It helps for Bsc first years to understand the basics of Confocal Microscopy

1. Confocal
Microscopy

2. INTRODUCTION
• An optical imaging technique for increasing optical
resolution and contrast of a micrograph.
• Radiations emitted from laser cause sample to
fluoresce.
• Uses pinhole screen to produce high resolution
images.
• Eliminates out of focus.
• So images have better contrast and are less hazy.
• A series of thin slices of the specimen are assembled
to generate a 3dimensinal image.
• Is an updated version of fluorescence microscopy.

3. HISTORY
• Two investigators at Cambridge, Brad Amos and John White
attempted to look at the mitotic divisions in the first few
divisions in embryos of C. elegans.
• They were doing antitubulin immunofluorescence and were
trying to determine the cleavage planes of the cells,
• But were frustrated in their attempt in that the majority of
the fluorescence they observed was out of focus.
• They looked at the technique called confocal imaging which
was first proposed by Nipkow and pioneered by a postdoc at
Harvard named Minsky.
• He made the first stage scanning confocal microscope in 1957.
• By illuminating single point at a time, Minsky avoided most of
the unwanted scattered light.
• For buiiding the image, Minsky scanned the specimen by
moving the stage rather than light rays.

4. COMPONENTS OF CONFOCAL
MICROSCOPY
• Laser
• Photomultiplier
• Filters

5. LASER

6. LASER
• Light Amplification by Stimulated Emission of
Radiation (Laser).
• Lasers are used because they are an intense
coherent monochromatic source of light, capable
of being expanded to fill an aperture or focused
to a spot.
• The laser beam is usually linearly polarized. The
main drawback in using lasers is that to cover a
large excitation range you will need several lasers.
• However a single laser line may not optimally
excite your molecule.

7. PHOTOMULIPLIER

8. PHOTOMULTIPLIER
• A photomultiplier tube, useful for light detection of very weak
signals,it is a photo emissive device in which the absorption of
a photon results in the emission of an electron.
• These detectors work by amplifying the electrons generated
by a photocathode exposed to a photon flux.

9. FILTERS
•
• Microscopy Filters are used in a variety of microscopy applications for
increasing contrast, blocking ambient light, removing harmful ultraviolet
or infrared light, or for selectively transmitting only wanted wavelengths.
• Most of the microscopes, are based on optical filters.
• A typical system has three basic filters: an excitation filter (or exciter), a
dichroic beamsplitter (or dichromatic mirror), and an emission filter (or
barrier filter).
• An excitation filter is a high quality optical-glass filter commonly used in
fluorescence microscopy and spectroscopic applications for selection of the
excitation wavelength of light from a light source.
• Barrier filters are filters which are designed to suppress or block (absorb) the
excitation wavelengths and permit only selected emission wavelengths to
pass toward the eye or other detector.
• A third version of the beam splitter is a dichroic mirrored prism assembly
which uses dichroic optical coatings to divide an incoming light beam into a
number of spectrally distinct output beams. The dichroic beam splitter
controls which wavelengths of light go to their respective filter.

10. DICHORIC MIRROR

11. INSTRUMENTAL DESIGN
Ray diagram for confocal microscope

12. CONFOCAL MICROSCOPE
Schematic diagram
confocal microscope

14. PRINCIPLE
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.
• Scanning of small sections is done and joined them
together for better view.

15. WORKING MECHANISM
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.

16. Contd….
• 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

17. LIGHT PATHWAY
• Light from
excitation filter
passes through
objective lens and
light absorbed by
specimen.
• Light emitted goes
back through
objective lens,
barrier filter, then
detector.

18. x y
z
OPTICAL SECTIONING AND 3-D RECONSTRUCTION

19. x y
z
OPTICAL SECTIONING AND 3-D RECONSTRUCTION

20. x y
z
=
OPTICAL SECTIONING AND 3-D RECONSTRUCTION

21. 3-D Imaging with Confocal Microscopy

22. ADVANTAGES
• The specimen is everywhere illuminated axially, rather than
at different angles, thereby avoiding optical aberrations.
• Entire field of view is illuminated uniformly.
• The field of view can be made larger than that of the static
objective by controlling the amplitude of the stage
movements.
• Image formed are of better resolution.
• Cells can be live or fixed. Serial optical sections can be
collected.
• Taking a series of optical slices from different focus levels in
the specimen generates a 3D data set.

23. DRAWBACKS
• Resolution : It has inherent resolution limitation due to
diffraction. Maximum best resolution of confocal microscopy is
typically about 200nm.
• Pin hole size : Strength of optical sectioning depends on the
size of the pinhole.
• Intensity of the incident light
• Fluorophores :
• a)The fluorophore should tag the correct part of the specimen.
• b)Fluorophore should be sensitive enough for the given
excitation wave length.
• c)It should not significantly alter the dynamics of the organism
in the living specimen.
• Photobleaching: photochemical alteration of a dye or a
fluorophore molecule such that it permanently is unable to
fluorescence.

24. APPLICATIONS
1.Confocal
microscopy allows
analysis of
fluorescent
labelled thick
specimens without
physical
sectioning.
Zebra fish embryo wholemount Neurons (green) and
Cell adhesion molecule (red)

25. APPLICATIONS
2. Three-Dimensional
Reconstruction of
Specimen .
3D shadow projection

26. 3. Improved Resolution
APPLICATIONS
An Intestine SectionKidney cells (fluorescence vs Confocal
microscope

27. 4. More Colour Possibilities
Because the images are
detected by a computer rather
than by eye, it is possible to
detect more color differences.
APPLICATIONS

28. IMAGE TAKEN BY CONFOCAL MICROSCOPE
Colour coded image of actin filaments in a cancer cell

29. IMAGE TAKEN BY CONFOCAL MICROSCOPE
Pancreatic islets of mouse

30. IMAGE TAKEN BY CONFOCAL MICROSCOPE
Drosophila brain
with triple
antibodies

31. IMAGE TAKEN BY CONFOCAL MICROSCOPE
Hepatocyte
sphenoid

32. Confocal vs. Widefield
Confocal Widefield
Tissue culture cell with 60x / 1.4NA objective
CONFOCAL VS. WIDEFIELD
Tissue culture cell with 60X /1.4NA Objective

33. CONFOCAL VS. WIDEFIELD
20 micrometer rat intestine section recorded with 60 x/1.4NA Objective

34. EXAMPLES
Drosophila S2 cell expressing GFP-H2B
and mChery-tubulin [ Nico suurname
and Ron vale ].
S.Cerevisae expressing mitochondrially
targeted RFP [Sussane Rafeilski
,Marshall lab ].

35. THANK YOU