There are many advantages to two-photon fluorescence microscopy
1) Long-wavelength light is less affected by scattering than short-wavelength light and can easily penetrate the specimen;
2) Fluorescent molecules outside the focal plane are not excited, allowing more excitation light to reach the focal plane, allowing the excitation light to penetrate deeper specimens;
3) Long-wavelength near-infrared light is less toxic to cells than short-wavelength light;
4) When observing specimens using a two-photon microscope, photobleaching and phototoxicity occur only on the focal plane. Therefore, two-photon microscopes are more suitable than single-photon microscopes for observing thick specimens, for observing living cells, or for performing fixed-point photobleaching experiments.
Knowledge about confocal fluorescence microscopy
The basic principle of confocal fluorescence microscopy: Use a point light source to illuminate the specimen to form a small, well-defined light spot on the focal plane. The fluorescence emitted by the spot after being illuminated is collected by the objective lens and returned to the dichroic mirror along the original illumination light path. constitute a beam splitter. The spectrometer sends the fluorescence directly to the detector. There is a pinhole in front of the light source and the detector, which are called the illumination pinhole and the detection pinhole respectively. The geometric dimensions of the two are consistent, about 100-200nm; relative to the light point on the focal plane, the two are conjugate, that is, the light point passes through a series of lenses and can ultimately be focused on the illumination pinhole and the detection pinhole at the same time. In this way, the light from the focal plane can be concentrated within the detection hole, while the scattered light from above or below the focal plane is blocked outside the detection hole and cannot be imaged. The sample is scanned point by point with the laser, and the photomultiplier tube behind the detection pinhole also obtains the confocal image of the corresponding light point point by point, which is converted into a digital signal and transmitted to the computer, and is finally aggregated on the screen into a clear confocal image of the entire focal plane. .
Each focal plane image is actually an optical cross-section of the specimen. This optical cross-section always has a certain thickness and is also called an optical thin section. Since the light intensity at the focus is much greater than the light intensity at the non-focus, and the non-focal plane light is filtered by the pinhole, the depth of field of the confocal system is approximately zero. Scanning along the Z-axis direction can realize optical tomography, forming the desired Observe the two-dimensional optical section at the focused spot of the sample. By combining X-Y plane (focal plane) scanning with Z-axis (optical axis) scanning, by accumulating continuous levels of two-dimensional images and processing them with specialized computer software, a three-dimensional image of the sample can be obtained.
That is, the detection pinhole and the light source pinhole are always focused on the same point, so that the fluorescence excited outside the focus plane cannot enter the detection pinhole.
The simple expression of the working principle of laser confocal is that it uses laser as the light source. On the basis of traditional fluorescence microscope imaging, a laser scanning device and a conjugate focusing device are added, and the system is controlled by a computer to collect and process digital images.
