How to choose the appropriate fluorescence microscope
Fluorescence microscope is a standard microscopic imaging equipment in laboratories and pathology departments, which uses fluorescence characteristics for observation and imaging. It is widely used in various fields such as cell biology, neurobiology, botany, microbiology, pathology, genetics, etc. Fluorescence imaging has the advantages of high sensitivity and specificity, making it very suitable for observing the distribution of specific proteins, organelles, etc. in tissues and cells, studying co localization and interactions, tracking life dynamic processes such as ion concentration changes, and so on.
Selection of microscopes
Fluorescence microscopes are mainly divided into three categories: upright fluorescence microscopes (suitable for slicing), inverted fluorescence microscopes (suitable for living cells and also for slicing), and stereo fluorescence microscopes (suitable for larger specimens, such as plants, zebrafish (adult/embryonic), medaka, mouse/rat organs, etc.).
Selection of fluorescent filter blocks
The selection of filter blocks should not only consider the excitation and emission wavelengths of fluorescent probes, but also consider whether there is non-specific excitation and whether there is color crosstalk for multi-color labeled samples. In the experiment, we will choose the wavelength closest to the excitation peak as much as possible for excitation, and the receiving range should include the peak emission. The excitation peak of Alexa Fluor 488 is 500nm, and a 480/40 excitation filter can be selected in a fluorescence microscope. The commonly used fluorescent filter blocks in fluorescence microscopy can be divided into two types: long pass (LP) and band pass (BP), which also need to be selected according to needs.
Confocal microscope
In traditional fluorescence microscopy observations, due to the overlap of fluorescent labeled substances and spontaneous fluorescence structures, they are tightly bound together. However, traditional falling fluorescence microscopy objectives not only collect light from the focal plane, but also scatter light up and down the focal plane, resulting in a significant reduction in image resolution and contrast.
Confocal imaging only detects the light reflected from the self focusing plane, thus solving the above problem. The light source forms a small and fine spot on the focal plane through a pinhole, and the light emitted from the focal plane is collected through the objective lens. The majority of the fluorescence emitted from points above or below the focal plane of the objective lens cannot converge to the pinhole. Only the fluorescence located in the focal plane and a small portion of defocused fluorescence can pass through the pinhole, while the light beam outside the focal plane converges in front or behind the pinhole plate and is blocked from entering the detector through the pinhole. The detected image is from the focal plane, so the final image quality is greatly improved.
Due to the various advantages and practicality of laser scanning confocal microscopy, it is currently an indispensable experimental assistant in high-precision cell biology, botany, and cell research fields. At the same time, in future scientific research centers, it will be the most basic and core research tool.
