Difference Between Ordinary Microscope and Fluorescence Microscope
Fluorescence microscopes use ultraviolet light as a light source to irradiate the object to be inspected, so that the object emits light, and then observe the object under the microscope. It is mainly used for immunofluorescence cells. It is mainly composed of a light source, a filter plate system and an optical system to observe the fluorescent image of the sample through the magnification of the eyepiece and objective lens. Let's take a look at the difference between this fluorescence microscope and an ordinary optical microscope.
1. In terms of lighting methods
The illumination method of the fluorescence microscope is generally episcopic, that is to say, the light source is projected on the test sample through the objective lens.
2. In terms of resolution
Fluorescence microscopes use ultraviolet light as the light source. The wavelength is relatively short, but the resolution is higher than that of ordinary optical microscopes.
3. The difference in the filter
The fluorescence microscope uses two special filters, which are used in front of the light source to filter out visible light, and used between the objective lens and the eyepiece to filter out ultraviolet light, which can protect human eyes.
Fluorescence microscope is also a kind of optical microscope, mainly because the wavelength excited by fluorescence microscope is short, so this leads to the difference in structure and use between fluorescence microscope and ordinary microscope. Most fluorescence microscopes have a good function of capturing weak light , so under extremely weak fluorescence, its imaging ability is also good. Coupled with the continuous improvement of fluorescence microscopes in recent years, the noise has also been greatly reduced. Therefore more and more fluorescence microscopes are used.
Knowledge about two-photon fluorescence microscopy
The basic principle of two-photon excitation is: in the case of high photon density, fluorescent molecules can absorb two long-wavelength photons at the same time, and emit a shorter-wavelength photon after a short period of so-called excited state lifetime. ; the effect is the same as using a photon whose wavelength is half the long wavelength to excite a fluorescent molecule. Two-photon excitation requires a high photon density. In order not to damage cells, two-photon microscopy uses high-energy mode-locked pulsed lasers. The laser emitted by this laser has high peak energy and low average energy, its pulse width is only 100 femtoseconds, and its frequency can reach 80 to 100 megahertz. When using a high numerical aperture objective lens to focus the photons of the pulsed laser, the photon density at the focal point of the objective lens is the highest, and the two-photon excitation only occurs at the focal point of the objective lens, so the two-photon microscope does not need a confocal pinhole, which improves the Fluorescence detection efficiency.
In general fluorescence phenomena, due to the low photon density of the excitation light, a fluorescent molecule can only absorb one photon at the same time, and then emit a fluorescent photon through a radiative transition, which is single-photon fluorescence. For the fluorescence excitation process using laser as the light source, two-photon or even multi-photon fluorescence may occur. At this time, the intensity of the excitation light source used is high, and the photon density meets the requirements of fluorescent molecules absorbing two photons at the same time. In the process of using a general laser as the excitation light source, the photon density is still not enough to produce the two-photon absorption phenomenon. Usually, a femtosecond pulsed laser is used, and its instantaneous power can reach the order of megawatts. Therefore, the wavelength of two-photon fluorescence is shorter than the wavelength of excitation light, which is equivalent to the effect produced by excitation at half the excitation wavelength.
Two-photon fluorescence microscopy has many advantages:
1) Long-wavelength light is less affected by scattering than short-wavelength light and easily penetrates the specimen;
2) The fluorescent molecules outside the focal plane are not excited, so that more excitation light can reach the focal plane, so that the excitation light can penetrate deeper specimens;
3) Long-wavelength near-infrared light is less toxic to cells than short-wavelength light;
4) When using a two-photon microscope to observe specimens, photobleaching and phototoxicity occur only in the focal plane. Therefore, two-photon microscopy is more suitable than single-photon microscopy for observing thick specimens, for observing living cells, or for spot photobleaching experiments.
Knowledge about confocal fluorescence microscopy
The basic principle of confocal fluorescence microscopy: a point light source is used to irradiate the specimen, and a well-defined small light spot is formed on the focal plane. composed of splitters. The beam splitter sends the fluorescence directly to the detector. There is a pinhole in front of the light source and the detector, respectively called the illumination pinhole and the detection pinhole. The geometric size of the two is the same, about 100-200nm; relative to the light spot on the focal plane, the two are conjugate, that is, the light spot passes through a series of lenses, and finally can 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 converged within the scope of 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 laser scans the sample point by point, and the photomultiplier tube after detecting the 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 finally aggregated into a clear confocal image of the entire focal plane on the screen .
Each focal plane image is actually an optical cross-section of the specimen. This optical cross-section always has a certain thickness, also known as an optical thin section. Since the light intensity at the focal point is much greater than that at the non-focus point, and the non-focal plane light is filtered by the pinhole, the depth of field of the confocal system is approximately zero, and scanning along the Z-axis can realize optical tomography, forming a Observe the two-dimensional optical section at the focused spot of the sample. Combining X-Y plane (focal plane) scanning with Z-axis (optical axis) scanning, the three-dimensional image of the sample can be obtained by accumulating two-dimensional images of continuous layers and processed by special computer software.
That is, the detection pinhole and the light source pinhole are always focused on the same point, so that the fluorescence excited outside the focal 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, and on the basis of traditional fluorescence microscope imaging, adds a laser scanning device and a conjugate focusing device, and is a system for digital image acquisition and processing through computer control.
