Biological applications of multimodal imaging involving Raman and 4Pi Raman microscopy

Raman microscopy is becoming an increasingly important label-free imaging technique. It proved to be a viable tool for life science applications allowing to analyze bacteria, cells, and tissues at the molecular level. Combining Raman microscopy with complementary imaging modalities and techniques is explored here to: (1) analyze mild traumatic brain injury (mTBI) in a combination with magnetic resonance imaging (MRI) for detecting mild, and invisible to medical imaging techniques, brain tissue damage; (2) reveal complementarity of Raman and fluorescence microscopy approaches for investigating and tracking bovine lactoferrin inside calf rectal epithelial cells in the presence of enterohemorrhagic Escherichia coli (EHEC); (3) apply Raman microscopy along-side the molecular analysis approaches (such as scanning transmission electron microscopy-energy dispersive X-ray (STEM-EDX), low energy X-ray fluorescence (LEXRF), nanoscale secondary ion mass spectrometry (Nano-SIMS)) to uncover the origin of the long-range conductance in cable bacteria; (4) develop multifunctional surface enhanced Raman scattering (SERS) platform based on calcium carbonate particles for enhancing a weak Raman scattering signal of biomolecules as well as to apply Raman microscopy for particle detection in vivo in Caenorhabditis elegans (C. elegans) worms; and (5) combine Raman microscopy and atomic force microscopy (AFM) to track Chlamydia psittaci in cells. Analysis of described above samples and phenomena is based on Raman molecular fingerprint images, where, similarly to fluorescence light microscopy, the resolution is limited by diffraction of light. Therefore, efforts are also put to enhance the resolution of Raman microscopy-based imaging by adding a 4Pi configuration to a confocal Raman microscope. As a result, a possibility to enhance the axial (also called longitudinal) resolution is investigated by constructing a 4Pi confocal Raman microscope, which is also applied to study bacteria inside cells. Results presented in this work emphasize the added value of multimodal microscopy approaches, particularly involving Raman microscopy, in a broad range of applications in bioengineering, biomedicine, and biology

Applications of AFM in pharmaceutical sciences

Atomic force microscopy (AFM) is a high-resolution imaging technique that uses a small probe (tip and cantilever) to provide topographical information on surfaces in air or in liquid media. By pushing the tip into the surface or by pulling it away, nanomechanical data such as compliance (stiffness, Young’s Modulus) or adhesion, respectively, may be obtained and can also be presented visually in the form of maps displayed alongside topography images. This chapter outlines the principles of operation of AFM, describing some of the important imaging modes and then focuses on the use of the technique for pharmaceutical research. Areas include tablet coating and dissolution, crystal growth and polymorphism, particles and fibres, nanomedicine, nanotoxicology, drug-protein and protein-protein interactions, live cells, bacterial biofilms and viruses. Specific examples include mapping of ligand-receptor binding on cell surfaces, studies of protein-protein interactions to provide kinetic information and the potential of AFM to be used as an early diagnostic tool for cancer and other diseases. Many of these reported investigations are from 2011-2014, both from the literature and a few selected studies from the authors’ laboratories