# Nanotechnology/Optical Methods#UV.2FVIS spectrometry

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# Optical Microscopy

## The Abbe diffraction limit

Observation of sub-wavelength structures with microscopes is difficult
because of the Abbe diffraction
limit.
Ernst Abbe found in 1873 that light with wavelength λ,travelling in a
medium with refractive index n and converging to a spot with angle φ
will make a spot with radius

$$d=\frac{0.61 \lambda}{n sin \phi}$$

The denominator nsinφ is called the numerical aperture (NA) and can
reach about 1.4 in modern optics, hence the Abbe limit is roughly d=λ/2.
With green light around 500nm the Abbe limit is 250nm which is large
compared to most nanostructures or biological cells with sizes on the
order of 1μm and their internal organelles being much smaller. To
increase the resolution, shorter wavelengths can be used such as UV and
X-ray microscopes. These techniques offer splendid resolution but are
expensive, suffer from lack of contrast in biological samples, and also
tend to damage the sample.

## Resources

-   Microscope
    optics
-   Microscopy
    Primer
-   Olympus Interactive Java Tutorials on
    Microscopy
-   Nikon MicroscopyU with interactive tutorials on microscopy
    techniques

## The optical microscope

!Sketch of an optical
microscope{width="300"}

### Bright Field

The light is sent to the sample in the same directions as you are
looking - most things will look bright unless they absorb the light.

### Dark Field

Light is sent towards the sample at an angle to your viewing direction
and you only see light that is scattered. This makes most images appear
dark and only edges and curved surfaces will light up.

### Polarized Light

### DIC vs H

# Laser Scanning Confocal Microscopy (LSCM)

 Confocal laser scanning
microscopy is a
technique that allows a much better resolution from optical microscopes
and three dimensional imaging. A review can be found in Paddock,
Biotechniques
1999

Using a high NA objective also gives a very shallow depth of focus and
hence the image will be blurred by structures above or below the focus
point in a classical microscope. A way to circumvent this problem is the
confocal microscope, or even better the Laser Scanning Confocal
Microscope (LSCM). Using a laser as the light source gives better
control of the illumintaion, especially when using fluorescent markers
in the sample. The theoretical resolution using a 1.4 NA objective can
reach 140nm laterally and 230nm vertically [^1] while the resolution
quoted in ref [^2] is 0.5×0.5×1μm. The image in the LSCM is made by
scanning the sample in 2D or 3D and recordning the signal for each point
in space on a PC which then generates the image.

# X-ray microscopy

 X-ray microscopy uses X-rays to image
with much shorter wavelength than optical light, and hence can provide
much higher spatial resolution and use different contrast mechanisms.
X-ray microscopy allows the characterization of materials with submicron
resolution approaching the 10\'s of nanometers. X-ray microscopes can
use both laboratory x-ray sources and synchrotron radiation from
electron accelerators. X-ray microscopes using synchrotron radiation
provide the greatest sensitivity and power, but are unfortunately rather
large and expensive. X-ray microscopy is usually divided into two
overlapping ranges, referred to as soft x-ray microscopy (100eV - 2keV)
and hard x-ray microscopy (1keV-40keV). All x-rays penetrate materials,
more for higher energy x-rays. Hence, soft x-ray microscopy provides the
best contrast for small samples. Hard x-rays do have the ability to pass
nearly unhindered through objects like your body, and hence also give
rather poor contrast in many of the biological samples you would like to
observe with the x-ray microscope. Nevertheless, hard x-ray microscopy
allows imaging by phase contrast, or using scanning probe x-ray
microscopy, by using detection of fluorescent or scattered x-rays.
Despite its limitations, X-ray microscopy is a powerful technique and in
some cases can provide characterization of materials or samples that
cannot be done by any other means.

# UV/VIS spectrometry

# Infrared spectrometry (FTIR)

vIdentification of the functional groups present in a nanomaterial is a
frequent requirement in nanoscience and nanotechnology research. Among
other tools, FT-IR has found much popularity among researches due to its
versatility, relative ease of use and ability to use as a quantification
tool.

Atoms in a chemical bonds constantly vibrate. This vibration can be
analogue to a system with two masses attached to a spring. The vibration
frequency depend upon the weight of the masses and the spring constant
of the connecting spring. In the same way, depending on the masses of
the atoms that contributes to a bond and cohesiveness of the bond,
frequency differ. Since bonds have atoms with different shapes and sizes
and different strength, each combination of atoms in an each type of
bond has a unique harmonic frequency. This natural frequency lies in the
range of infrared region and therefore a spectroscopic method that use
IR can be devised to analyze bond vibrations.

When the IR radiation with the same harmonic frequency of the bond
shines upon the bond. The bond vibration is amplified by increased
transfer of energy from the IR radiation. When range of IR frequencies
given to the material, it only absorb IR frequencies that corresponds to
the natural frequencies of the bonds that exist in the sample. Others
are not absorbed and can be analyzed using an Infrared spectrometer,
which tells you the frequencies that are absorbed by the sample. This
provides important information about the functional groups present in
the sample. This is exactly what FT-IR does.

As FT-IR can be used to get information about functional groups present
in nanomaterials. This is particularly useful in cases such as when one
attempts to surface modify nanomaterials to increase affinity,
reactivity or compatibility. Analyzing the FT-IR of a nanomaterial would
tell you what groups present and then appropriate surface modification
strategy be decided based on the groups present. Further, it can also be
useful in characterizing the surface modification has taken place, as
new groups should emerge if the reaction is successful.Identification of
the functional groups present in a nanomaterial is a frequent
requirement in nanoscience and nanotechnology research. Among other
tools, FT-IR has found much popularity among researches due to its
versatility, relative ease of use and ability to use as a quantification
tool.

Atoms in a chemical bonds constantly vibrate. This vibration can be
analogue to a system with two masses attached to a spring. The vibration
frequency depend upon the weight of the masses and the spring constant
of the connecting spring. In the same way, depending on the masses of
the atoms that contributes to a bond and cohesiveness of the bond,
frequency differ. Since bonds have atoms with different shapes and sizes
and different strength, each combination of atoms in an each type of
bond has a unique harmonic frequency. This natural frequency lies in the
range of infrared region and therefore a spectroscopic method that use
IR can be devised to analyze bond vibrations.

When the IR radiation with the same harmonic frequency of the bond
shines upon the bond. The bond vibration is amplified by increased
transfer of energy from the IR radiation. When range of IR frequencies
given to the material, it only absorb IR frequencies that corresponds to
the natural frequencies of the bonds that exist in the sample. Others
are not absorbed and can be analyzed using an Infrared spectrometer,
which tells you the frequencies that are absorbed by the sample. This
provides important information about the functional groups present in
the sample. This is exactly what FT-IR does.

As FT-IR can be used to get information about functional groups present
in nanomaterials. This is particularly useful in cases such as when one
attempts to surface modify nanomaterials to increase affinity,
reactivity or compatibility. Analyzing the FT-IR of a nanomaterial would
tell you what groups present and then appropriate surface modification
strategy be decided based on the groups present. Further, it can also be
useful in characterizing the surface modification has taken place, as
new groups should emerge if the reaction is successful.

# Terahertz Spectroscopy

# Raman Spectroscopy

-    Raman scattering
-    Resonance Raman
    spectroscopy

## Surface Enhanced Raman Spectroscopy (SERS)

-    Surface Enhanced Raman Spectroscopy
    (SERS)
-   Surface-enhanced Raman spectroscopy: a brief
    retrospective

# References

See also notes on editing this book
Nanotechnology/About#How_to_contribute.

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[^1]: Confocal laser scanning microscopy, Paddock SW, Biotechniques ,
    vol. 27 (5): 992 NOV 1999

[^2]: A new UV-visible confocal laser scanning microspectrofluorometer
    designed for spectral cellular imaging, Favard C, Valisa P,
    Egret-Charlier M, Sharonov S, Herben C, Manfait M, Da Silva E, Vigny
    P, Biospectroscopy , vol. 5 (2): 101-115 1999
