Fluorescence microscopy is not totally different from light microscopy since it also employs ultraviolet light, but fairly different since its process of imaging is totally different from light microscope since it employs light emitted from the molecules or atoms after being excited through light, or by mechanical means or also by chemical means. (more…)
The Laser scanning confocal microscope stimulator (LSCM) is perhaps the most significant advance in optical microscopy during the past decade has been the refinement of mainstream laser scanning confocal microscope techniques using improved synthetic fluorescent probes and genetically engineered proteins, a wider spectrum of laser light sources coupled to highly accurate acousto-optic tunable filter control, and the combination of more advanced software packages with modern high-performance computers. (more…)
Here is an article shows the conjugation or attachment of fluorescent proteins, natural or synthetic to the proteins of specimen under study, and observed through fluorescent microscope. It is noteworthy to mention here that in the process of attachment, these proteins can be studied as to its location, its gene, and even the ions, and elements like calcium and others inside the cell can be read. (more…)
The highest intensity of the excitation light is at the focal point of the lens, but nonetheless, the other parts of the sample do get some of this light and they do fluoresce. This contributes to a background haze in the resulting image. Adding a pinhole/screen combination solves this problem. Because the focal point of the objective lens of the microscope forms an image where the pinhole is, these two points are known as conjugate points or alternatively, the sample plane and the pinhole/screen are conjugate planes. The pinhole is conjugate to the focal point of the lens, thus it is a confocal pinhole. (more…)
Before we get into how a confocal microscope works, it is better to refresh you with the terms used in fluorescent microscopy for better understanding and the mechanics by which images are formed in fluorescent microscope. What is then fluorescence? If you shine light on some molecules, you may see light of a different color emitted from those molecules. This is known as fluorescence or luminescence. Those molecules absorb high energy light (blue, for example as reported). This increases the energy of the molecules, and they are called excited molecules. Some of the energy from the blue photon is lost internally. The molecules then emit a photon with less energy, green in this example. Fluorescence is a common dye that acts in exactly this way, emitting green light when hit with blue excitation light. The color of light emitted is material dependent, and likewise the excitation light wavelength depends on the material. There are other forms of inelastic scattering; fluorescence is particularly strong. (more…)