CONFOCAL
MICROSCOPE
·
A Confocal microscope is a type of microscope
that uses laser light to produce high-resolution images of samples at different
depths within the sample.
·
The basic principle of Confocal Microscopy is
that the Illumination and Detection optics are focused on the same Diffraction-limited
spot, which is moved over the sample to build the complete image on the
detector.
·
The modern Confocal microscope has all the
possible integration of technology and mechanical components including optical
components, which perform the primary function of the configuration by use of
electronic detectors, a computer, and laser systems.
·
Over the past three decades, Confocal
microscopy has evolved as a useful, non-invasive, imaging technology that has
diagnostic and prognostic implications.
·
Confocal microscopes are widely used in many
different fields, including Biology, Medicine, and Materials science, to study
the structure and function of cells, tissues, and molecules at the microscopic
level.
·
Currently, the Laser Scanning Confocal
Microscope (LSCM) is the most used confocal version for biomedical research.
Advantages of Confocal microscopy over
conventional Optical microscopy
·
One of the main advantages of Confocal
microscopy is its ability to produce high-resolution, three-dimensional images
of samples. This is achieved by scanning the sample in a series of layers or
optical sections and reconstructing the image using computer algorithms.
· Confocal
microscopy can also be used to visualize multiple different Fluorophores within
a single sample, allowing for the simultaneous visualization of multiple
processes or pathways.
· Some of the
advantages of Confocal microscopy over conventional Optical microscopy are
a) Short depth
of field.
b) Elimination
of out-of-focus glare.
c) Ability to
collect optical slices serially from thick specimens.
d) Ability to
produce high-resolution, three-dimensional images of samples.
e) Used to
visualize multiple different Fluorophores within a single sample.
History
of Confocal microscope
·
The concept of Confocal microscopy was
initially developed by Marvin Minsky in the 1955 at Harvard University with an
aim of viewing the Neural network without staining the tissues but it did not
bear fruit due to lack of enough light source and a computerized system to
store the large data.
· The work of Marvin
Minsky was later adapted by David Egger and Mojmir Petran, forming a Multiple-beam
Confocal microscope in the late 1960s. They used a Spinning disk known as
Nipkow which they used to examine brain tissues and ganglion cells that were
unstained. The technique was later modified and published by Egger forming a
mechanical Scanned Confocal Laser Microscope, that was able to visualize images
of cells.
·
The first practical Confocal microscope was
developed by Marvin Minsky and a team of researchers in the 1970s. The first
Confocal microscope used a Helium-neon laser and a Photomultiplier tube to
detect the Fluorescence emitted by the sample.
· The first
commercial Confocal microscope was developed in 1987 with improved optics and
electronics, powerful lasers with high scanning efficiency. The development of
Solid-State Lasers and Charge-Coupled Device (CCD) cameras led to the
widespread adoption of Confocal microscopy as a research tool.
·
In the 1990s, the development of Multi-photon
excitation techniques and other advanced imaging techniques further expanded
the capabilities of Confocal microscopy.
Parts
of Confocal microscope
a)
Objective lens: The
objective lens is a high-resolution lens that is used to collect and focus
light from the sample.
b) Laser light
source: The Laser light source is used to produce a focused beam of light
that is used to scan the sample and excite the Fluorophores within the sample.
It can be chosen via a selection device and is matched with the fluorophores
used in your experiment.
c) Scanning
system: The scanning system is responsible for moving the laser beam over
the sample in a raster pattern to produce an image. It is typically based on a
Galvanometer or Acousto-optic modulator.
d) Beam
splitter: It separates the excitation from the emitted light in the
Fluorescence beam path of the microscope.
e) Detectors: The
detectors are used to detect the Fluorescence emitted by the sample at
different depths. There are typically multiple detectors in a Confocal
microscope, including Detectors for different wavelengths of Fluorescence.
f) Control and
data acquisition system: The control and data acquisition system is
responsible for controlling the various components of the microscope and
acquiring and storing the data from the detectors. It typically includes a
computer and specialized software for controlling the microscope and analyzing
the data.
g) Stage: The stage
is a platform that holds the sample and allows it to be moved in order to scan
the sample.
h) Z-control: This allows
the user to focus the laser beam on any focal plane within the specimen. The
motorized Z-stepper allows us to move around the axial direction in small step
sizes (approximately >10 nm) with high precision.
i) Eyepieces: The
eyepieces are used to view the image produced by the microscope.
j) Pinhole: Pinhole is
a type of adjustable iris. Pinhole allows the exclusion of most of the
out-of-focus light from the acquired image and thus provides optical sectioning
capacity. The size of the pinhole can be
set by using the software on the user’s computer.
k)
Photomultiplier tube (PMT): It converts
the photons into an electrical signal which is then used up by the computer to
create an image of the specimen.
Working
principle of Confocal microscope
·
Confocal microscope uses laser beams instead
of lights. The laser beams are released from their source and then focused onto
a fluorescent stained sample.
· Neutral
density filters and a set of Scanning mirrors control the intensity of the
laser light by moving them very precisely and quickly.
· One mirror
tilts the beam within the X route, the opposite within the Y route. Together,
they tilt the beam in a raster style.
· Then an
Objective lens focuses it onto the Sample.
· The
Fluorochrome stained sample will be excited and then it will emit Fluorescent
lights. These Fluorescent lights will travel back into the Objective lens
through the same path that the laser travels.
· The main
effects of these Scanning mirrors are on this light is to generate a spot of
light which is not scanning, but standing still. Then, a semi-transparent
mirror reflects this Fluorescent light away from the Laser and toward the
Detection system.
· Before
entering into the detection system, the Fluorescent light passes through a
Pinhole. This Pinhole allows only a small central portion of the light through
to the light Detectors.
· Confocal
microscope produces a very low-intensity light, so the light is amplified by a
Photomultiplier tube (PMT) (Photomultipliers have the ability to amplify a
faint signal around one million times without introducing a single noise).
·
After that, the Photomultiplier tube (PMT)
releases an electrical signal, which is then converted into an image by using a
Computer.
Types
of Confocal microscope
a)
Single-photon confocal microscopes: These
microscopes use a single photon detector and a laser to produce high-resolution
images of a sample.
b) Two-photon
confocal microscopes: These microscopes use a two-photon
excitation process to produce high-resolution images of a sample. They are
typically used to image deep within tissues, as the two-photon excitation
process allows for deeper penetration of the sample.
c) Multiphoton
confocal microscopes: These microscopes use a multiphoton
excitation process to produce high-resolution images of a sample. They are
similar to two-photon confocal microscopes, but they are able to produce even
higher-resolution images and can be used to image even deeper within tissues.
d) Laser
scanning confocal microscopes: These microscopes use a laser beam to scan
over the sample and produce a high-resolution image. They are commonly used to
image live cells and tissues.
e) Fluorescence
lifetime imaging microscopes (FLIM): These
microscopes use the fluorescence lifetime of a sample to produce images. They
are often used to study biochemical processes within cells and tissues.
f) Stimulated
emission depletion (STED) microscopes: These
microscopes use stimulated emission to selectively deplete the fluorescence of
a sample, resulting in high-resolution images. They are able to achieve a
higher level of spatial resolution than other confocal microscopes.
g) Super
resolution microscopes: These microscopes use techniques such as
STED, structured illumination microscopy, or single-molecule localization
microscopy to achieve a spatial resolution that is beyond the diffraction limit
of light. They are able to produce images with a higher level of detail than
other confocal microscopes.
h) Hybrid
Scanning Confocal Microscopes: The slit-scanning confocal, which replaces
the circular aperture with a rectangular slit to reject out-of-focus light, is
an intermediate approach between single and multi-point scanning confocal
microscopes. Slit-scanning devices cover a larger portion of the sample in a
single field of view and greatly increase imaging speed at the expense of quick
photobleaching and reduced resolution.
i) Swept field
confocal microscope (SFC) - The SFC can be operated in either a
pinhole or slit scanning mode, in which the apertures stay immobile while
galvanometer- and piezo-controlled mirrors sweep the image of the illuminated
apertures across the sample. The emitted photons are directed to a
Charge-coupled device (CCD) camera via a set of complementary pinholes
or slits. The primary benefits of this method are its speed, increase in light
collection efficiency, and decrease in artefacts caused by the apertures’
movement, as in a spinning disc system.
j) Programmable
array microscope (PAM): This confocal microscope type employs a
Spatial Light Modulator (SLM) (SLM – an object that imposes some form of
spatially-varying modulation on a beam of light). The SLM is equipped with a
series of moveable apertures (pinholes) and arrays of pixels with opacity,
reflectivity, or optical rotation. In addition to microelectrochemical mirrors,
the SLM is equipped with a Charge-coupled device (CCD) camera for image
capturing.
k) Spinning
disk: Spinning disc, also known as the Nipkow disc, is a form of
confocal microscope that uses several moveable apertures (Pinholes) on a disc
to scan for light spots in a parallel way over a given plane for an extended
length of time. Compared to a Confocal laser scanning microscope, the longer
the exposure period, the less excitation energy is required for illumination.
Reduced excitation energy minimizes phototoxicity and photobleaching; hence,
live cell imaging is its primary application.
l)
Dual spinning Disk: Dual
spinning Disk or Microlens enhanced confocal Microscope. It functions similarly
to the spinning disc, with the exception that it has a second spinning disc
with micro-lenses that is located before the spinning disc with pinholes. The
microlenses capture a broad spectrum of light and focus it into each pinhole,
so increasing the quantity of light directed into each pinhole and decreasing
the amount of light obstructed by the spinning disc. These Confocal Microscopes
with improved Microlenses are far more sensitive than spinning discs.
Comparison
between Confocal Microscopy and Conventional Fluorescence Microscopy
Parameters |
Confocal Microscopy |
Conventional
Fluorescence Microscopy |
Light source |
Monochromatic laser |
Mercury arc lamp-white light |
Pinholes |
Two pinholes – one each at
conjugate focal planes |
Absent |
Filters |
Not required |
Emission and Excitation filters required |
Scanning |
The specimen is scanned to create an image
mosaic. |
Widefield microscopy – whole specimen is
illuminated. |
Resolution |
Superior – as focused and point
source of light illuminates a tiny bit of tissue at a time, eliminating
background illumination |
Blurring effect due to background
illumination |
In vitro/In vivo utility |
Confocal microscope can be used
to examine both Live (Reflectance Confocal Microscopy) and Excised tissues
(Reflectance Confocal Microscopy and Fluorescence Mode Confocal Microscopy) |
Tissues have to be excised, processed,
fixed, and stained before viewing |
Time requirement |
Few minutes to hours. |
Hours to day. |
Applications
of Confocal microscope
a)
Biology and Medicine: Confocal
microscopes are often used to study cells and tissues, including live cells.
They are commonly used to study the structure and function of cells and
tissues, as well as to identify and characterize specific proteins or other
molecules within cells. Used for the examination of various eye diseases. Used
for qualitative analysis, and quantification of endothelial cells of the
cornea. Used for localizing of filamentary fungal elements in the corneal
stroma in cases of keratomycosis.
b) Materials
science: Confocal microscopes are used to study the structure and
properties of materials, including polymers, ceramics, and metals.
c) Microelectronics: Confocal
microscopes are used to study the structure and properties of microelectronic
devices, including transistors and integrated circuits.
d) Art
conservation: Confocal microscopes are used to study the
structure and composition of artwork, including paintings and sculptures. It is
also used for optical scanning and recovery of damaged historical audio.
e) Geology: Confocal
microscopes are used to study the structure and composition of geological
samples, including minerals and rocks.
f) Agriculture: Confocal
microscopes are used to study the structure and function of plants, including
their cells and tissues.
g) Industrial inspection: Confocal
microscopes are used to inspect the quality and consistency of industrial
products, including food, pharmaceuticals, and consumer goods. It also widely
used in the pharmaceutical industry to control the quality and uniformity of
the drug distribution.
h)
3D Optical data: Used in 3D
optical data storage systems.
Overall,
confocal microscopes are an important tool for researchers and scientists who
need high-resolution images of small samples and structures.
Advantages
of Confocal microscope
·
High spatial resolution: Confocal
microscopes are able to produce high-resolution images of small samples and
structures, making them ideal for studying cells, tissues, and other small
samples.
· Increased
depth of field: Confocal microscopes use a pinhole to block
out-of-focus light, which increases the depth of field of the image. This
allows for a clearer image of samples with varying depths, such as tissues.
· Reduced
photobleaching: Confocal microscopes use a laser to excite
the sample, which reduces the amount of light needed to produce an image. This
reduces the photobleaching of samples, which can occur when samples are exposed
to high levels of light for extended periods of time.
· Reduced
background noise: The pinhole in a confocal microscope blocks
out-of-focus light, which reduces the background noise in the image. This
allows for a clearer, more detailed image of the sample.
· Live cell
imaging: Confocal microscopes are often used to image live cells, as they
do not produce a lot of heat and do not damage the sample.
· Multiplexing
capabilities: Confocal microscopes can be used to study
multiple samples or multiple fluorescence labels at the same time, allowing
researchers to study multiple aspects of a sample simultaneously.
· Accuracy: Because the
Confocal microscope analyses the image from one optical point to the next,
there is no interference from scattered light from other portions of the
specimen, resulting in a more accurate image.
· Living and
Dead Cell: It can be used to examine both living and dead cells
· Confocal
microscope can be used to collect Optical portions in series.
· Confocal
microscope illuminates the focus points consistently.
· Using a
factor known as the Zoom factor, Confocal microscope electronically adjust the
magnification without changing the objectives.
·
Confocal microscope produces 3D picture sets.
Overall, Confocal microscopy is a powerful
tool for producing high-resolution, detailed images of small samples and
structures, and is widely used in a range of research and industrial
applications.
Limitations
of Confocal microscope
·
Complexity and cost: Confocal
microscopes are more complex and expensive than other types of microscopes,
such as light microscopes. This can make them difficult for some researchers to
access or afford. It is costly to create the UV light that Confocal Microscopes
employ. They are also expensive to produce and acquire.
· Limited
penetration depth: Confocal microscopes are not able to image
very deep within tissues, as the laser beam used to excite the sample cannot
penetrate very far into the sample.
· Low light
sensitivity: Confocal microscopes are less sensitive to low levels of light
than other types of microscopes, such as widefield microscopes. This can make
it difficult to image samples with low levels of fluorescence.
·
Sample preparation: Confocal
microscopy requires the sample to be labeled with a fluorescent dye or protein,
which can be time-consuming and may alter the sample’s properties.
Limited field of view: Confocal microscopes have a small field of view compared to other types of microscopes, which can make it difficult to study large samples or structures.
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