Confocal microscopy produces sharp images from specimens that would otherwise appear blurred. It excludes out-of-focus light using pinholes to illuminate and receive light only from the focal plane. By assembling thin slices, it can generate 3D images with better contrast than conventional microscopy. Confocal microscopy was pioneered by Minsky in 1955 and works by scanning a laser point-by-point across the specimen while detecting only in-focus light through a pinhole before the detector. It allows imaging of live or fixed cells without processing and can collect serial optical sections.

1. CONFOCAL MICROSCOPY
Dr R.Jayaprada

2. 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-
dimensinal image.

3. 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 reconstruted
using a long persistance screen.
For builiding the image, Minsky scanned the
specimen by moving the stage rather than light
rays.

4. 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
 Need to scan point onto the
sample
CONDENS ER
LENS
OBJECTIVE
LENS
BIOLOGICAL
S AMPLE
OUT-OF-FOCUS PLANE
OUT-OF-FOCUS PLANE
"POINT"
S OURCE
OF LIGHT "POINT"
DETECTOR
APERTURE
IN-FOCUS (OBJECT) PLANE
CONTAINING ILLUMINATED S POT

6.  . 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.

8. PROCEDURE
 The frozen tissue was thawed and
confocally imaged.
 The thawed tissue specimen was washed
in phosphate buffered saline and 5%
acetic acid (3 minutes each solution) prior
to confocal imaging.
 The acetic acid causes the aggregation of
chromatin within the cell nuclei and
enhances contrast in confocal images.

9. 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.

10. 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

11. Excitation and Emission
 Stokes Shift/Law
 Florescence emission
wave length is longer
 Excitation wave length
is shorter

12. Light Path
 Light from excitation
filter thru objective
lens; light absorbed
 Light emitted goes
back thru objective
lens, barrier filter, then
detector

13. Immunolabeling for Fluorescence
 1.Block with PBST+5% milk 1 hr
 2.Incubate with primary antibody in PBS or
blocking solution 1-2hr, @ r.t
 3.Wash with PBST+5% milk 3x3 min
 4.Incubate with 2ndary antibody in PBS 1hr
r.t
 5.Wash with PBST+5% milk 5 min
 6.Wash with PBS no milk 2x5 min
 7.Wash with dH20 2x10 min
 8.Coverslip withVectashield & view with
fluorescence/confocal microscope

14. Confocal Microscope
 Better resolution
 Cells can be live or fixed
 Serial optical sections can be collected

15. Laser Beam
 Laser goes thru aperture,
then objective lens; pixel
by pixel scanning
 Light is reflected back
thru objective lens, beam
splitter allows laser thru,
and reflects fluorescence
 To the detector, pic can
be viewed on the
computer

16. Fluorochromes
 FITC: fluorescein isothiocyanate absorption
maximum at 495 nm, 488nm excitation
wavelength
 TEXAS RED: 595nm excitation wavelength, 615
max absorption, red dye, marks protein.

17. 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.

18. HOW DOES A CONFOCAL MICROSCOPE WORK
 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.

19. 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

20. LIMITATIONS OF CONFOCAL MICROSCOPY
 1.Resolution : It has inherent resolution limitation due to
diffraction. Maximum best resolution of confocal microscopy is
typically about 200nm.
 2.Pin hole size : Strength of optical sectioning depends on the size
of the pinhole.
 3.Intensity of the incident light.
 4.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.
 5.Photobleaching

21. FAST CONFOCAL MICROSCOPY
 Most confocal microscopes generate a single
image in 0.1-1 second.
 Two commonly used designs that can capture
image at high speed are :
 Nipkow disk confocal microscope:This builds an
image by passing light through a spinning mask
of pinholes ,thereby simultaneously illuminating
many discrete points.
 Confocal microscope that uses an acousto-optic
deflector (AOD) for steering the excitation light.
Fast horizontal scans can be achieved with AOD.

22. TWO PHOTON MICROSCOPY
 This microscopy is related to confocal microscopy.
 It provides excellent optical sectioning.