How to Choose the Right Fluorescence Microscope
Fluorescence microscope is a standard microscopic imaging equipment for laboratories and pathology departments. It uses fluorescence characteristics for observation and imaging. It is widely used in cell biology, neurobiology, botany, microbiology, pathology, genetics and other fields. 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.
Choice of Microscope
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), stereoscopic fluorescence microscopes (suitable for larger specimens, such as plants, zebrafish (adult/embryo) , medaka, mouse/rat organs, etc.).
Selection of Fluorescence Filter Cubes
In addition to considering the excitation and emission wavelengths of fluorescent probes, the selection of filter blocks also needs to consider whether there is non-specific excitation and cross-color for multi-color labeled samples. In the experiment, we will try to choose the wavelength closest to the excitation peak for excitation, and the receiving range must include the emission peak. For example, the excitation peak of Alexa Fluor 488 is 500nm, and a 480/40 excitation filter can be selected in a fluorescence microscope. Fluorescence filter cubes commonly used in fluorescence microscopes can be divided into two types: long pass (LP for short) and band pass (BP for short), which also need to be selected according to needs.
Choice of fluorescent light source
Currently commonly used fluorescent light sources include mercury lamps, metal halide lamps, and LED light sources that have developed rapidly in recent years. The spectrum of the fluorescent light source is continuous and discontinuous, and the energy will be different in different bands. Due to its relatively narrow spectral band, more stable energy output, long lifespan, safety and environmental protection and many other advantages, LED light source is gradually becoming the main light source of fluorescence microscope.
confocal microscope
In traditional fluorescence microscope observation, due to the overlapping of fluorescent marker substances and autofluorescence structures, they are closely combined, while the traditional epi-fluorescence microscope objective lens not only collects light from the focal plane, but also collects scattered light above and below the focal plane , resulting in greatly reduced image resolution and contrast.
Confocal imaging solves this problem by only detecting the portion of the light reflected from the focal plane. The light source passes through a pinhole to form a small and fine light spot on the focal plane. The light emitted from the focal plane is collected by the objective lens. Most of the fluorescence emitted from the point above or below the focal plane of the objective lens cannot converge to the small hole. Only the fluorescence located in the focal plane and a small part of the off-focus fluorescence can pass through the pinhole, while the light beams outside the focal plane converge at the front or back of the pinhole plate and are blocked from entering the detector through the pinhole. The detected image is the image from the focal plane, so the final image quality is greatly improved.
Due to the advantages and practicability of the functions of the laser scanning confocal microscope, the confocal microscope is an indispensable experimental assistant in the field of high-precision cell biology, botany and cell research. At the same time, In the future scientific research center, it will be the most basic and core scientific research tool.
