Confocal laser scanning microscopy

" Agriculture is an art,
Try to feel & make a love"

1. FUNDAMENTALS OF AGRICUTURAL MICROBIOLOGY (1+1) LASER SCANNING CONFOCAL MICROSCOPE BY TO BASKAR S Dr.S PARTHASARATHY BATCH-A 19AGM111 CB.AG.U4AGR19018 ( PLANT PATHOLOGIST)

2. CONFOCAL MICROSCOPY An optical microscopy technique for increasing optical resolution and contrast of a microscope. The principle of confocal microscopy was patented by Marvin Minsky in 1957. It works on the principle of fluorescence optics. It is a modified version of fluorescent microscope.

3. MARVIN CLSM MINSKY

4. Why it is invented? Due to the drawbacks in fluorescent microscope. The continuous exposure of samples to high intensity UV light causes photo bleaching (fading).

5. MODIFICATIONS There are two major modifications are done in fluorescent microscope. Pinhole aperture is placed to avoid the out of focus in which focus on above and below focal planes are cancelled. Laser light is used as light source instead of mercury arch lamp.

6. MODIFICATIONS & COMPARISON

7. LASER SCANNING CONFOCAL MICROSCOPE It is similar to the principle of confocal microscopy. Because of using monochromatic laser beam (He-Ne) as a light source, confocal microscope is known as laser scanning confocal microscope. In this, detector (PMT) and scanner are placed to analyse the different focal planes (3D).

8. COMPONENTS OF CLSM Laser beam Beam splitter Scanner Objective lens Z-control Pinhole Photomultiplier tube (PMT)

9. RAY DIAGRAM OF CLSM

10. WORKING PRINCIPLE OF CLSM Laser beam is focused on the smaller region of specimen. The intensity of laser can be adjusted by beam splitter (neutral filter). Then the laser passes through 2 scanning mirrors which rotates in an opposite X & Y direction to focus on another region.

11. The laser beam is then brought to the focal plane of objective lens which focuses onto sample. If the sample is fluorescent , light will pass back into objective lens ( travels on same path that laser travels). Then it passes through the semi transparent mirror.

12. Due to rotating mirrors, laser scans the whole surface of the specimen. Through pinhole , detector (PMT) captures the images of particular regions of whole specimen (light signal is converted into electrical signal). Atlast captured individual images are compiled by software which is connected to detector and gives real time image .

13. WORKING PRINCIPLE OF CLSM (DIAGRAM)

14. SCANNING AND COMPILING

15. ADVANTAGES Produces 3D images Provides internal structures at different levels within the specimen. Captured analog image data is readily available for printout. Ability to produce thin optical sections (0.5 – 1.5 micrometer).

16. DISADVANTAGES Monochromatic LASER is harmful to humans. It is very costlier (around 1-1.5 crores ).

17. REFERENCES Confocal Laser Scanning Microscopy – Colin J. R. Sheppard Microbiology – P.D.Sharma https://www.umassmed.edu/globalassets/maps/documents/confocal-explanation.pdf https://bitesizebio.com/19958/what-is-confocal-laser-scanning-microscopy/

18. THANK YOU