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CONFOCAL MICROSCOPY
Dr R.Jayaprada
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INTRODUCTION A confocal microscope creates sharp images of a
specimen that would appear otherwise blurredwith the conventional microscope this isachieved by excluding most of the light from thespecimen, but not from the microscopes focal
plane. The image obtained has better contrast & less
hazy .
In confocal microscopy, a series of thin slices ofthe specimen is assembled to generate a 3-dimensinal image.
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HISTORY Confocal microscopy was pioneered by Marvin
Minsky in 1955.
By illuminating single point at a time, Minskyavoided most of the unwanted scattered light
that obscures an image when the entirespecimen is illuminated at the same time.
Additionally, the light returning from the specimenpasses through a second pin-hole aperture.
Remaining desirable light rays are collected by aphotomultiplier & the image is reconstruted
using a long persistance screen.For builiding the image, Minsky scanned the
specimen by moving the stage rather than lightrays.
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Principle of confocal
microscopy
In confocal microscopy two pinholesare typically used:
A pinhole is placed in front ofthe illumination source to allowtransmission only through asmall area
This illumination pinhole isimaged onto the focal plane ofthe specimen, i.e. only a pointof the specimen is illuminatedat one time
Fluorescence excited in thismanner at the focal plane isimaged onto a confocal pinhole
placed right in front of thedetector
Only fluorescence excitedwithin the focal plane of thespecimen will go through thedetector pinhole
Need to scan point onto thesampleCONDENSERLENS OBJECTIVELENSBIOLOGICALSAMPLE
OUT-OF-FOCUS PLANE
OUT-OF-FOCUS PLANE
POINT"OURCE
OF LIGHT "POINT"DETECTORAPERTURE
IN-FOCUS (OBJECT) PLANECONTAINING ILLUMINATED SPOT
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. Confocal microscopy is unique because itcan rapidly produce images of cellularmorphology without the need to process thetissue (i.e., without freezing, sectioning and
staining). A confocal microscope images have refractive
index variation within the epithelial andstromal compartments of the tissue. These
refractive index variations are due to thechemical variations within the tissue.Structures that backscatter more light appearbrighter than less scattering structures.
Because the source of image contrast is notdue to exogenous stains, confocal imagesappear different than those from tissue thathas been histologically processed and
stained.
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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.
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MODERN CONFOCAL MICROSCOPY
Modern confocal microscope have taken the key
elements of Minskys design;i.e; pinholeapertures & point-by-point illumination of thespecimen.
Majority of the confocal microscopes imageeither by reflecting the light off the specimen orby stimulating fluorescence from dyes(fluorophores) applied to the specimen.
Advances in the optics & electronics have beenincorporated into the current designs andprovide improvements in speed, image quality &
storage of generated images.
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Alexander Jablonski Diagram
Light from the
excitation filterexcites thefluorochoromes to ahigher energy state
From the high stateit declines slowlyreleasing energy
Transition betweenabsorption &emission
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Excitation and Emission
Stokes Shift/Law Florescence emission
wave length is longer
Excitation wave length
is shorter
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Light Path
Light from excitation
filter thru objectivelens; light absorbed
Light emitted goes
back thru objectivelens, barrier filter, thendetector
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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 with Vectashield & view withfluorescence/confocal microscope
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Confocal Microscope
Better resolution
Cells can be live or fixed Serial optical sections can be collected
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Laser Beam
Laser goes thru aperture,then objective lens; pixelby pixel scanning
Light is reflected backthru objective lens, beamsplitter allows laser thru,and reflects fluorescence
To the detector, pic canbe viewed on thecomputer
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Fluorochromes FITC: fluorescein isothiocyanate absorption
maximum at 495 nm, 488nm excitationwavelength
TEXAS RED: 595nm excitation wavelength, 615max absorption, red dye, marks protein.
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HOW DOES A CONFOCAL MICROSCOPE WORKConfocal 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 usedZirconium arc
lamp in Minskys design & laser light source in moderndesign.
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 scanningmirrors.
c)These motor driven mirrors scan the laser beam acrossthe specimen.d) The specimen is scanned by moving the stage back &
forth in the vertical & horizontal directions and opticsare kept stationary.
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HOW DOES A CONFOCAL MICROSCOPE WORK
Dye in the specimen is excited by the laser light
& fluoresces. The fluorescent (green) light isdescanned by the same mirrors that are used toscan the excitation (blue) light from the laser
beam then it passes through the dichoric
mirror then it is focused on to pinhole thelight passing through the pinhole is measuredby the detector such as photomultiplier tube.
For visualization, detector is attached to thecomputer, which builds up the image at the rateof 0.1-1 second for single image.
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ADVANTAGES OF CONFOCAL MICROSCOPY
1.The specimen is everywhere illuminated axially,rather than at different angles, thereby avoiding
optical aberrations entire field of view isilluminated uniformly.
2.The field of view can be made larger than that of
the static objective by controlling the amplitude ofthe stage movements.
3.Better resolution
4.Cells can be live or fixed 5.Serial optical sections can be collected
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LIMITATIONS OF CONFOCAL MICROSCOPY
1.Resolution : It has inherent resolution limitation due todiffraction. 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
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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 animage by passing light through a spinning maskof pinholes ,thereby simultaneously illuminatingmany discrete points.
Confocal microscope that uses an acousto-opticdeflector (AOD) for steering the excitation light.Fast horizontal scans can be achieved with AOD.
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TWO PHOTON MICROSCOPY This microscopy is related to confocal microscopy.
It provides excellent optical sectioning.
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