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Biomedical Signal and Image Processing
18.5 NEAR-FIELD SCANNING OPTICAL MICROSCOPY
NSOM circumvents the diffraction-limited imaging of the entire sample caused by
the lens. This is achieved by the scanning of the sample using a subwavelength aperture. The subwavelength aperture is usually a fiber optic that has been stretched and
etched to form a tip whose diameter is in the nanometer range. This fiber optic functions as the point source for scanning with the transmitted or reflected light captured
with a highly sensitive light radiance detector.
The sample is moved with respect to the probe on the x–y plane while maintaining a constant distance with the surface of the sample. The NSOM measures the
attenuation or reflection of each data point to perform either a density measurement
or a reflectivity measurement. The transmission density measurement is similar to
basic x-ray imaging in many ways, while the reflection NSOM resembles ultrasound
density imaging. The NSOM, as described earlier, gives an intensity profile that is
representative of the local tissue density combined with a topographic profile.
In addition to collecting the intensity of the detected light, the scanning stage
also has a feedback mechanism that measures the force between the fiber-optic
probe and the sample. This principle is known as atomic force microscopy, which is
not very useful in biological imaging on its own generally. In the case of the NSOM,
the fiber-optic probe is within nanometers from the surface of the sample, hence the
name near field. Due to the close proximity of the probe to the sample, great care
needs to be taken not to disturb the sample itself. The sample will have a topography
of its own that has greater variations than the separation between the sample and the
probe. The feedback mechanism applies a voltage to a motor to maintain the exact
same distance between the sample and the probe during the entire planar scan by
adjusting the position of the probe until the force has returned to the default value.
The distance is logged for the entire scan by means of the feedback voltage for
height adjustment, and the sample height distribution can be plotted from these data.
The most common imaging mode of NSOM is the transmission mode. In this
mode, the light launched through the probe is transmitted through the sample and
detected by a detector. The detector records the intensity variations over the sample as
it is raster scanned. These are converted into voltage values and a 2-D intensity image
is built up in addition to the topography image from the “atomic force” microscope.
A third and rather novel measurement mode of NSOM has been inspired by a
more general imaging technique called phase-contrast microscopy. In the phase
mode of NSOM, by measuring the phase delay in transmission at each (x, y) coordinates, the optical density can be analyzed with greater detail than based on attenuation only. The optical phase is an indication of the local index of refraction and
can thus reveal details about the chemical composition of the sample. The phase
information is obtained with the use of interferometry.
As any other microscopic imaging discussed so far, the resolution of the NSOM
is restricted by the wavelength used for imaging. Specifically, the resolution of an
NSOM is identified by the size of the point light source used, which is typically in
the order of 50–100 nm. Figure 18.8 shows the NSOM image of a red blood cell.
Standard microscopy has the disadvantage of giving only a view of the contents of a slide averaged over the thickness of the slide. Furthermore, almost all the
Biomedical Signal and Image Processing
18.5 NEAR-FIELD SCANNING OPTICAL MICROSCOPY
NSOM circumvents the diffraction-limited imaging of the entire sample caused by
the lens. This is achieved by the scanning of the sample using a subwavelength aperture. The subwavelength aperture is usually a fiber optic that has been stretched and
etched to form a tip whose diameter is in the nanometer range. This fiber optic functions as the point source for scanning with the transmitted or reflected light captured
with a highly sensitive light radiance detector.
The sample is moved with respect to the probe on the x–y plane while maintaining a constant distance with the surface of the sample. The NSOM measures the
attenuation or reflection of each data point to perform either a density measurement
or a reflectivity measurement. The transmission density measurement is similar to
basic x-ray imaging in many ways, while the reflection NSOM resembles ultrasound
density imaging. The NSOM, as described earlier, gives an intensity profile that is
representative of the local tissue density combined with a topographic profile.
In addition to collecting the intensity of the detected light, the scanning stage
also has a feedback mechanism that measures the force between the fiber-optic
probe and the sample. This principle is known as atomic force microscopy, which is
not very useful in biological imaging on its own generally. In the case of the NSOM,
the fiber-optic probe is within nanometers from the surface of the sample, hence the
name near field. Due to the close proximity of the probe to the sample, great care
needs to be taken not to disturb the sample itself. The sample will have a topography
of its own that has greater variations than the separation between the sample and the
probe. The feedback mechanism applies a voltage to a motor to maintain the exact
same distance between the sample and the probe during the entire planar scan by
adjusting the position of the probe until the force has returned to the default value.
The distance is logged for the entire scan by means of the feedback voltage for
height adjustment, and the sample height distribution can be plotted from these data.
The most common imaging mode of NSOM is the transmission mode. In this
mode, the light launched through the probe is transmitted through the sample and
detected by a detector. The detector records the intensity variations over the sample as
it is raster scanned. These are converted into voltage values and a 2-D intensity image
is built up in addition to the topography image from the “atomic force” microscope.
A third and rather novel measurement mode of NSOM has been inspired by a
more general imaging technique called phase-contrast microscopy. In the phase
mode of NSOM, by measuring the phase delay in transmission at each (x, y) coordinates, the optical density can be analyzed with greater detail than based on attenuation only. The optical phase is an indication of the local index of refraction and
can thus reveal details about the chemical composition of the sample. The phase
information is obtained with the use of interferometry.
As any other microscopic imaging discussed so far, the resolution of the NSOM
is restricted by the wavelength used for imaging. Specifically, the resolution of an
NSOM is identified by the size of the point light source used, which is typically in
the order of 50–100 nm. Figure 18.8 shows the NSOM image of a red blood cell.
Standard microscopy has the disadvantage of giving only a view of the contents of a slide averaged over the thickness of the slide. Furthermore, almost all the