Confocal microscopy by prabhat

Confocal microscopy by prabhat

1. CONFOCAL MICROSCOPY By Prabhat Kumar M.S. Brain & Cognition sciences Submitted to Dr . Sarfraj Ahmad Siddiqui

2. CONFOCAL MICROSCOPY Introduction Principle Components Applications Advantage Disadvantage References

3. Introduction Marvin Minsky made the first confocal microscope in 1957, he was postdoc at Harvard University. Also known as C onfocal laser microscopy . This microscopy basically aim to solve the problem that is in different microscopy techniques that is the lack of resolution. Confocal microscopy making balance between optical microscopy and Marvin Minsky electron microscopy techniques in term of resolution. By using this technique we get better resolution when we comparison other optical microscopy. Uses pinhole to produce high resolution images because its exclude out of the focus light. So images have better contrast and are less hazy.

4. Z-stack : A set of confocal images taken from the specimen so that the image area along the x- and y-axis remains the same but the distance from the objective (z-axis) is different for each image. As the distance between adjacent images is precisely controlled in the z-stack, it can be used to form a 3-dimensional image . A series of thin slices of the specimen are assembled to generate a 3- dimensional image .(Optical Sectioning ) Fluorescence - emission of light by substance that has absorbed light & remission of light stop as soon as incident light is cutoff. It occurs when electrons move from their ground state to an excited state. These electrons keep the same spin as in the ground state, but when they return to the ground state they emit energy. This energy has a longer wavelength than the originally absorbed energy. If this longer wavelength is within the visible spectrum , then we can see a glowing light . Example of fluorescence are :- DAPI, ALEXA , FITC , GFP etc.

5. Principle Similar to the wide field microscope, the confocal microscope uses fluorescence optics. Instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. A pinhole inside the optical pathway cut off signals that are out of focus , thus allowing only the fluorescence signals from the illuminated spot to enter the light detector . 3D objects can be visualized by scanning several optical planes and stacking them using a suitable microscopy software (z-stack ).

6. Components Light Source Laser :- laser light can be chosen via selection device and are matched with fluorophore used in experiment. Pinhole :- A pertures placed near the light source and detector enable the microscope to produce thin optical sections of focal planes in the specimen. Its exclude the out of focus light rays . Dichromic Mirror :- This is a filter which separates the excitation from the emitted light in fluorescence beam path of microscope, also called as beam splitter . Photomultiplier Detector :- Highly sensitive detectors tube used in confocal microscopes to gain an amplified electrical signal from a weak light source.

7. Applications C onfocal microscopy has been adapted into numerous fields such as Cell biology , Life sciences, Semiconductor inspection, M aterials science, Neuroanatomy , and Neurophysiology . Confocal Microscopy is a powerful tool for studying signaling mechanisms. Key applications include : Detail structure of specific part of cell Localizations of proteins. Imaging two or more fluorescence stains. Acquisition of high quality images for presentation.

8. Advantage It is non-invasive (no requirement of much effort ) microscopy techniques. We can do Qualitative as well as quantitative studies . We can do function and dynamic analysis. No requirement of vacuum . E xamine live or fixed cell. We get 3-D image in confocal microscopy. Setup cost and maintenance cost are comparatively very low when we comparison with electron microscopy. Precise and focus. Provides deep analysis of any part of specimen .

9. Disadvantage Damage to specimen in case of live cell. Decrees in Intensity of the incident light. The fluorophore should tagged properly to part of the specimen. Fluorophore should be sensitive enough for the given excitation wave length, so that they can emit the light. Photo bleaching : photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluorescence.

10. Image by Confocal Microscope Fig : An Intestine Section Fig : Kidney cells (fluorescence vs Confocal microscope)

11. References Wilson and Walker's Principles and Techniques of Biochemistry and Molecular  Biology. Confocal Microscopy Denis Semwogerere Eric R. Weeks, Emory University, Atlanta, Georgia, U.S.A. https :// www.olympuslifescience.com/en/microscoperesource/primer/techniques/confocal/glossary/ http://www.physics.emory.edu/faculty/weeks//confocal / https:// ibidi.com/content/216-confocal-microscopy http://microscopy.berkeley.edu/courses/tlm/clsm/index.html