CLSM is a scanning imaging technique in which the resolution obtained is best explained by comparing it with another scanning technique like that of the scanning electron microscope (SEM). Do not confuse CLSM with phonograph-like imaging—AFM or STM, for example, where the image is obtained by scanning with an atomic tip over a surface.
In CLSM a fluorescent specimen is illuminated by a point laser source, and each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning volume is determined by the spot size (close to diffraction limit) of the optical system because the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the numerical aperture of the system's objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using wide-field illumination. However, with confocal microscopy it is even possible to improve on the resolution limit of wide-field illumination techniques because the confocal aperture can be closed down to eliminate higher orders of the diffraction pattern. For example, if the pinhole diameter is set to 1 Airy unit then only the first order of the diffraction pattern makes it through the aperture to the detector while the higher orders are blocked, thus improving resolution at the cost of a slight decrease in brightness. In practice, the resolution limit of confocal microscopy is often limited by the signal to noise ratio caused by the small number of photons typically available in fluorescence microscopy. One can compensate for this effect by using more sensitive photodetectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination later risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required."
In conclusion, it's all about the pinhole baby.
And that is this week's science blag. Stay tuned next week where we wil discuss the most heated question in science: Was T. rex primarily a hunter or a scavenger?
Peace.
The dogmas of the quiet past are inadequate to the stormy present. The occasion is piled high with difficulty, and we must rise with the occasion. As our case is new, so we must think anew, and act anew. We must disenthrall ourselves, and then we shall save our country.
Readers underscore value of life(Authors names omitted)
We have a deep concern about the upcoming presidential election. It is a critical election in the history of our nation.
The most important issue when choosing a candidate is their character and morality. Both major presidential candidates have given their stance on abortion and homosexuality, and they are at opposite sides. If we allow human life to be devalued and destroyed in the womb, and if we allow sexual perversion to redefine marriage, then all other issues will eventually cease to matter as society crumbles and falls. We must refuse to vote for a person or political party that endorses abortion and/or the giving of protected (or even privileged) status to homosexuality. This is more important than the economy, taxes, healthcare, social security, immigration and even national defense.
We urge you to pray for our nation and this important election.
