Correlated Fluorescence and Atomic Force Microscopy (AFM)

Life Sciences Atomic Force Microscopy (bio - AFM)

Atomic force microscopy allows direct visualization of the three-dimensional structure of a sample surface with nanoscale resolution. Unlike many other high-resolution imaging techniques, AFM does not require staining or coating of the sample, leaving native surfaces unaltered. Together with the ability to operate in a fluid environment, this makes AFM an unparalleled technique for studying biomolecules and the dynamics of biological processes in situ and in almost real-time.

The BioScope Resolve's seamless integration of atomic force microscopy (AFM) and a recently upgraded inverted fluorescence microscope reveals even more detail and information of biological samples including mechanical properties. The combined optical and AFM instrument design allows simultaneous access to the sample for both modalities on a single instrument setup for correlated high-resolution data collection at high-speed in near physiological conditions.

Example Application 1:

Correlative fluorescence and Atomic Force microscopy -
High resolution imaging and mechanical mapping with nanometer resolution on a mouse bone/collagen tissue section

AFM probe is guided to a relevant tissue section for correlative high resolution imaging and mechanical mapping with nanometer resolution. Left image: optical brightfield image with AFM cantilever visible as bright area in the top part. Right image: fluorescence image is revealing FITC dye labeled cells within the tissue section. Tissue: Mouse bone/collagen sections.

The resulting AFM images and the nano-mechanical analysis from the selected area in the fluorescence microscope are shown in the AFM images above. Left image: topography images of the selected area shows individual cells embedded in the tissue. Right image: Simultaneous Young’s modulus mapping identifies highly calcified cells with a higher stiffness/modulus within the tissue. (Tissue from Joan Richtsmeier group, Anthropology)

Example application #2:

FastTapping mode for fast scanning of biological samples in correlative microscopy mode -
Fast Tapping of live BHK-21 cells (hamster kidney fibroblasts) for analysis of induced structural and cytoskeletal modifications in mammalian host cells.

The optical phase contrast image allows observation and selection of relevant cells and targeted simultaneous AFM imaging. Live adhered BHK-21 cells (hamster kidney fibroblasts) at 37°C in media and an AFM probe (in the top part of image) are visible. (Cells from Joyce Jose group, BMB)

The integration of the optical microscope with the high-speed, fast scanning AFM mode allows a directed approach to specific areas of a cell. The membrane structures of a part from a live BHK cell is visible in the topographical AFM video. The high temporal resolution shows the dynamic reorganization of the cytoskeleton of the cell under induced mechanical stress. Detailed analysis (e.g. membrane retraction profiling) can be performed in almost realtime and can be correlated to optical/fluorescence images.

Technique Advantages:

  • High-resolution multimodal AFM imaging for all scan ranges from cells and tissues to single molecules
  • Lateral AFM scan range up to 100µm, vertical range (height) up to 15 µm. Vertical resolution typical < 0.5 nm,
  • Comprehensive Package for live cell and tissue imaging and nano-mechanical mapping
  • Small volume capabilities (~60µl) with sample perfusion for molecular biology and single molecule applications
  • Fast scanning options for live cell imaging and single molecular scanning (up to 98Hz) in liquid
  • Fully integrated inverted fluorescence microscope with live cell imaging capabilities
    • Gas (e.g. 5% CO5 balanced air) and liquid perfusion available (ml to µl volume)
    • Software controlled heating stage from ambient temperatures to ~60°C
  • Semi-automated fluorescence image acquisition and device control and a wide range of image analysis options and correlation with AFM images
  • Wide range of suitable for sample and substrates: Glass coverslips up to 75x25mm, plastic and glass bottom petri dishes from 35 - 60mm.
  • Easy sample access and navigation by a long range fully motorized X-Y-Z stage a top view camera for optical brightfield observation of opaque samples
  • Low noise design with a full acoustic isolation enclosure and anti-vibration table

Typical Applications:

  • High resolution imaging of biological samples in buffer solution (tissues, cells, viral particles, hydrogels applications, biomedical devices, and individual biomolecules)
  • High resolution investigation of local mechanical properties (i.e. stiffness, adhesion, friction) of biological samples
  • Time resolved imaging of biological samples up to 98 scan lines per second
  • Studies of nano-scale forces between ligands in the single molecule range (Molecular interaction mapping, Single Molecule Force Spectroscopy (SMFS)
  • Correlated fluorescence and atomic force microscopy (AFM) operation for high-resolution data collection and functional targeting of samples of interest (e.g. tissues, surfaces, cells)