What is the Working Principle of Confocal Microscopy? and
What is the Advantage of Confocal microscope over the Normal Microscope?
If I am exciting one sample with high intense laser beam (514nm) using Normal microscope set up having an objective 100X with a N.A of ~1 . If I am doing the same experiment with same objective but using the help of Confocal Microscope, then what will be the advantage over Normal microscopy? or How it will differ from the Normal microscopy???
The clue lies in the "confocality" of this probing technique. It allows you to collect signal from a chosen region of interest while as a depth of focus on the sample can also be adjusted depending on the level of advancement of your confocal microsocope.
Thank you Dr. Zubair Buch for your answer..
But How we can improve the confocality of microscope...What are the parameters affects the confocality of the microscope??
or How we can convert a Normal microscope to a confocal one?
Dear A K Sivadasan, I am a PhD student so i would be grateful for not being called a Doctor. Regarding the confocality, we enable the detected signal to pass through a pinhole placed in the detection path which eliminates the out of focus signal from the sample. If you want to know about the optics parameters of some of the state of art confocal microscopes please refer to www.witec.de.
Sadasivan,
Following link might be of use to you on understanding basic concepts of confocal microscopy
http://www.microscopyu.com/articles/confocal/index.html
Good luck,
Rajesh
Dear Dr. Sandra Cristina Raimundo
thanks for your detailed description of confocal microscopy...
I enquired about confocal microscopy for understanding How we can focus exactly the different layers of a multilayer thin film and taking the Raman spectra for each specific layers... I may not be interested on imaging....!!
anyway thanks for your valuable feedback...!
Dear A.K. There are several advantages and disadvantages to confocal light microscopy, when compared to more conventional forms of LM, and other forms of microscopy, or imaging. The 2 main advantages to confocal are the elimination of out-of-focus light, and the ability to do serial, optical sections (including reconstructing those serial sections).
The confocal system uses laser light (usually monochromatic or close to it) to image, which eliminates chromatic aberations (not all wavelengths of light focusing at the same cross-over point.
The confocal system allows one to access focal plains within a sample, which will be in full focus. Any plain outside of that one in-focus plain are eliminated in the image (being out-of-focus). One can choose just about any plain within the sample, depending upon where the object of interest is.
One can also do serial "optical" sectioning, meaning one can focus on and image connecting plains within the sample. This is interesting from an imaging point of view in itself, but using appropriate software allows one to reconstruct all of those serial optical sections into a unified single image, which can be rotated, or tumbled in any direction in 3-D computer screen space. One can also expose the internal structure of this intact reconstruction by optically slicing or splitting the 3-D object in any direction including the sectional direction, thus opening the opject and exposing the morphology where ever one chooses to make the split.
One of the main limits of confocal is that of sample size, which is usually very small. The system was origionally designed for biological application, but both food and material applications are also being done. Other methods can be employed to do related imaging techniques, which do not have the sample size limits associated with confocal, such as Computer-assisted x-ray tomography (or CT).
Hope this helps...
Var
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