Hyperspectral imaging of nanoparticles in biological samples: Simultaneous visualization and elemental identification

Abstract

While engineered nanomaterials (ENMs) are increasingly incorporated into industrial processes and consumer products, the potential biological effects and health outcomes of exposure remain unknown. Novel advanced direct visualization techniques that require less time, cost, and resource investment than electron microscopy (EM) are needed for identifying and locating ENMs in biological samples. Hyperspectral imaging (HSI) combines spectrophotometry and imaging, using advanced optics and algorithms to capture a spectrum from 400 to 1000 nm at each pixel in an enhanced dark-field microscopic (EDFM) image. HSI-EDFM can be used to confirm the identity of the materials of interest in a sample and generate an image "mapping" their presence and location in a sample. Hyperspectral mapping is particularly important for biological samples, where ENM morphology is visually indistinct from surrounding tissue structures. While use of HSI (without mapping) is increasing, no studies to date have compared results from hyperspectral mapping with conventional methods. Thus, the objective of this study was to utilize EDFM-HSI to locate, identify, and map metal oxide ENMs in ex vivo histological porcine skin tissues, a toxicological model of cutaneous exposure, and compare findings with those of Raman spectroscopy (RS), energy-dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Results demonstrate that EDFM-HSI mapping is capable of locating and identifying ENMs in tissue, as confirmed by conventional methods. This study serves as initial confirmation of EDFM-HSI mapping as a novel and higher throughput technique for ENM identification in biological samples, and serves as the basis for further protocol development utilizing EDFM-HSI for semiquantitation of ENMs.

Biodistribution of inhaled metal oxide nanoparticles mimicking occupational exposure: a preliminary investigation using enhanced darkfield microscopy

“…Paraffin-embedded tissues were sectioned (6 μm thickness) using a rotary microtome with a clean blade for each cut to minimize cross-contamination and mounted onto glass microscopy slides. Deparaffinized and rehydrated tissue sections were stained with hematoxylin and eosin (H&E), then dehydrated through graded alcohols to xylene and coverslipped [9,10,12,13]. Tissue slides were scanned and imaged using BF microscopy, using a Nikon E200 brightfield microscope, and EDFM, using a CytoViva EDFM-HSI system (Auburn, AL) [9,10].…”

“…The current best-known method for direct visualization of NMs is electron microscopy (EM), often coupled with energy dispersive X-ray spectroscopy (EDS) for elemental identification [8]. However, rapid identification and characterization of NMs in biological samples and other forms of complex media, such as environmental media, is hindered by the associated cost and time required to perform EM [9,10]. This analytical bottleneck is slowing progress in many disciplines where nanoscale visualization and characterization are critical.…”

“…This analytical bottleneck is slowing progress in many disciplines where nanoscale visualization and characterization are critical. Enhanced darkfield microscopy (EDFM) offers a more rapid and less costly alternative to EM for direct visualization of NMs in complex matrices [9,10]. The CytoViva enhanced darkfield microscope has a patented condenser that improves alignment and focus of oblique angle illumination, thereby enhancing the signal-to-noise up to seven times over standard darkfield optics and allowing for improved visualization of nanoscale materials.…”

“…Coupled with hyperspectral imaging (HSI), EDFM provides a method for visualization and nanoparticle (NP) identification based on hyperspectral data. While EM is capable of higher magnification and resolution than EDFM-HSI, there is also considerable time-to-knowledge, cost-of-ownership ($1-4M for transmission EM vs. $155,000 for EDFM-HSI), and intensive training for tool operation that is not the case with EDFM [10]. For example, rapid scanning of a 1.5 cm × 1.5 cm tissue sample with EDFM at 10-100x magnification takes a trained microscopist less than 1 hr, while it would take a minimum of 5 months (8 hr/day, 5 days/week) to scan the same sample area with transmission EM at “low” 1,000x magnification [10].…”

“…While EM is capable of higher magnification and resolution than EDFM-HSI, there is also considerable time-to-knowledge, cost-of-ownership ($1-4M for transmission EM vs. $155,000 for EDFM-HSI), and intensive training for tool operation that is not the case with EDFM [10]. For example, rapid scanning of a 1.5 cm × 1.5 cm tissue sample with EDFM at 10-100x magnification takes a trained microscopist less than 1 hr, while it would take a minimum of 5 months (8 hr/day, 5 days/week) to scan the same sample area with transmission EM at “low” 1,000x magnification [10]. Further, samples for EDFM need only be prepared as for light microscopy, which is commonly performed at most histology cores; special handling and preparation, including stains, fluorescent markers or creation of thin sections for EM, are not required.…”