Difference between a fluorescence microscope and a normal microscope
I recently tried to make some frozen sections of mice. Next, I will use a fluorescence microscope to see if the virus I injected is in the brain area I want. Some basic principles of fluorescence microscopy need to be learned briefly, and I will share them here.
Fluorescence microscopes use ultraviolet light as a light source to illuminate the object being inspected, causing the object to emit 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. The fluorescent image of the sample is observed through the magnification of the eyepiece and objective lens. Let's take a look at the difference between a fluorescence microscope and an ordinary optical microscope.
1. Look at the lighting method
The illumination method of fluorescence microscope is generally epi-illumination, which means that the light source is placed on the test sample through the objective lens.
2. Look at the resolution
Fluorescence microscopes use ultraviolet light as a light source, which has a shorter wavelength but higher resolution than ordinary optical microscopes.
3. Differences in filters
Fluorescence microscopes use two special filters, one used in front of the light source to filter out visible light, and one used between the objective lens and the eyepiece to filter out ultraviolet rays, which can protect human eyes.
Fluorescence microscope is also a type of optical microscope. The main reason is that 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 good function of capturing weak light. , so its imaging ability is also good under extremely weak fluorescence. 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 being used.
Knowledge about two-photon fluorescence microscopy
The basic principle of two-photon excitation is: under the condition of high photon density, fluorescent molecules can absorb two long-wavelength photons at the same time, and after a short so-called excited state lifetime, emit a photon with a shorter wavelength. ;The effect is the same as using a photon with a wavelength half the long wavelength to excite fluorescent molecules. Two-photon excitation requires a high photon density. In order not to damage cells, two-photon microscopes use high-energy mode-locked pulse 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 using a high numerical aperture objective lens to focus the photons of the pulsed laser, the photon density at the focus of the objective lens is the highest. Two-photon excitation only occurs at the focus of the objective lens, so the two-photon microscope does not require a confocal pinhole, which improves 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 one fluorescence photon through a radiative transition. This is single-photon fluorescence. For the fluorescence excitation process using laser as the light source, two-photon or even multi-photon fluorescence phenomena may occur. In this case, the intensity of the excitation light source used is high, and the photon density meets the requirement for the fluorescent molecules to absorb two photons at the same time. In the process of using ordinary lasers as excitation light sources, the photon density is still not enough to produce two-photon absorption. Femtosecond pulse lasers are usually used, and their instantaneous power can reach the megawatt level. Therefore, the wavelength of two-photon fluorescence is shorter than the wavelength of the excitation light, which is equivalent to the effect produced by half-excitation wavelength excitation.
