Near Field Scanning Optical

Microscope (NSOM)
 NSOM includes the
following basic elements

• the probe
• scanning system to
move a probe
towards the
sample surface
• detecting system
• optical excitation
system

Figure ____. A Near-Field Microscope and its parts (Lifted from https://www.olympus-
lifescience.com/en/microscope-resource/primer/techniques/nearfield/nearfieldintro/).
Figure ____. Actual image of NSOM (Lifted from http://eiselegroup.com/nsom-picture/).
Near Field Scanning Optical Microscope Principle
Conventional microscopes utilize wavelengths of light which
restricts to 200 nm only. In contrast, NSOM is specialized to
attain higher resolution (around 50 nm) by overcoming
diffraction limit of conventional optical microscopy.

NSOM allows light to pass through a sub-wavelength diameter

aperture and illuminates a sample that is placed within its
near field. Within this proximity, sub-wavelength details can
be observed.

Figure 1. Diagram of the basic principle of NSOM ( Fu, 2010).

 General Use
NSOM is used to:
• provide higher resolution (50 nm)
than conventional microscopes
• Provide simultaneous
measurements of topography
and optical properties
• Study material surfaces like
nanoparticles, polymer blends,
porous silicon, and biological
systems

Figure 2. NSOM imaging of β2-microglobulin amyloid fibril. Topographic

flourescence imaging (top) and 3D overlay of optical signals (Uversky &
Lyubchencko, 2013).
A Study employing NSOM
Migration mechanism of mesenchymal stem cells studied
by QD/NSOM
Ke, C., Chen, J., Guo, Y., Chen, Z.W., Cai, J., 2015. Biochimica
et Biophysica Acta (BBA)-Biomembranes, 1848(3), pp.859-
868.
Utilized NSOM combined with fluorescent quantum dot (QD)-
based nano-technology to capture the functional relationship
between CD44 receptor and CD29 receptor adhesion molecules
on mesenchymal stem cells and the effect of their spatial
rearrangements.

Ke et al. (2015) developed a NSOM/QDs-based nanotechnology

using dual-color imaging to visualize the nanometer scale
distribution and organization of CD44 and CD29, as well as their
spatial organization on the cell surface.
Fig 3. In situ NSOM/QD-based dual color fluorescence imaging in MSCs induced
by VEGF for 30 min. a, Control MSCs; b and c, NSOM images of MSCs after stimulation
by VEGF for 10 min and 30 min respectively. (A) Topographic images of the cell surface.
(B) Near-field fluorescence images of CD44 labeled using QD565. (C) Near-field
fluorescence images of CD44 labeled using QD655.
Figure 3. (D) Superimposition of (B) and (C), yellow indicates colocalization between CD44
andCD29. (E) The marked area in (D) is magnified by B-spline interpolation. Few CD44
molecules colocalize with CD29 in the merged image (a). As VEGF stimulation time
increased, both CD44 and CD29 formed nanodomains or even microdomains (> 500 nm)
and colocalization increased between the two molecules (c). d, Histograms of the
frequency distribution of all the CD44 and CD29 fluorescent spots, blue: control group;
green: 10 min VEGF stimulation; and red: 30 min VEGF stimulation.
Results of using NSOM technique

On one hand, Laser Scanning Confocal microscopy (LSCM)

showed the expression levels of CD44 and CD29 aggregates on
the cell membranes. However the technique was not able to
produce nanometer-scale information about how these
molecules are distributed. In addition, no obvious changes
detected by LSCM during a short simulation.

On the other hand NSOM/QD-based nanoscale imaging system

directly visualized the changes of molecular distribution and
organization on the membrane of MSCs
Migration mechanism of mesenchymal stem cells
studied by QD/NSOM (Ke et al., 2015)

Highlights of NSOM techniques in the study:

• NSOM can provide simultaneous optical and topographic
lateral resolution beyond the diffraction limit of light

• QD/NSOM can be used to elucidate the molecular

mechanism behind VEGF-induced migration of MSCs
References:

Fu, Y., 2010. Subwavelength Optics: Theory and Technology.

Bentham Science Publishers.

Uversky, V., Lyubchenko, Y. eds., 2013. Bio-nanoimaging: Protein

Misfolding and Aggregation. Academic Press.