applied a droplet-chip-time-resolved ICP-MS single-cell analysis system to quantify the number of GNP uptakes by a single cell and draw conclusions regarding the heterogenicity of the cellular uptake of GNP among cells ^[15]. In another recent report, single-cell isotope dilution analysis using laser ablation ICP-MS was used to quantify the content of silver nanoparticles inside a single microphage. The reported approach resulted in a limit of detection inside a single cell as low as 0.2 fg Ag per cell ^[16].

ICP-MS was used to evaluate the biological distribution of pharmaceutical polymeric carriers loaded with metal-based anticancer therapeutics (e.g., cisplatin) or tagged with metal-based molecular probes. Among the few available examples, PLGA nanoparticle distribution into vital organs in rats after intravenous administration was quantified using ICP-MS upon labeling with palladacycles (pallidum-containing) tags ^[17]. It has been reported on the efficient encapsulation of GNP into PLGA, in which ICP-MS is used to determine the average number of encapsulated GNP per single PLGA nanocarrier which was then used to determine the number of PLGA nanoparticles per cell utilizing the low detection limit of ICP-MS analysis for gold ^[5].

Raman spectroscopy is a powerful analytical tool but suffers from weak sensitivity. GNP are excellent enhancers of Raman scattering for nearby Raman-active tags ^[18] due to the enhanced electrical field near the surface of the excited GNP ^{[19][20][21][22][23][24]}. This phenomenon is called surface enhanced Raman scattering (SERS), which enables the detection of analytes with high sensitivity. Upon laser excitation, the oscillating electrons in the conduction band of the metal nanoparticle generate an electrical field that dramatically improves Raman scattering of nearby molecules ^{[25][26][21][24]}. In addition to the electrical field-based enhancement, the chemical enhancement that originates from direct interaction with the surface of the metallic nanostructure contributes to the overall signal enhancement. In fact, GNP and other metallic nanostructures were able to support SERS-based detection to the level of a single molecule ^[22][27]. It is worth mentioning that SERS signal enhancement is a function of the nanoparticle’s size, shape and the distance of the SERS tags from the nanoparticle surface.

“SERS-tagging” is an emerging labeling technique where a “Raman-active molecule” with a unique scattering fingerprint is attached to GNP to allow the tracking and detection of the tagged nanoparticles ^{[22][28][29][30][31][32][33]}. For example, Zavaleta et al. used silica nanoparticles encapsulating GNP that are decorated with a monolayer of Raman active molecules to detect the hybrid nanoparticle in vivo using fiber optic-based Raman endoscopy ^[28][30]. Despite the exciting SERS phenomena of GNP and other metallic nanostructures (silver and copper), little has been done to encapsulate these SERS tags into pharmaceutical polymeric carriers to allow their tracking and detection using Raman spectroscopy, microscopy or endoscopy ^[34]. In this direction, Strozyk et al. reported on the efficient encapsulation of GNP that are functionalized with Raman active tags into PLGA microparticles and films to enable SERS-based imaging at high spatial resolution within the encapsulating host. The research team did not evaluate the in vitro and in vivo capability of the prepared PLGA-GNP; however, they concluded that this SERS-active nanocomposite can be used to enable biological long-term monitoring with exceptional stability when compared to conventional fluorescence-based imaging alternatives ^[35].