1. Dr R.Jayaprada
CONFOCAL MICROSCOPY

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

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 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

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 :
Point-by-point illumination of the specimen.
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 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 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.

23. THANK YOU