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Pre-Ph.D. Course Work Presentation on
Submitted toDr. Vandna Soni
Submitted ByNishi Mody
CONFOCAL LASER ELECTRON MICROSCOPY
After the presentation, we will be having an idea of
Introduction and Historical Perspective
Principle and Basic Concept
Configuration of CLSM
Optical pathway in CLSM
How does CLSM works
Advantages of CLSM
Applications
Labs where we can access CLSM
Introduction and Historical Perspective
The basic concept of confocal microscopy was originally
developed by
Marvin Minsky in the mid-1950s and patented in 1961.
Fortuitously, shortly after Minsky’s patent had expired,
practical laser
scanning confocal microscope designs were translated
into working
instruments by several investigators.
Dutch physicist G. Fred Brakenhoff developed a scanning
confocal
microscope in 1979.
Tony Wilson, Brad Amos, and John White nurtured the
concept and
later (during the late 1980s) demonstrated the utility of
confocal
imaging in the examination of fluorescent biological
specimens.
The first commercial instrument appeared in 1987.
Modern confocal microscopes can be considered as
completely
integrated electronic systems where the optical microscope
plays a
central role in a configuration that consists of one or more
electronic
detectors, a computer (for image display, processing, output,
and
storage), and several laser systems combined with
wavelength selection
devices and a beam scanning assembly.
It can be considered as digital or video imaging system
capable of
producing electronic images.
These microscopes are now being employed for routine
investigations
on molecules, cells, and living tissues that were not possible
just a few
years ago.
Principle and basic concept
Confocal microscopy is an optical imaging technique used to
increase
optical resolution and contrast of a micrograph by using
point
illumination and a spatial pinhole to eliminate out of- focus
light in
specimens that are thicker than the focal plane.
Coherent light emitted by the laser system (excitation
source) passes
through a pinhole aperture that is situated in a conjugate
plane
(confocal) with a scanning point on the specimen and a
second pinhole
aperture positioned in front of the detector (a
photomultiplier tube).
As the laser is reflected by a dichromatic mirror and scanned
across the
specimen in a defined focal plane, secondary fluorescence
emitted from
points on the specimen (in the same focal plane) pass back
through the
dichromatic mirror and are focused as a confocal point at
the detector
pinhole aperture.
It enables the reconstruction of three-dimensional structures
from the
obtained images
Configuration of CLSM
The confocal fluorescence microscope consists of
Multiple laser excitation sources.
Scan head with optical and electronic
components.
Electronic detectors usually photomultipliers.
Computer for acquisition, processing,
analysis, and
display of images.
The scan head is at the heart of the
confocal system and is responsible for
rasterizing the excitation scans, as
well as collecting the photon signals
from the specimen that are required
to assemble the final image.
A typical scan head contains inputs
from the external laser sources,
fluorescence filter sets and
dichromatic mirrors, a galvanometer-
based raster scanning mirror system,
variable pinhole apertures for
generating the confocal image, and
photomultiplier tube detectors tuned
for different fluorescence
wavelengths.
The excitation laser beam is connected to the scan unit
with a
fiber optic coupler followed by a beam expander that
enables the
thin laser beam wrist to completely fill the objective
rear aperture (a
critical requirement in confocal microscopy).
One of the most important components of the scanning
unit is
the pinhole aperture, which acts as a spatial filter at the
conjugate
image plane positioned directly in front of the
photomultiplier.
The aperture serves to exclude fluorescence signals
from out-of-
focus features positioned above and below the focal
plane.
The pinhole aperture also serves to eliminate much of
the stray light
passing through the optical system.
Schematic diagram of the optical pathway in a laser scanning confocal microscope.
Coherent light emitted by
the laser system (excitation
source) passes through a
pinhole aperture that is
situated in a conjugate
plane (confocal) with a
scanning point on the
specimen and a second
pinhole aperture positioned
in front of the detector (a
photomultiplier tube).
As the laser is reflected
by a dichromatic mirror
and scanned across the
specimen in a defined
focal plane, secondary
fluorescence emitted
from points on the
specimen (in the same
focal plane) pass back
through the
dichromatic mirror and
are focused as a confocal
point at the detector
pinhole aperture.
How does CLSM works A laser is used to provide the excitation light in order to
get very high
intensities.
The laser light reflects off a dichromatic mirror.
From there, the laser hits two mirrors which are mounted
on motors
These mirrors scan the laser across the sample.
Dye in the sample fluoresces, and the emitted light gets
descended by
the same mirrors that are used to scan the excitation
light from the
laser. The emitted light passes through the dichromatic
mirror and is
focused onto the pinhole.
The light that passes
through the
pinhole is measured by a
detector,
a photomultiplier tube.
There never is a complete
image of
the sample at any given
instant,
only one point of the sample
is
observed.
The detector is attached to a
computer which builds up
the
image.
Advantages of CLSM
With regular fluorescence microscopy the sample is
completely
illuminated by the excitation light, so all of the sample is
fluorescing at
the same time. Of course, the highest intensity of the
excitation light is
at the focal point of the lens, but nonetheless, the other
parts of the
sample do get some of this light and they do fluoresce. This
contributes
to a background haze in the resulting image.
Adding a pinhole/screen combination solves this problem.
Because the
focal point of the objective lens of the microscope forms an
image
where the pinhole is, these two points are known as
"conjugate points“
or conjugate planes. The pinhole is conjugate to the focal
point of the
lens, thus it is a confocal pinhole.
Laser scanning confocal microscopy has the ability to
serially produce
thin (0.5 to 1.5 micrometer) optical sections.
Image information is restricted to a well-defined plane,
rather than
being complicated by signals arising from remote locations
in the
specimen.
Contrast and definition are dramatically improved over
widefield
techniques due to the reduction in background
fluorescence and
improved signal-to-noise.
Optical sectioning eliminates artifacts that occur during
physical
sectioning and fluorescent staining of tissue specimens for
traditional
forms of microscopy.
The non-invasive confocal optical sectioning technique
enables the
examination of both living and fixed specimens under a
variety of
conditions with enhanced clarity.
Vertical sections in the x-z and y-z planes (parallel to
the
microscope optical axis) can be readily generated by
most confocal
software programs. Thus, the specimen appears as if it
had been
sectioned in a plane that is perpendicular to the lateral
axis.
Many software packages enable investigators to conduct
measurements of length, volume, and depth, and
specific
parameters of the images, such as opacity, altered to
reveal internal
structures of interest at differing levels within the
specimen.
Widefield versus confocal microscopy illumination volumes,
demonstrating the difference in size between point scanning
and widefield excitation light beams.
Applications
Conjugated Antibodies
DNA/RNA
Organelle Structure
Cytochemical Identification
Cellular Functions
Dynamic Observation of pH Change within polymer matrix
In characterization of chemical enhancers in drug- in-
adhesive
transdermal patches
Tracking nanoparticles in three-dimensional tissue-
engineered
models using.
Tracking nanoparticle drug delivery through skin.
Cellular Function
Enzyme Activity
Oxidation Reactions
Intracellular pH
Intracellular Calcium
Phagocytosis & Internalization
Apoptosis
Membrane Potential
Cell-cell Communication (Gap Junctions)
A Gram-positive Bifidobacterium strain was labeled with two pH-
sensitive fluorophores, which could be independently excited. The
pixel intensity ratio of images taken by confocal laser-scanning
microscopy (CLSM) can be color coded, which allows the
visualization of pH within formulations during exposure to acidic
solutions. This method could be applied to any system in which the
bacteria may be suspended within a matrix.
Dynamic Observation of pH Change within Polymer
Examples from Bio-Rad Web site
Projection of 25 optical
sections of a triple-labeled
rat lslet of Langerhans,
acquired with a
krypton/argon laser.
This image shows a
maximum brightness
projection of Golgi stained
neurons.
Examples from Bio-Rad web site
Paramecium labeled with an anti-tubulin-antibody showing thousands of cilia and internal microtubular structures. Image
Whole mount of Zebra Fish larva stained with Acridine Orange, Evans Blue and Eosin.
Confocal vs. Widefield
Confocal
WidefieldTissue culture cell with 60x / 1.4NA objective
Confocal vs. Widefield
Confocal
Widefield
20 mm rat intestine section recorded with 60x / 1.4NA objective
Higher z-resolution
and reduced out-of-
focus-blur make
confocal pictures
crisper and clearer.
Labs where we can access CLSM
Banaras Hindu University, Varanasi
National Institute of Plant Genome Research, New
Delhi
National Centre for Cell Science, Pune
Central Drug Research Laboratory, Lucknow
Jawaharlal Nehru University, New Delhi
Centre for Cellular and Molecular Platforms, Bangalore
Indian Institute of Technology, Delhi
Indian Institute of Technology, Bobmay
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