confocalmicroscopy-121115061847-phpapp02

7/27/2019 confocalmicroscopy-121115061847-phpapp02

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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.
http://en.wikipedia.org/wiki/Fluorescein_isothiocyanatehttp://en.wikipedia.org/wiki/Fluorescein_isothiocyanate


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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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