Advances in Microscopy
and Their Applications in
Biomedical Research
Microscopy
We perceive the physical world around us using our eyes, but only down to
a certain limit.
Objects with a diameter smaller than 75 µm cannot be recognized by the
naked eye,
and due to this reason, they remained undiscovered for the most of human
history.
Entities which belong to this category include cells (diameter of 10 µm),
bacteria (1 µm), viruses (100 nm), molecules (2 nm), and atoms (0.3 nm).
In fact, the importance of these micro/nano entities in almost every aspect
of our life cannot be sufficiently appreciated.
Microscopes are the tools which enable us to extend our vision to the micro
world and, despite the prefix micro- in the name, to the nano-world, too.
Microscopy as an integral part of biomedical research
The ability to view and analyze specimens under high
resolution has always been crucial for biomedical sciences.
Various imaging modalities are used for viewing and
analysis of biologic specimen, which includes imaging in
both clinical and research settings.
Clinical modalities consist of ultrasound, X-ray, computed
tomography (CT), magnetic resonance imaging (MRI),
endoscopy, and optical coherence tomography (OCT),
among others,
while electron microscopy, mass spectrometry imaging, and
various optical microscopy techniques (e.g., confocal, total
internal reflection, multiphoton, superresolution
fluorescence microscopy) are included in research
modalities.
Microscopy as an integral part of
biomedical research
A combination of both clinical imaging and research
microscopy is required for interpretation of clinical datasets.
Combination of engineering sciences, chemical
engineering, and computational studies together continue
to contribute toward the advancement of microscopy in
biomedical research.
Since the invention of the first microscope in the 16th
century, various types of microscopes have been
developed, which can be broadly categorized
according to the illumination source used for imaging,
namely electrons and photons. The microscopy
techniques that employ photons as their illumination
source are grouped under the category of photon-
based imaging,
Photon-based imaging
Photon-based microscopy includes techniques that involve
. photons as their illumination source, and these microscopes are
known as optical microscopes or light microscopes.
Photon-based microscopic imaging modalities used in biomedical
research mainly include :
bright field microscope
dark field microscope
phase contrast microscope
fluorescence microscope
near-field scanning optical microscopy (NSOM)
These modalities have the ability to provide an image of a
given sample of tissue at cellular level.
They also provide adjustable magnification and depth of
field that ensures a well-focused image.
bright field microscopy
bright field microscopy:
the source of light is situated beneath the specimen
which illuminates the specimen placed above,
so the image depends upon the light being transmitted by the
specimen. The specimen to be observed absorbs part of the
light and transmits the remaining through the ocular lens as a
result of which the specimen appears darker than the
surrounding field.
The area around the specimen is brightly illuminated, thus this
technique's name.
This type of microscopy is generally used to study the
morphology of cells, but it requires staining to increase the
contrast of the image.
Microscopy as an integral part of
biomedical research
Stains can be generally divided into two types:
basic dyes having positive charge and acidic dyes that are
negatively charged.
Cell components exhibiting negative charge, like nucleic acids
bind to basic dyes.
Positively charged components, in turn, are attracted to acidic
dyes.
The positively charged stains (i.e., basic dye) attach promptly to
the specimen and are used more often than the acidic dyes as
negative stains are readily repelled by the negative charge on
the surface of the cells.
Acidic dyes are often employed to stain surrounding field to
increase contrast. Some of the widely used positive stains are
methylene blue and Rose bengal.
Microscopy as an integral part of
biomedical research
These stain the whole cell, which can be visualized against a
bright unstained background.
Differential stains involve two stains: namely primary stain and
counter stain. The most widely used differential staining was
developed by Hans Christian Gram, known as Gram staining.
It utilizes two stains, crystal violet and safranin. The difference
in staining occurs due to the difference in the components of
the cell walls of gram-positive and gram-negative
In an attempt to increase the contrast and resolution of the
standard bright field microscope, several techniques have
been developed with slight differences (e.g., dark field
microscopy, phase contrast microscopy, and differential
interference contrast
Dark field microscopy
Dark field microscopy has only slight changes to bright
field microscopy.
In this type of microscope, a central stop is attached just
above the condenser lens, which allows only a limited
amount of light to reach the objective lens.
Therefore only the light scattered through the edges of
the specimen is used to view the image.
In this way the surrounding field is obtained dark and the
specimen appears bright. This technique ensures better
contrast than the standard bright field microscopy. Dark
field microscopes are generally used to study
morphology and motility of cells.
Phase contrast microscopy
light absorption is responsible for image formation.
Objects which absorb light are called amplitude objects
since they affect light amplitude.
Transparent objects, on the other hand, hardly alter the
amplitude of light.
They, however, delay light wave introducing a phase shift,
and thus, they are given the name phase objects.
Typical light detectors such as retina in our eyes can
recognize amplitude variations but they are insensitive to
phase distortion. In the 1930s, the Dutch physicist Frits
Zernike came up with a brilliant trick for converting the
invisible phase shift to a visible amplitude change using
an optical filter. His contribution is the basis for a long-
established technique in laboratories today known as
phase contrast
Phase contrast microscopy
Phase contrast microscopy is another attempt to increase the
contrast of the transparent samples.
This technique is based on the concept that all the transparent
cellular components have different densities. Since light interacts
differently with varying amounts of densities, a contrast is observed
between the cellular components and the outside surrounding area.
To focus the light wave on single focal plane, Kohler illumination is
put into use. The light is focused onto the specimen as hollow cone. A
diffraction plate is situated at the back focal point of the objective
lens. The diffraction plate alters the phase of the ray of light that
comes through the specimen.
This results in varying degrees of retardation that causes either
darkening or lightening of the sample to be viewed.
Differential interference contrast microscopy
Differential interference contrast microscopy gives three-
dimensional (3D), colored, and highly contrasting images.
In this technique the light ray that will illuminate the specimen is
divided into two different beams of light:
The first beam reaches the specimen and passes through it,
while the other beam does not.
This results in a phase difference between the two beams of light.
Both the beams of light are then combined to cause interference.
With such images, we can detect minor elevations or variations in
depth in the specimen that gives the perception of 3D image.
The abovementioned types of photon-based microscopes have
their fair share of contribution majorly involving analysis of
morphology of cells.
Two optical microscopes have stood out and made
exceptional contributions to the field of biomedical
sciences: fluorescence microscope and confocal
microscope.
Fluorescence microscopy: Working, advancements,
and biomedical applications
Fluorescence microscopy has become a major tool to analyze cell
physiology.
The phenomenon of absorption and subsequent reemission of
photons, formally called fluorescence, has had a major impact on
biomedical research as it not only enables visualization with good
spatial resolution but also allows researchers to track single
molecules in vivo and replace microscopy to age-old radioactive
assays.
Some special materials, when illuminated with light having a
specific wavelength, emit light with another wavelength. an
excitation filter is required to select a part of the
electromagnetic spectrum for exciting the fluorescent materials
in specimen. An other filter is then utilized to separate the
emitted light from that used in the excitation process.
These microscopes offer better contrast, resolution, and variety in
design when compared with the standard light microscopes.
Fluorescence microscopy
The excitation light from a suitable light source is made
to focus on the specimen via the objective lens.
The emitted fluorescence coming from the specimen is
directed toward the detector.
Fluorescence microscopy involves the use of various
fluorescent dyes such as fluorescein or acridine orange.
These dyes are used to stain the specimen, which has to
be observed under fluorescence microscope.
The underlying principle involved in using such dyes is
Stokes shift.
Fluorescence microscopy/Stokes
shift
When photon of a specific wavelength hits the fluorescent molecule, an
electron is transferred from its ground state to excited state.
Electron cannot stay in the excitation band for long as it is in a very
unstable state, so it travels back to its ground state in just a matter of
some nanoseconds.
While traveling back, a photon with lesser energy than the incident
photon is released, which is detected as fluorescence.
Therefore the wavelength of light emitted from the fluorescent dye is
always higher than the wavelength of light incident on the stain.
Since a major part of the excitation light is transmitted through the
specimen, the main aim of fluorescence microscopy is to separate the
excitation light from the emitted light.
Therefore indicators or fluorescence dyes that exhibit large Stokes shifts
are preferred.
Stokes shift
In fluorescence spectroscopy, the Stokes shift is the difference
between the spectral position of the maximum of the first absorption
band and the maximum of the fluorescence emission and can be
expressed in either wavelength or wavenumber units
Fluorescence microscopy/Stokes shift
The Stokes Shift is named after Irish physicist George Gabriel Stokes
and is the spectral shift to lower energy between the incident light
and the scattered or emitted light after interaction with a sample.
This separation of the bright excitation light from the comparatively
dimmer emitted light is done by using optical filters.
The fundamental key to obtaining a good image lies with choosing
the right filter.
Therefore the choice of filter is a compromise between emitted light
(passing through) and excitation light (blocking rays).
Configuration of a fluorescence
microscope.
Advancements and biomedical applications of fluorescence
microscopy
New advancements have been introduced to fluorescence microscopy,
which include employing
photobleaching-based techniques like fluorescence recovery after
photobleaching (FRAP), fluorescence localization after photobleaching
(FLAP), fluorescence resonance energy transfer (FRET), and the related
fluorescence loss in photobleaching (FLIP).
shortcomings of fluorescence
imaging
There are at least two shortcomings of fluorescence imaging:
Firstly, staining may cause some undesired effects on the sample
under study. For instance, it was shown that the dyes used in cell
viability detection affect cell stiffness.
Secondly, what we see under fluorescence microscopy is the activity
of fluorescent dyes which, in general, does not reveal structural
information.
Moreover, these fluorescent dyes do not always cover the entire
imaged object. These two factors lead to incomplete shape
information.
Confocal microscopy: Working, advancements, and biomedical
applications
Confocal microscopy provides high-resolution images of thin sections
of the specimen both ex vivo and in vivo. This technique allows the
user to obtain sectioned images of normal tissue and lesions for their
structural and morphologic analysis.
These structural features are then used to differentiate between
normal and abnormal cells.
In the case of point illumination, a single point on the focal plane is
illuminated. This is achieved by keeping the focal point of both the
objective and condenser lenses the same, hence the name confocal.
Confocal microscopy
This arrangement of lenses ensures a single point focus and imaging
A pinhole is located at the focus point right above the detector, which
allows only the in-focus rays to reach the detector.
The light rays are focused on a small area of the specimen, and the
condenser lens (having the same focal length as the objective lens) is
focused on the same area.
Thus the imaging is done only through the light reflected by the focal
plane as adjusted by the user. Such arrangement of the lenses and
subsequent focusing offers better resolution than the conventional
light microscope
Working of a confocal microscope.
Objective
lens
Confocal microscopy
A pinhole is located at the focus point right above the detector,
which allows only the in-focus rays to reach the detector.
The light rays are focused on a small area of the specimen,
and the condenser lens (having the same focal length as the
objective lens) is focused on the same area.
Thus the imaging is done only through the light reflected by
the focal plane as adjusted by the user.
Such arrangement of the lenses and subsequent focusing
offers better resolution than the conventional light microscope
This kind of optical sectioning facilitates imaging the interiors
of both the translucent and opaque biologic specimens.
confocal microscopy
The whole image construction is done by scanning the
specimen.
Three types of scanning designs have been set up to
date:
stage scanning,
beam scanning,
and tandem scanning
confocal microscopy/Light sources
Light sources employed for confocal microscopy can be
both monochromatic and polychromatic.
Polychromatic: Tungsten, xenon, mercury, carbon, quartz-
halogen, and zirconium lamps that emit white light, which
includes light rays of many wavelengths, are known as
polychromatic light sources
These light sources are easily available, cheap, and offer
wavelength flexibility when used with various wavelength
filters.
These are widely used for tandem scanning confocal
microscopy.
Lasers exhibit some unique properties, which makes
them a better alternative over polychromatic light
sources:
such as high coherence,
and low degree of divergence which allows the user to
focus the beam at a point,
high brightness allows for better and clearer image,
and high degree of monochromaticity enables the work
to be done at a specific wavelength
Advancements and biomedical applications of confocal
microscopy
Confocal microscopes majorly employ software for image
acquisition and further image processing.
Images can be processed both during image acquisition
and post image acquisition
To date, several image processing methodologies are
available all of which generally include three procedures:
image manipulation,
display,
and analysis
confocal microscopy
Image manipulations involve contrast enhancement,
smoothing, image alignment. But the most widely used
image manipulation is the 3D reconstruction.
Image analysis can be carried out using numerous
operations like calculation of distances, angles, volumes,
and surfaces. The collected information can then in turn
be projected in several ways: rotation of object, coloring
of different light intensities, choice of background color,
projection on a specific surface, dye labeling, among
others
confocal microscopy
Various fields of life sciences have benefited from the quality of
images provided by confocal microscopy.
It is mostly considered as a better alternative to biopsy and
subsequent conventional microscopy analysis.
For instance, detection of melanoma involves histologic examination
of lesions,
but the whole procedure requires biopsies and can cause unnecessary
sampling errors or distortion of tissue.
The procedure is time consuming, and dynamic observation with time
cannot be performed.
Confocal microscopy, on the other hand, offers noninvasive slice-by-
slice sectioning of tissue and does not require slide preparation as in
the case with normal bright field microscopic observations.
Confocal microscopy has been widely applied for investigative analysis of
skin as the depth range of the instrument is around 200 to 300 μm, which
allows for viewing the different layers of the skin.
homogeneous layers like exhibit low contrast and thus look darker,
meanwhile heterogeneous layers like viable epidermis exhibit higher contrast
and look brighter.
An accuracy of 89.7% of confocal microscopy has been reported for
detecting cancerous growth in colon, esophagus, and stomach
While confocal microscopy has found its usage majorly for immediate biopsy,
the real advantage that it holds is its property to noninvasively image a
specimen tissue, especially that of gastrointestinal tract, eye, and the skin.
in a case where premalignant and benign lesions in the oral cavity could not
be clearly distinguished, a laser confocal endomicroscopy was used in vivo,
which revealed the difference.
Confocal microscopy has also been useful for studying drug delivery
by combining it with fluorescence techniques.
For instance, a pH-sensitive fluorescence dye was used to visualize
and measure the surrounding pH during the time of degradation of
poly(lactic-co-glycolic acid) (PLGA) microspheres, which is primarily a
controlled drug release system.
Characterization of pharmaceutical formulations like microspheres,
pellets, colloidal systems, hydrophillic matrices and film coatings
Electron-based imaging: electron microscopy (EM)
Electron microscopy delivers detailed high-resolution
images of tissues, cells, cell components, and even
macromolecules.
The source of illuminating radiation in this type of
microscopy is a beam of electrons, which exhibit a very
short wavelength.
The high resolving power of the microscope is because of
the shorter wavelength and focusability of electron
beam.
EM offers a magnification up to 1,000,000X, which allows
for detailed observation of very fine structures of cellular
components.
Comparison between optical microscope and electron
microscope.
In an electron microscope, an electron beam is applied onto the
specimen placed in a vacuum chamber.
This electron beam is focused by using a series of electromagnets.
Images obtained with EM are always in shades of gray,
1. Scanning electron microscope (SEM)
2. Transmission electron microscope (TEM)
Scanning electron microscope: Working and biomedical
applications
SEM is used to scan the surface of the specimen under
observation and produces a 3D image of the surface
characteristics of the cells.
Before viewing, the specimen has to undergo a relatively
long sample preparation step, which involves the following:
Fixation of the specimen using aldehyde solution
Dehydration of specimen (as it has to be kept in vacuum)
c. Mounting the sample onto a metal stub
d. Coating of the specimen with electrically conductive
heavy metal (gold, palladium, or carbon)
Once the sample is prepared, they are kept into a vacuum chamber,
and an electron beam is bombarded on it.
The electron beam is given off by an electron gun consisting of a
tungsten filament that is heated to around 2700 K.
The electron beam is focused onto the specimen with the help of
condenser lenses that are essentially electromagnets.
Voltage used for accelerating electrons ranges between 60 and 100
kV.
After being hit with the electron beam, the specimen gives off
secondary electrons, Auger electrons, backscattered electrons, X-rays,
and photons of various energies.
The backscattered electrons and the secondary electrons are used as
signals to produce a 3D image of the surface topography of the
specimen.
electrons reach the detector, they strike on a scintillator (a
luminescence material that fluoresces when struck by electrons or
any high energy particle).
This emits rays of light, which get converted into an electric current
by a photomultiplier, sending a signal to the cathode ray tube.
This produces an image in the similar way a television photograph is
generated.
SEM has been resourceful to a wide variety of biomedical research.
One of the initial biologic entities to be viewed under SEM were red
blood cells.
They compared the smooth normal cells with pathologic samples,
which could be identified by their granular appearance by using SEM
The morphology and structural properties of dentine have also been
revealed by SEM
There have been various studies of the skin as well.
The ultrastructure of surfaces of endothelium and epithelium of
cornea have been studied,
SEM has also contributed to characterization of micro- and
nanostructures, which are slowly becoming major players in health
and diagnostic industry.
By conjugating proteins, antibodies, or different small biomolecules to
nanoparticles, scientists have been able to develop novel drug delivery
approaches as well as diagnostic techniques for various diseases
It has been used to view the microstructures of various bacterial
species
Exosomes obtained from different types of human cells have also been
characterized using SEM.
SEM are now also available as new generation benchtop versions,
which can be used for cell biology and pathology settings
Focused ion beam SEM (FIB-SEM) has been quite instrumental in
providing good quality images to study the 3D architecture of cells.
Recently this technique was combined with ice embedding
methodology to ensure that the biologic specimens remain as near to
their native state as possible
SEM has proved to be an immensely versatile instrument available for
the study and examination of morphology of micro- and
nanostructures, and it continues to contribute to the field of
biomedical sciences.
SEM image of polycaprolactone nanoparticles.
Transmission electron microscope: Working and biomedical
applications
TEM involves interaction of electron beam with not the surface of the
specimen (as in the case of SEM) but it allows for transmission of the
electrons through the sample
Preparing the sample for TEM is more extensive than
SEM and involves the following steps:
a. Sample fixation using formaldehyde or glutaraldehyde
b. Dehydration of the sample using ethanol
c. Embedding, where ethanol is replaced by an
embedding agent, which is a highly miscible plastic
d. Curing at a high temperature (70°C) ensures that the
embedding agent polymerizes and becomes solid
e. Thin sections (~90 nm) are then made using glass or
diamond knife
Among all the microscopes used for biomedical purposes,
TEM is the most powerful with a resolution limit of 0.5
nm.
TEM has contributed and has had a huge impact on
research in biomedical sciences.
The profound increase in resolution power has allowed
viewing of viruses as well.
Different families of viruses exhibit morphologic
variations, which have facilitated the identification of
viruses.
TEM has also facilitated discovery of several viruses,
notably the viruses linked with gastroenteritis.
It is already being employed to examine the new age
viruses like SARS and human monkeypox virus.
TEM has also been immensely resourceful in
characterization studies of nanoparticles and
nanoparticle systems.
High-resolution TEM imaging is now being combined with
atomic resolution electron energy loss spectroscopy and
nanodiffraction.
Together these instruments provide critical information
that is fundamental to the study of nanoscience and
nanotechnology.
Combination of electron energy loss spectroscopy
microanalysis with TEM has been applied to detect calcium
during the mineralization process in the bone tissue
This methodology can be applied for the identification of a
target drug
TEM
TEM has also been used to examine the subcomponents like
proteins, lipids, polysaccharides, and nucleic acids of biofilms
Recently introduced techniques like automated scan
generators, scanning transmission electron microscopy
(STEM), and high-resolution columns have allowed for
resolution at subnanometer level.
STEM has been used for various pathologic diagnosis like
brain, lung, renal tissues.
TEM has also been widely used in the field of
nanobiotechnology for studying the morphology of various
nanoparticles and Nano formulations
With the advancements in biomedical research technologies to
analyze biomolecules at nanoscale, TEM has been quite
instrumental in the whole development process. With the
entry of nanotechnology to biomedical sciences in the form of
drug delivery tools, molecule tracking, diagnostic tools, among
many more,
Resolution for light microscopy and
electron Microscopy
Resolution of the Electron
microscopy