The LCIMB’s Nanoinstrumentation and Force Spectroscopy (NFS) Section develops specialized instrumentations and their applications at macromolecular, cellular and tissue level in areas of biomedical research and medicine. NFS scientists collaborate closely with other intramural and extramural investigators to provide innovative approaches through biophysical modeling, mathematical analysis, and custom instrumentation primarily for nanoscale characterizations. Our current focus includes the development and applications of high-resolution and high-speed atomic force microscopy (AFM) for force spectroscopy and nanometric bioimaging of biological and soft materials. We also develop related technologies such as laser and optical technologies for spectroscopic analysis of biochemical reaction kinetics, multimodal instrumentations, and broader biomedical characterizations.
NFS Section shares a core biological atomic force microscopy (AFM) facility at NIBIB and extends the functionality of several commercial and developing AFM platforms toward higher speed, higher resolution, and multimodal measurements to yield better insights on biomolecules, their complexes, multifunctional theranostics, cells, and tissues. One example is an integration of optical spectroscopes with an AFM platform for tip-enhanced Raman scattering (TERS) and other multimodal characterizations. In another example, we have started an international collaboration dedicated to development and biomedical applications of a next-generation high-speed AFM. Over the years, our staff has had extensive experience improving such instrumentations to investigate a broad range of biomedical samples and systems.
NFS Section continues to develop our biophysical measurement systems based on AFM imaging and single molecule force spectroscopy (SMFS), quartz crystal microbalance-dissipation (QCM-D), and other technologies, and to apply these technologies to a number of biomedical and biological investigations in collaboration with NIH intramural and extramural researchers. Major collaborations with notable results include: (A) Macromolecular structure and nanomechanical properties of multiple malaria vaccine candidates in collaboration with Dr. David Narum (Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH) and other co-investigators. These protein antigen constructs are being produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up productions. We have focused on using AFM and QCM-D to understand the structural properties of these developing vaccines toward enhanced immunological response and eventual eradication of malaria. (B) Multifunctional nanomedicine probes with Dr. Shawn Chen (LOMIN, NIBIB), Dr. Ashwin Bhirde (LOMIN, NIBIB), Dr. Peng Huang (LOMIN, NIBIB), and many co-investigators. New results on several multi-functional nano-drug, photothermal therapy (PPT), siRNA delivery and imaging systems have been published and developed further toward cellular and medical applications. (C) Protein domain structure and interactions clathrin and clathrin assemblies in receptor-mediated endocytosis and intracellular trafficking in collaboration with Dr. Ralph Nossal (NICHD, NIH), Prof. Eileen Lafer (Univ.TexasHealthSciencesCenter, San Antonio), and coworkers. (D) A number of other intramural and extramural collaborations involving Bio-AFM, QCM-D, and related nanotechnologies for macromolecular and cellular studies.
Bacteriorhodopsin (BR) is a bacterial, membrane-bound proton pump that is a prototype for mammalian cytochrome oxidase of respiratory chains. It is a well characterized protein with spectral signatures in both the visible and infra-red (IR) regions of the electromagnetic spectrum. Previously in collaboration with NHLBI/NIH and NIST investigators, we produced a multi-channel visible spectrum analyzer that had a time resolution of 5 microseconds, which enabled characterization of changes to the spectral signature that occurred following initiation of the BR photocycle by an actinic laser photolyzing pulse. Using this new instrumentation and a linear algebraic approach following a singular value decomposition of a matrix of the raw data, we have isolated the absolute visible and infrared spectra of all of the photocycle intermediates. Recently, our team has developed a new system to perform the same analyses and compare the behavior BR in its native purple membrane environment (PM) with that in isolated crystals. This new mutli-channel analyzer, which offers sub-microsecond time resolution, incorporates a fiber optically coupled spectrograph comprising a frame-shift ccd camera, and a time-gated image intensifier, with an IR/visible microscope that has its own IR detector. The sample, whether PM or a crystal is contained in a defined 50 micron by 50 micron space. The ultimate goal is two-fold. First, to validate the biological relevance of BR crystals; and secondly to use the same linear algebra deconvolution approach to isolate the X-ray diffraction maps and real structures of the photocycle intermediates. This should help in understanding how protein conformational changes during the pumping of protons across the membrane form the electrochemical potential that drives ATP synthesis. In a parallel study, we are combining optical spectroscopy with AFM imaging to better understand conformational changes and polymerization of amyloid beta protein in Alzheimer's disease (AD).
Biological AFM capabilities
Combining high-resolution AFM under physiological conditions with sensitive force measurements and mathematical modeling to gain greater understanding of complex biological systems.
Imaging and force measurement at atomic resolution
0.1 nm special resolution under physiological conditions, dynamic changes, and temperature studies; 5 pN force and force modulation resolution.
- Multimode PicoForce AFM
- TIRF AFM (Bioscope Catalyst)
- Raman (LabRam) AFM (XE-120)
- Single Molecule Force Spectroscopy (SMFS) AFM (ForceRobot)
- Open-source platforms for nanotechnologies (AFM Workshop & developing High-speed AFM)
- Multi-modality (Raman, TIRF, NSOM, etc.)
- Environmental Control
- AFM Tip Functionalization
- Surface Modifications Staff