What are the advantages of confocal laser microscopy
Compared with traditional wide-field microscopes, laser scanning confocal microscopy has the characteristics of high definition, high resolution, and high sensitivity. It is widely used in the field of biomedicine and has become an important imaging tool in this field. Why can confocal get so much love, and what advantages does it have? This article lists three aspects to discuss the advantages of confocal.
Only collect the signal on the focal plane, making the image clear from blur
So how does confocal microscopy achieve this advantage? This is due to the key confocal hardware—the pinhole, as shown in Figure 2. It is a small hole in front of the detector and placed on the optical plane conjugate to the focal plane of the sample. When the excitation light irradiates the sample, both the focal plane and the non-focal plane of the sample area will be excited to generate fluorescence signals, adjust the appropriate pinhole size (usually 1 Au), so that the fluorescence signal on the focal plane (green dotted line) can be Fluorescent signals (red and blue dashed lines) that pass through the pinhole to reach the detector out of the focal plane cannot reach the detector through the pinhole. Through the addition of hardware pinholes, the confocal can obtain clear and high-quality images without changing the method of making ordinary fluorescence microscopes.
Due to the existence of the pinhole, we can only acquire the signal on the focal plane and obtain a high-resolution two-dimensional image. By continuously adjusting the Z-axis position, two-dimensional images at different Z-axis positions can be obtained, and a series of continuous "optical slice" images can be obtained. After obtaining these optical slices, the 3D module equipped with the instrument software can be used to reconstruct the 3D image to construct a clear 3D image. This non-destructive, continuous collection of optical slice images realizes the function of "cell CT".
The accuracy of colocalization results is closely related to the resolution of the image, so we need to choose a higher resolution imaging method when performing colocalization experiments. Compared with traditional microscopes, confocal microscopy has become a powerful tool for colocalization research due to its high-resolution advantages.
Due to the existence of the diffraction limit, the conventional confocal optical resolution is around 200nm. When we need to observe structures larger than 200nm, confocal can accurately determine the degree of colocalization. If colocalization analysis is required, the colocalization analysis module equipped in the confocal software can be selected.
