In: Physics
'' Given a fixed fluorescently labeled sample with a thickness of 80 mm which technique would you use to obtain in depth images with the best resolution? Describe the basic principles of the technique''
Please answer well so I can understand cause im not good at this topic. thank you.
For a fluorescent labelled sample, the fluorescent light microscopy is the most suited one since it has far better resolution than optical microscopy. The working principle of fluorescent light microscopy is: the fluorescent labelled sample are excited by high intensity light source. This incident light excites the electron in the sample to higher energy level. When this excited electron comes back to ground state,it emits photons. The emitted photon and the excitation photon are of different frequency because of stokes shift. Generally the emitted photons has lower frequency compared to excitation photons. Using the filter which can pass only the emitted photons such as low pass filter if the emitted photon has lower frequency, the emitted photons are collected for further investigation.
More specifically,the Confocal fluorescent microscopy is used to obtained the 3D image of sample. It works in the same principle as explained above. It is done by sources like laser which can focus in very small region of the sample. The focusing of the laser in the sample is changed level by level in the depth of the sample. For example, at first the laser in focused in 1mm inside the sample ,second time laser will be focused on 2mm and then 3mm so on. Once the entire thickness (80mm in this case) is covered, then this multilevel image data is then processed computationally to get the depth image.
For translucent material, some part of the incident light are reflected back and some part will help to generate the fluorescence. By filtering this reflected and the fluorescence light, the sample can be imaged.
The main advantage of fluorescent light microscopy compared to optical microscopy is that the resolution of the fluorescent light microscopy is much better. For example, Rhodamine B excited at 509 nm and with objective numerical aperture of 1 NA ,the optical microscopy gives the resolution of 254nm whereas the fluorescent light microscopy gives the resolution of around 30nm.