Difference Between Fluorescence Microscope and Normal Microscope
1. Look at the lighting method
The illumination method of fluorescence microscope is generally epi-type, that is to say, the light source is placed on the test sample through the objective lens.
2. Look at the resolution
Fluorescence microscopes use ultraviolet light as the light source, with a relatively short wavelength, but the resolution is higher than that of ordinary optical microscopes.
3, the difference in the filter
Fluorescence microscope uses two special filters, used in front of the light source to filter out visible light, and used between the objective lens and eyepiece to filter out ultraviolet light, which can protect human eyes.
Fluorescence microscope is also a type of optical microscope, mainly because the wavelength excited by the fluorescence microscope is short, so this leads to the difference in the structure and use of the fluorescence microscope and the ordinary microscope. Most of the 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 microscopy in recent years, noise has also been greatly reduced. Therefore, more and more fluorescence microscopes are used.
Knowledge of 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 after a very short so-called excited state lifetime, emit a short-wavelength photon ; the effect is the same as using a photon with a wavelength half the long wavelength to excite a fluorescent molecule. Two-photon excitation requires high photon density, and in order not to damage cells, two-photon microscopy uses high-energy mode-locked pulsed lasers. This laser emits laser light with high peak energy and low average energy, with a pulse width of only 100 femtoseconds and a frequency of 80 to 100 MHz. When a high numerical aperture objective lens is used 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 occurs only 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 the general fluorescence phenomenon, 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 the radiation transition, which is called single-photon fluorescence. For the fluorescence excitation process with laser as the light source, two-photon or even multi-photon fluorescence phenomenon may occur. At this time, the intensity of the excitation light source used is high, and the photon density meets the requirement that the fluorescent molecule absorbs 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 two-photon absorption. Usually, a femtosecond pulsed laser is used, and its instantaneous power can reach the order of megawatts. Therefore, the wavelength of the two-photon fluorescence is shorter than the wavelength of the excitation light, which is equivalent to the effect produced by the half-excitation wavelength excitation.
Two-photon fluorescence microscopy has many advantages:
1) Long-wavelength light is less affected by scattering than short-wavelength light and can easily penetrate 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 viewing specimens with two-photon microscopy, photobleaching and phototoxicity are only present at the focal plane. Therefore, two-photon microscopy is more suitable for viewing thick specimens than single-photon microscopy, for viewing living cells, or for performing fixed-point photobleaching experiments.
Knowledge of confocal fluorescence microscopy
The basic principle of confocal fluorescence microscopy: using a point light source to illuminate the specimen, a small light spot with a well-defined outline is formed on the focal plane. constituted of the beam splitter. 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 dimensions of the two are the same, about 100-200 nm; 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 concentrated within the range of the detection hole, while the scattered light from above or below the focal plane is blocked out of the detection hole and cannot be imaged. The sample is scanned point by point with the laser, 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 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, and this optical cross-section always has a certain thickness, also known as an optical slice. Since the light intensity at the focal point is much greater than that at the non-focal point, and the non-focal plane light is filtered out 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 the Observe a two-dimensional optical section at the focused spot of the sample. Combining the X-Y plane (focal plane) scanning with the Z-axis (optical axis) scanning, by accumulating two-dimensional images of successive layers and processing by special 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 focusing plane cannot enter the detection pinhole.
The simple expression of the working principle of confocal laser is that it uses a laser as the light source, and on the basis of traditional fluorescence microscope imaging, a laser scanning device and a conjugate focusing device are attached, and the digital image acquisition and processing system is controlled by a computer.
