True/Functional Techniques and Technology
Essentially, super-resolution microscopy (SRM)
refers to any optical technique that is used for the purposes of resolving
structures beyond the diffraction-limited resolution of conventional light
The techniques of super-resolution microscopy allows for images to
be viewed under higher resolution for fine mapping of such cellular structures
as neural synapses and Golgi apparatus among others.
Super-resolution can be achieved through a number of methods including:
- 4Pi microscopy
- structured illumination microscopy
- stimulated emission depletion
- reversible saturable optical fluorescence transition
A Brief on Diffraction Limit
Although a typical microscope allows for the
visualization of the specimen by magnifying the image, ultimately it fails to
see infinitely small details. This is largely due to the fact that such light
microscopes are heavily limited by the fact that light is a wave that is subject
In a light microscope, the rays of light from
the object converge onto a single point at the image plane during the imaging
process. However, as a result of diffraction, the exact convergence of these
rays is prevented which causes a sharp point on the object to blur in to a
finite-sized spot on the image.
Whereas diffraction limit of resolution may not
affect imaging at the organ or tissue level in light microscopy, it becomes a
problem when zooming in to cells where a large number of the sub-cellular
structures tend to be smaller than the wavelength. Here, it becomes difficult
to use a light microscope to study cell structures in detail.
techniques overcome the challenges of diffraction limit making it possible to
view and study the molecular structure of cells.
is broadly classified into two main categories. These include:
True Super-Resolution Techniques - This
category of super- resolution microscopy captures information within the
Functional Super-Resolution Techniques - This
category of super-resolution microscopy employs experimental techniques for the
reconstruction of the super-resolution image.
True Super-Resolution Techniques
True sub wavelength techniques include the techniques that
Near field scanning optical microscopy (NSOM)
Also referred to as scanning near-field optical
microscopy, this technique uses fiber optic probe to funnel light to the
nanometric dimension. Here, it becomes possible to take fluorescence measurements
with a spatial resolution of tens of nanometers by scanning the probes near the
surface of the sample. This is achieved by focusing the excitation laser
through an aperture with a smaller diameter than the excitation wavelength,
which results in an evanescent field on the other side of the aperture.
this technique, users obtain an order of magnitude improvement far above what
can be achieved using a conventional fluorescent microscope. For instance,
using NSOM, lateral resolution of 20 nm and vertical resolution of between 2
and 5 nm has been attained. Therefore, the technique proves unique and
beneficial in that it helps bridge the gap between spacial resolution between
the optical approaches commonly used and such high resolution techniques as
One of the biggest advantage of ASOM is that it can provide
both optical and spectroscopic data at high spatial resolution in combination
with simultaneous topographic information.
It also has a number of
limitations that include:
- zero working distance and
very small field depth
- very long scan times for
high resolution images/large specimen areas
- very low transmissity of apertures
smaller than the incident light wavelength
4Pi-Microscope is a laser scanning fluorescence
microscope that uses two high resolution objective lenses for illuminating the
sample. Here, the sample is illuminated from both the back and front side.
Using this technique, the problem of blurred images is partially resolved since
both focal light spots are superimposed, which helps remove the blur to a
certain extent. Resolution largely depends on
the effective 4Pi-spot, which is 3 to 5 times sharper than the spot of a
regular confocal microscope. This allows the microscope to be used for studying
various mitochondrial proteins and morphology.
Structured Illumination Microscopy Technologies
This microscopy technique uses a laser-based
wide-field microscopy set-up in which a movable diffraction granting has been
inserted into the excitation beam path. In this set-up, only the Zero order or
first order diffracted laser beams are allowed to pass through the objective.
At the focal plane of the objective, these beams interfere with each other to
create an illumination in stripe. Through superimposition with the sample, the
stripe pattern generates a Moire effect. This technique offers significant
advantages in that it enhances resolution beyond conventional resolution
associated with diffraction, which in turn provides sufficiently high
resolution to get dynamic images of live cells.
Using this technique, users can
capture 85-100 nm distinction, which is very useful in biological sciences.
Some of the benefits of SIM include:
- 2x increase in spacial
resolution compared to wide-field microscopy
- 4D imaging at fast frame
- Using this technique, there
is no need to employ photoactivable or photoswitchable fluorophores.
While it has a number of advantages, it also has
its limitations which include:
- Artifacts may be generated
during image reconstruction.
- It is sensitive to
out-of-focus light. This makes it difficult to use with thick/very densely labeled
For most part, true super-resolution techniques
are used in nano-science largely due to the fact that they provide
super-resolution images directly. A good example of this is with NFOM which captures
information contained in the evanescent wave close to the object.
images are obtained as long as the evanescent field around the object is
directly measured. While these techniques have their strengths,
super-resolution technique requires special equipment, which makes the techniques
complex and hard to operate.
Functional Super-Resolution Microscopy
Functional super-resolution techniques include
microscope techniques that use experimental techniques to reconstruct a
This category is further divided into two groups:
Deterministic Super-Resolution - The techniques used here often use fluorophores, which is the
most common emitter in biological microscopy. This shows a non-linear response
to excitation that is used to enhance resolution
Stochastical Super-Resolution - These techniques are
dependent on the chemical complexities of various molecular light sources that
gives them a complex temporal behaviour. This factor makes several close-by
fluorophores emit light at given separate times and become resolvable in time.
Stimulated Emission Depletion Microscopy
Essentially, this technique is used to create
super-resolution images through the selective deactivation of fluorophores and
the minimization of the area of illumination at the focal point to enhance an
achievable resolution for the system.
Stimulated emission depletion microscopy (STED)
microscopy uses two laser beams to illuminate the specimen. Here, an excitation
laser pulse that is generally created by a multiphoton laser is followed
closely by a doughnut-shaped red-shifted pulse (STED beam).
fluorosphores that are exposed to this beam (STED beam) are instantaneously
returned to the ground state by stimulated emission. Here, non-linear depletion
of the fluorescent state by the STED beam is the basis for super-resolution.
Although the two laser pulses may be diffraction-limited, the STED pulse in
this microscope technique is modified in a manner that allows it to feature
zero-intensity point at the center of focus with strong intensity at the
Once the two laser pulses are superimposed, it is only the molecules
that reside at the center of the STED beam that are able to emit fluorescence.
Through this action, the point spread function in effectively narrowed
ultimately increasing resolution beyond the diffraction limit. In order to
generate a complete image, the center zero is raster-scanned across the
specimen in a similar manner to single-photon confocal microscopy.
In STED, the high the intensity of the STED
beam, the more the molecules will emit a stimulated photon (than the
spontaneous photon). STED microscopy offers 20nm of lateral resolution and 40
to 50 nm axial resolution. This makes it particularly useful in structural analysis,
viewing life cells (when combined with other methods such as fluorescence
One of the main problems associated with STED
microscopy is with regards to photo bleaching, which can occur as a result of
excitation in higher excited states of excitation in the triplet state.
Ground State Depletion Microscopy
For this microscopy technique, fluorescent
markers are typically used depending on given conditions. For instance, the
marker may be freely excited from the ground state and return spontaneously
through emission of the fluorescence photon. On the other hand, if light of
appropriate wavelength is additionally applied, then the dye can be excited to a
long lived state (where fluorescence does not occur).
Being a RESOLFT technique
(REversible Saturable Optical Linear Fluorescence Transitions) ground state
depletion exhibits a time-sequential readout from within the diffraction zone
at given defined coordinates by using reversible saturable/photoswitchable
It also requires lower laser intensities employed in such
techniques as STED given that it employs a metastable triplet state that has a
lifetime range in the milliseconds to microseconds. With GSD microscopy, users
will benefit from an acquisition time of between 2 and 10 minutes with a
maximum resolution of 20 nm.
Saturated Structured Illumination Microscopy
Like SPEM (saturated pattern excitation
microscopy) saturated structured illumination microscopy (SSIM) is a non-linear
method that depletes the fluorophore ground state by saturated excitation
thereby generating a sinusoidal emission pattern to be recorded onto the
area-array CCD detector.
Here, the saturated excitation illumination produces
narrow line-shaped regions on the zero nodes surrounded by high levels of
fluorescence signal and generates a negative imprint of features that are being
Photo-Activated Localization Microscopy
This technique is a form of localization
microscopy that depends on the ability to isolate, detect and image individual
Nanometer scale resolution in this case is achieved by exploiting
properties of photoswitchable fluorophores. Here, exposing the fluorophores to
given wavelength brings about a change in emission spectra. Here,
photoactivable and reversible photoswitchable fluorophores become fluorescent
while the reversible photoswitchable fluorophores change from a fluorescent
state to another.
With this technique, the basic principle is that
low-power activating laser beam stochastically converts fluorophores with only
a few of these being in their active state. The molecules are then imaged using
a high-power illuminating laser beam so as to immediately convert then back to their
This process (activation and inactivation) is repeated over
thousands of frames in order to ensure that all the molecules have been imaged.
With PALM, it is only by exciting the fluorophores stochastically in a
controlled manner that each dot of fluorescence comes from one fluorophore.
With high labeling efficiencies and specimen
with good signal to noise ratio, it is possible to achieve precisions down to
10 nm using this technique.
Fluorescence Photo-Activated Localization Microscopy
Fluorescence Photoactivation Localization
Microscopy (FPALM) brings together a number of existing technologies that produce
images based on fluorescence of individual molecules. For this particular
technique, lasers are used for the purpose of exciting dye molecules on the
surface of the sample. Here, the lasers cause portions of the molecules to
fluoresce giving off a light that creates the image to be captured digitally.
Like in PALM, this process is repeated with new sets of molecules being
excited. The resolution of images using this technique has been shown to be
about 20 times better compared to other traditional light microscopes. As such,
the microscope is capable of creating images with a as low as 10 to 20 nm
Functional super-resolution techniques have
become very important in biological imaging. This is largely due to the fact
that they are relatively easy to realize as is the case with SSRM. Although
they present various advantages, there are also a number of challenges with
these techniques given that some techniques like STED may prove complicated
with relatively slow image acquisition.
There are a number of differences between
super-resolution microscopy and electron microscopy. For instance, whereas
super-resolution microscopy is a form of light microscopy (where light has to
be manipulated to overcome the problems of light diffraction) electron
microscopy is different in that it relies on electrons for imaging. On the
other hand, super-resolution can be used to view and study cellular processes
as they happen. This is to say that it can be used for viewing live cells and
thus various cell structures or other molecules.
When it comes to
electron microscopy, the sample is usually placed in a vacuum, which means that
it is difficult to use electron microscopes to view and study live cells.
Electron microscopy has an advantage over super-resolution microscopy, however, in that it is capable of much greater resolution that has allowed for better
study of various molecular structures and protein molecules (EM spatial
resolution approaches molecular and atomic levels).
has been shown to be relatively easy to use as is the case with methods like
structured illumination microscopy (SIM). Here, sample preparation has been
shown to be more similar to such methods as wide field/confocal microscopy.
This is not the case with electron microscopy, which requires more complicated
Applications and Benefits of Super-Resolution Microscopy
With super-resolution microscopy, scientists and
researchers are no longer limited to the type of resolution that only relied on
fixed tissue. Through such techniques as STED, it has now become possible to
view and study live specimens and reorganization of dynamic protein. This is
due to the fact that the technique can penetrate the tissue sample and allow
for fast image acquisition.
While electron microscopy has a lot to offer in medical research and biology, it proves less beneficial when it comes to live specimen visualization in addition to the fact that electron microscopes are very costly with complicated specimen preparation procedures.
Super-resolution microscopy also allows for detailed close-up images of such microbes as malaria parasites and viruses to be viewed and studied. As such, it overcomes the challenges of the typical microscopes making it a valuable tool in clinical research and biology.
Some of the benefits of
super-resolution microscopy include:
- It can be used for studying
sub-cellular architecture and dynamics at nano scale. This makes it particularly
beneficial when it comes to studying cell structures.
- Super-resolution microscopy
combines intrinsic optic sectioning with fast data acquisition and dual color
super-resolution to provide quality images in a timely fashion for further
- It is capable of capturing
molecules in actions, which can help study and understand their behaviour,
interaction and other characteristics.
- It is possible to carry out
live cell imaging and colorization studies using super-resolution microscopy
- Details that are smaller
than 50 nm can be quickly resolved.
- It can work with standard
- It can minimize drift for
accurate localization of molecules when combined with suppressed motion technology.
Microscopy Imaging Techniques
Brightfield Microscopy - the most elementary microscopy technique but important to understand and apply correctly.
Oil Immersion Microscopy - when used properly increases the refractive index of a sample/specimen. With only a few disadvantages, slides prepared with oil immersion techniques work best under higher magnification where oils increase refraction despite short focal lengths.
The Confocal Microscope - check out how image details can be viewed through state of the art technology and lasers are impossible to view using a conventional microscope.
Phase Contrast Microscope - learn about an entire new world that has opened up in the field of microscopy. Once limited to bright field illumination phase contrast observation is now a standard feature on almost all modern microscopes.
Fluorescence Microscope - study the most used microscope in medical/biological fields which uses high powered light waves to provide unique image viewing options.
Dark Field Microscope - learn more about how when the light source is blocked off, light scatters as it hits the specimen and is then able to reveal details otherwise difficult to see.
Polarizing Microscope - discover its use in a wide range of applications in fields such as geology, metallurgy and medicine. Essential in obtaining information about the color intensity, structure and composition of a sample.
Kohler Illumination - broaden your knowledge of this technique which evenly illuminates the viewing field providing a bright specimen image and eliminates glare.
Differential Interference Contrast - a microscopy techique which benefits from differences in the light refraction by various sections of living cells and transparent specimens allowing for better visibility during microscopic imaging.
And how it all applies to Life Sciences.
Return from Super-Resolution Microscopy to MicroscopeMaster Research Home
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Wenhui Wang, Junnan Gu, Ting He, Yangbin Shen, Shaobo Xi,
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super-resolution microscopy and its applications in nano-catalysis.