Holotomography and atomic force microscopy: a powerful combination to enhance cancer, microbiology and nanotoxicology research

Modern imaging strategies are paramount to studying living systems such as cells, bacteria, and fungi and their response to pathogens, toxicants, and nanomaterials (NMs) as modulated by exposure and environmental factors. The need to understand the processes and mechanisms of damage, healing, and cell survivability of living systems continues to motivate the development of alternative imaging strategies. Of particular interest is the use of label-free techniques (microscopy procedures that do not require sample staining) that minimize interference of biological processes by foreign marking substances and reduce intense light exposure and potential photo-toxicity effects. This review focuses on the synergic capabilities of atomic force microscopy (AFM) as a well-developed and robust imaging strategy with demonstrated applications to unravel intimate details in biomedical applications, with the label-free, fast, and enduring Holotomographic Microscopy (HTM) strategy. HTM is a technique that combines holography and tomography using a low intensity continuous illumination laser to investigate (quantitatively and non-invasively) cells, microorganisms, and thin tissue by generating three-dimensional (3D) images and monitoring in real-time inner morphological changes. We first review the operating principles that form the basis for the complementary details provided by these techniques regarding the surface and internal information provided by HTM and AFM, which are essential and complimentary for the development of several biomedical areas studying the interaction mechanisms of NMs with living organisms. First, AFM can provide superb resolution on surface morphology and biomechanical characterization. Second, the quantitative phase capabilities of HTM enable superb modeling and quantification of the volume, surface area, protein content, and mass density of the main components of cells and microorganisms, including the morphology of cells in microbiological systems. These capabilities result from directly quantifying refractive index changes without requiring fluorescent markers or chemicals. As such, HTM is ideal for long-term monitoring of living organisms in conditions close to their natural settings. We present a case-based review of the principal uses of both techniques and their essential contributions to nanomedicine and nanotoxicology (study of the harmful effects of NMs in living organisms), emphasizing cancer and infectious disease control. The synergi