Dr R.

Jayaprada

CONFOCAL MICROSCOPY

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

Principle of confocal In confocal microscopy two pinholes are typically used: microscopy A pinhole is placed in front of

OUT-OF-FOCUS PLANE IN-FOCUS (OBJECT) PLANE CONTAINING ILLUMINATED S POT OUT-OF-FOCUS PLANE

"POINT" S OURCE OF LIGHT CONDENS ER LENS BIOLOGICAL S AMPLE OBJECTIVE LENS

"POINT" DETECTOR APERTURE

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

 . 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.

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.

MODERN CONFOCAL MICROSCOPY

Modern confocal microscope have taken the key

elements of Minskys 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

Excitation and Emission

Stokes Shift/Law Florescence emission wave length is longer Excitation wave length is shorter

Light Path
Light from excitation filter thru objective

lens; light absorbed Light emitted goes back thru objective lens, barrier filter, then detector

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 with fluorescence/confocal microscope

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

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

Fluorochromes
FITC: fluorescein isothiocyanate absorption

maximum at 495 nm, 488nm excitation wavelength TEXAS RED: 595nm excitation wavelength, 615 max absorption, red dye, marks protein.

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 usedZirconium arc lamp in Minskys 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.

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.

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

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

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.

TWO PHOTON MICROSCOPY

This microscopy is related to confocal microscopy. It provides excellent optical sectioning.