Dark-field/hyperspectral microscopy for detecting nanoscale particles in environmental nanotoxicology research

Nanoscale contaminants (including engineered nanoparticles and nanoplastics) pose a significant threat to organisms and environment. Rapid and non-destructive detection and identification of nanosized materials in cells, tissues and organisms is still challenging, although a number of conventional methods exist. These approaches for nanoparticles imaging and characterisation both inside the cytoplasm and on the cell or tissue outer surfaces, such as electron or scanning probe microscopies, are unquestionably potent tools, having excellent resolution and supplemented with chemical analysis capabilities. However, imaging and detection of nanomaterials in situ, in wet unfixed and even live samples, such as living isolated cells, microorganisms, protozoans and miniature invertebrates using electron microscopy is practically impossible, because of the elaborate sample preparation requiring chemical fixation, contrast staining, matrix embedding and exposure into vacuum. Atomic force microscopy, in several cases, can be used for imaging and mechanical analysis of live cells and organisms under ambient conditions, however this technique allows for investigation of surfaces. Therefore, a different approach allowing for imaging and differentiation of nanoscale particles in wet samples is required. Dark-field microscopy as an optical microscopy technique has been popular among researchers, mostly for imaging relatively large specimens. In recent years, the so-called "enhanced dark field" microscopy based on using higher numerical aperture light condensers and variable numerical aperture objectives has emegred, which allows for imaging of nanoscale particles (starting from 5 nm nanospheres) using almost conventional optical microscopy methodology. Hyperspectral imaging can turn a dark-field optical microscope into a powerful chemical characterisation tool. As a result, this technique is becoming popular in environmental nanotoxicology studies. In this Review Article we introduce the reader into the methodology of enhanced dark-field and dark-field-based hyperspectral microscopy, covering the most important advances in this rapidly-expanding area of environmental nanotoxicology.