PRINCIPLES OF CONFOCAL

MICROSCOPY AND SAMPLE

PREPARATION

SUBMITTED BY: SUBMITTED TO:

ADITYA RUDRA Dr. ANJANA SAGAR

2017LSC1075 DEPT. OF BOTANY
INTRODUCTION

 A confocal microscope creates sharp images of a

specimen that would appear otherwise blurred
with the conventional microscope –this is
achieved by excluding most of the light from the
specimen, but not from the microscope’s focal
plane.
 The image obtained has better contrast & less
hazy .
 In confocal microscopy, a series of thin slices of
the specimen is assembled to generate a 3-
dimensional image.
HISTORY
 Confocal microscopy was pioneered by Marvin
Minsky in 1955.
 By illuminating single point at a time, Minsky
avoided most of the unwanted scattered light
that obscures an image when the entire
specimen is illuminated at the same time.
 Additionally, the light returning from the specimen
passes through a second pin-hole aperture.
 Remaining desirable light rays are collected by a
photomultiplier & the image is reconstructed
using a long persistence screen.
 For building the image, Minsky scanned the
specimen by moving the stage rather than light
rays.
 PRINCIPLE OF CONFOCAL MICROSCOPY
In confocal microscopy two pinholes
are typically used:
• A pinhole is placed in front of
the illumination source to allow
transmission only through a
small area
• This illumination pinhole is
imaged onto the focal plane of
the specimen, i.e. only a point
of the specimen is illuminated
at one time
• Fluorescence excited in this
manner at the focal plane is
imaged onto a confocal pinhole
placed right in front of the
detector
• Only fluorescence excited
within the focal plane of the
specimen will go through the
detector pinhole
• Scan point onto the
Sample.
• Confocal microscopy is unique because it
can rapidly produce images of cellular
morphology without the need to process the
tissue (i.e., without freezing, sectioning and
staining).
• A confocal microscope images have refractive
index variation within the epithelial and
stromal compartments of the tissue. These
refractive index variations are due to the
chemical variations within the tissue.
Structures that backscatter more light appear
brighter than less scattering structures.
• Because the source of image contrast is not
due to exogenous stains, confocal images
appear different than those from tissue that
has been histologically processed and
stained.
MODERN CONFOCAL MICROSCOPY

• Modern confocal microscope have taken the key

elements of Minsky’s design i.e. pinhole
apertures & point-by-point illumination of the
specimen.
• Majority of the confocal microscopes image
either by reflecting the light off the specimen or
by stimulating fluorescence from dyes
(fluorophores) applied to the specimen.
• Advances in the optics & electronics have been
incorporated into the current designs and
provide improvements in speed, image quality &
storage of generated images.
ALEXANDER
JABLONSKI DIAGRAM

• Light from the

excitation filter
excites the
fluorochoromes to a
higher energy state
• From the high state
it declines slowly
releasing energy
• Transition between
absorption &
emission
HOW DOES A CONFOCAL MICROSCOPE
WORK

Confocal microscope incorporates 2 ideas :

1. Point-by-point illumination of the specimen.
2. Rejection of out of focus of light.
Light source of very high intensity is used—Zirconium arc
lamp in Minsky’s design & laser light source in modern
design.
a)Laser provides intense blue excitation light.
b)The light reflects off a dichoric mirror, which directs it to
an assembly of vertically and horizontally scanning
mirrors.
c)These motor driven mirrors scan the laser beam across
the specimen.
d) The specimen is scanned by moving the stage back &
forth in the vertical & horizontal directions and optics
are kept stationary.
• Dye in the specimen is excited by the laser light
& fluoresces. The fluorescent (green) light is
descanned by the same mirrors that are used to
scan the excitation (blue) light from the laser
beam then it passes through the dichoric
Mirror then it is focused on to pinhole the
light passing through the pinhole is measured
by the detector such as photomultiplier tube.
• For visualization, detector is attached to the
computer, which builds up the image at the rate
of 0.1-1 second for single image
 ADVANTAGES OF CONFOCAL
MICROSCOPY

1. The specimen is everywhere illuminated

axially,
rather than at different angles, thereby avoiding
optical aberrations entire field of view is
illuminated uniformly.
2. The field of view can be made larger than
that of
the static objective by controlling the amplitude
of
the stage movements.
3. Better resolution
4. Cells can be live or fixed
5. Serial optical sections can be collected
Processing for confocal microscopy is broken down into 4 basic steps
where each step may have several sub-steps and we are presented
with a large variety of opportunities to ruin the entire process! In
general, these are the steps one must follow:

1. Fixation
2. Permeabilisation
3. Staining
4. Mounting
Tissue Fixation
Processing of most samples begins with fixation to preserve morphology. A fixation method
must be chosen that balances two characteristics:

"Good" preservation of cellular 3-D structure

"Adequate" access to antigenic sites.

The goal is to preserve sufficient cellular organization to allow identifying the features of
interest, but yet not destroy the antigenicity of the target.

Commonly used histological method of fixation and permeabilization often consist of

treating the cells or tissues with solvents, such as alcohol or acetone. While these methods
are quick acting precipitating fixatives, and also are good permeabilizing agents, they have
one significant negative consequence: cellular shrinkage. The degree of shrinkage may be
almost insignificant for monolayers of cells, but will distort tissue samples dramatically.

Glutaraldehyde is a commonly used fixative used for electron microscopy, and does a
terrific job of preserving microscopic structure - and ultrastructure. Unfortunately,
glutaraldehyde has the unfortunate side effect of cross-linking (and thereby destroying)
many antigenic sites.
A good compromise is the use of paraformaldehyde (PF) as a fixative.
Permeabilization

If a component of the cytoplasm or nucleus is to be effectively labeled, the plasma

membrane must be made permeable to staining solutions. There are several ways to
do this, and in part they depend on the fixation method chosen. Cells fixed with
solvents do not usually need any additional permeabilizing - the solvent has already
extracted enough of the membrane. So, solvent fixation is doubly efficient for this
reason. However, cells fixed with crosslinking aldehydes do need to have the plasma
membrane integrity breached by the use of chemical agents. Commonly used reagents
include DMSO and detergents like Triton X-100, saponin or deoxycholate.

Permeabilization is another area, like fixation where fine tuning is necessary.

Different detergents solubilize different molecules within the membrane, so it's
necessary to know that what you are interested in isn't being washed away in the
permeabilization step! Concentration of detergent is another detail that should be
adjusted carefully. The idea is to selectively remove plasma membrane constituents
to allow access to the cytoplasm without altering either the antigenicity or
morphology of the sample
Protocols for Labeling

Two basic techniques are used: direct labeling and indirect

labeling. Both labeling methods are suitable for confocal
microscopy.
Direct labeling consists of using a fluorescently labeled primary
antibody or chemical ligand to cause the structure of interest to
become fluorescent. Advantages of this method include speed and
ease of application. A potential disadvantage is lack of sensitivity
(low signal intensity).
The indirect method involves binding a primary antibody to the
epitope of interest, followed by a fluorescently labeled secondary
antibody. The primary advantage of using this technique is the great
amplification of signal possible through an antibody cascade.
Disadvantages include increased complexity, more time consuming,
and often problems with non-specific antibody reactions.
Choosing a Fluorescent Label
Label choice depends on available equipment (laser lines, emission
filters, transmitted light detectors) and the availability of certain fluors conjugated
to required antibodies for use in multiple labeling schemes. In general, the laser lines
available dictate which fluorophores may be used. Recent advances in biochemistry
have created new families of fluorophores with very favorable signal-to-noise and
quantum efficiency (QE) properties. In particular, the Cy dyes and the Alexa dyes are
particularly useful. Both families have high QEs, are very resistant to photobleaching,
and are available in a variety of excitation/emission wavelengths.

Fluorescence detection is not the only way to use a confocal, however. The same light
that passes through a specimen while imaging a fluorophore may be used to image the
specimen by the Nomarski technique. Combining the emitted fluorescence and
transmitted Nomarski signal is a particularly powerful technique for illustrating details
of a cell layer that may not be fully fluorescent. An additional non-fluorescence based
technique is reflection-mode confocal microscopy. Light reflected from the point of
focus is collected and used as the source of signal for generating the image. Common
samples used for reflection mode confocal microscopy are silver-enhanced gold-
conjugated antibodies, or materials science samples.
Mounting Samples

Fluorescently labeled cells and tissues exhibit a characteristic photo

bleaching curve in response to excitation by the laser. Much of the
photo bleaching can be attributed to generation of free radicals
during production of the electronically excited fluor species. Use of
free radical scavengers has been shown to greatly decrease the rate of
photo bleaching. Common scavengers include n-propyl gallate, p-
phenylenediamine and DABCO (1,4-diazobicyclo-(2,2,2)-octane). Live
systems have reported reduced photo bleaching in the presence of
vitamin C.