Fluorescence observation of optical microscope
Fluorescence refers to the process in which a fluorescent substance emits light with a longer wavelength almost simultaneously when it is irradiated with light of a specific wavelength (Figure 1). When light of a specific wavelength (excitation wavelength) strikes a molecule, such as those in a fluorophore, the photon energy is absorbed by the molecule's electrons. Next, the electrons transition from the ground state (S0) to a higher energy level, the excited state (S1'). This process is called excitation①. The electron stays in the excited state for 10-9–10-8 seconds, during which the electron loses some energy②. During the process of electrons leaving the excited state (S1) and returning to the ground state③, the remaining energy absorbed during the excitation process is released.
Fluorescent Jablonski Diagram
The residence time of the fluorescent molecule in the excited state is the fluorescence lifetime, which is generally on the nanosecond level, and is an inherent characteristic of the fluorescent molecule itself. Fluorescence Lifetime Imaging (FLIM), which uses fluorescence lifetime imaging technology, can perform more in-depth functional measurements in addition to fluorescence intensity imaging, and obtain molecular conformation, intermolecular interactions, and the microenvironment of molecules, etc. Information that is difficult to obtain with conventional optical imaging.
Another important property of fluorescence is the Stokes shift, the wavelength difference between the excitation and emission peaks (Figure 2). Typically the emission wavelength is longer than the excitation wavelength. This is because electrons will lose part of their energy through the relaxation process after the fluorescent substance is excited and before releasing photons. Fluorescent substances with larger Stokes shifts are easier to observe under a fluorescence microscope.
fluorescence microscope
Fluorescence microscope is an optical microscope that uses fluorescence properties for observation and imaging, and is widely used in various fields such as cell biology, neurobiology, botany, microbiology, pathology, and genetics. Fluorescence imaging has the advantages of high sensitivity and high specificity, and is very suitable for the observation of the distribution of specific proteins and organelles in tissues and cells, the study of colocalization and interaction, the tracking of life dynamic processes such as ion concentration changes, etc.
Most molecules in cells do not fluoresce, and if you want to see them, you label them fluorescently. There are many methods of fluorescent labeling, such as direct labeling (such as using DAPI to label DNA), or immunostaining using the antigen-binding properties of antibodies, or using fluorescent proteins (such as GFP, green fluorescent protein) to label target proteins, and reversible binding. synthetic dyes (such as Fura-2), etc.
Inverted Fluorescence Microscope MF53-N
At present, the fluorescence microscope has become the standard imaging equipment of various laboratories and imaging platforms, and is a good helper for our daily experiments. Fluorescence microscopes are mainly divided into three categories: upright fluorescence microscopes (suitable for sectioning), inverted fluorescence microscopes (suitable for living cells, taking into account sectioning), fluorescent stereoscopes (suitable for larger specimens, such as plants, zebrafish (adult/embryo) , medaka, mouse/rat organs, etc.).
Fluorescence microscopy imaging technology is widely used and rich in types, and new technologies are still emerging. You can choose the technology to complete your own research.
