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