Scanning Probe Microscopy Part II

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Scanning Probe MicroscopyPart II

• BMEN 489/689

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AFM in Biological Samples

• AFM can work well in liquid samples, so it is
  well suited to biological studies
• Electrolytes in the media and pH must be
  considered because they can alter the
  probe/material interaction
• Silicon/silicon nitride will degrade in water
  environment
• Probe tips can become negatively charged and skew
  the experimental results

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AFM in Biological Samples

• Even with minimal tracking forces, contact mode
  can damage or distort biological samples.
  Additionally, adsorbed gas at surface pulls the
  probe toward the sample.
• Non-contact mode provides low resolution in
  liquid as the attractive force is weak and can be
  further screened by surface contaminants
• Tapping mode allows measurement of samples that
  are easily damaged or loosely held to surface.
  High frequency makes surface stiff (viscoelastic)
  greatly reducing the tip/sample adhesion forces
  (non-stick)

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AFM in Biological Samples

• AFM imaging of biological samples offers
  significant advantages compared to other
  techniques such as electron microscopy
• Little or no sample preparation
• Imaging occurs under near native conditions
• Media exchange/modification allows imaging of
  specific interactions
• However, AFM does not yield molecular resolution

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AFM in Biol Probes

• Often the AFM probe tips are functionalized to
  preserve them (hydrophobic coating) or allow
  attachment of bio-molecules
• Biotinylated
• PEG with amine or carboxyl
• Oligonucleotides for RNA/DNA
• Cells (grown on beads)

• The cantilevers resonance freq is much lower in
  water
• Viscous damping
• Need to move fluid greater effective mass

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AFM in Biol Applications

• AFM micrographs of DNA on a surface. AFM opened
  the door for imaging DNA in aqueous environments.
  Tapping mode allows such images with minimal
  sample damage

Angel Rivera, Department of Microbiology,
University of Illinois at Urbana-Champaign
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AFM in Biol Applications

• AFM image of aggregated Protein (ß2M) Fibril
  implicated which has been implicated in diseases
  like Alzheimer's and Parkinson's

Note periodicity of spiral pattern (left-handed)
Scale bar is 200nm
http//www.astbury.leeds.ac.uk/Facil/SPM/SPM.htm
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AFM in Biol Applications

• Researchers also take advantage AFMs ability to
  apply/control small forces (pN scale)

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Near-field Scanning Optical Microscopy (NSOM or
SNOM)

• Using near-field light presents the opportunity
  to perform spectroscopy below the diffraction
  limit (resmin 0.61 ?)
• Near-field refers to the fact that the light is
  put through a subwavelength aperture
• The emerging light contains a large fraction of
  non-propagating, evanescent field
• the decays exponentially away
• from the aperture
• ( aperture/p)

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NSOM Probes

• In reality, optical fibers are used as the probe
• These are normally designed to propagate light
  and have core diameters of gt5µm
• The fiber is heated and pulled down until it
  breaks (50nm in diameter)
• The taper is coated with metal to prevent light
  loss

www.monos.leidenuniv.nl/smo/index.html?basics/micr
oscopy.htm
physics.nist.gov/Divisions/Div844/facilities/nsom/
nsom.html
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NSOM Probes

• The fiber is then attached to a tuning fork
• The system has a resonant frequency and
  interactions between the tip and the surface are
  sensed through atomic interactions
• Distance from surface 5-50nm

www.olympusmicro.com/primer/techniques/nearfield/n
earfieldintro.html
www.azonano.com
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NSOM Detection

• There are four standard modes of operation
• Transmission incident light through NSOM
  detected through sample with standard optics
• Reflection incident light through NSOM detect
  light from sample surface
• Collection illuminate sample with standard
  optics and collect with NSOM
• Illumination/collection NSOM used for
  illumination and collection

www.nanonics.co.il
www.olympusmicro.com/primer/techniques/nearfield
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NSOM Images

• Here is an image of aluminum bumps on a slide.
  Note the topographic image and the NSOM image are
  correlated

Topographic Image
NSOM Image
electron.mit.edu/gsteele/mirrors/elchem.kaist.ac.
kr/jhkwak/TopometrixWeb/lumstd.htm
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NSOM Images

• Images of muscle tissue showing the ability to
  image tissue in culture

Sid Ragona and Phil Haydon, Laboratory of
Cellular Signaling Dept. of Zoology and Genetics,
Iowa State University, Ames, IA 50011
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NSOM Images

• Epi-illumination and near-field image of cell
  loaded with Fluo-3 (Ca2 indicator). Note the
  single molecule imaging in NSOM

Sid Ragona and Phil Haydon, Laboratory of
Cellular Signaling Dept. of Zoology and Genetics,
Iowa State University, Ames, IA 50011
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NSOM Images

• Can also combine the technique with SERS to see
  individual R6-G molecules