How fluorescence microscopy differs from conventional microscopy
I recently tried to make some frozen sections of mice, and now I need to use a fluorescence microscope to see if the virus I injected is in the desired brain area. Some basic principles of fluorescence microscopy need to be studied briefly, and I will also share them here.
Fluorescence microscope uses ultraviolet light as a light source to illuminate the object being tested, causing the object to emit a light source, and then observe the object under the microscope. Mainly used for immunofluorescence cells, it is composed of a light source, filter plate system, and optical system to observe the fluorescence image of the sample through magnification of the eyepiece and objective lens. Let's take a look at the difference between this fluorescence microscope and a regular optical microscope.
1. In terms of lighting methods
The lighting method of a fluorescence microscope is generally using a falling beam method, which means that the light source is projected onto the test sample through the objective lens.
2. In terms of resolution
Fluorescence microscopy uses ultraviolet light as the light source, with a relatively short wavelength but higher resolution than ordinary optical microscopes.
3. Differences on the filter
A fluorescence microscope uses two special filters, one in front of the light source to filter out visible light, and the other between the objective and eyepiece to filter out ultraviolet light, which can protect the eyes.
Fluorescence microscopy is also a type of optical microscope, mainly because the wavelength excited by fluorescence microscopy is short, which leads to differences in the structure and use of fluorescence microscopy and ordinary microscopy. Most fluorescence microscopes have a good function of capturing weak light, so their imaging ability is also good under extremely weak fluorescence. In addition, with the continuous improvement of fluorescence microscopy in recent years, the noise has also been significantly reduced. Therefore, more and more fluorescence microscopes are being applied.
Knowledge related to two-photon fluorescence microscopy
The basic principle of two-photon excitation is that at high photon density, fluorescent molecules can simultaneously absorb two long wavelength photons, and after a short period of so-called excited state lifetime, emit a shorter wavelength photon; Its effect is the same as using a photon with a wavelength of half the long wavelength to excite fluorescent molecules. Two photon excitation requires a high photon density, and to avoid damaging cells, a high-energy mode-locked pulse laser is used in a two-photon microscope. The laser emitted by this type of laser has high peak energy and low average energy, with a pulse width of only 100 femtoseconds and a frequency of up to 80 to 100 megahertz. When using a high numerical aperture objective to focus the photons of a pulsed laser, the photon density at the focal point of the objective is the highest, and two-photon excitation only occurs at the focal point of the objective. Therefore, a two-photon microscope does not require confocal pinholes, which improves the efficiency of fluorescence detection.
In general fluorescence phenomena, due to the low photon density of excitation, a fluorescent molecule can only absorb one photon at the same time, and then emit one fluorescent photon through radiation transition, which is called single photon fluorescence. For the fluorescence excitation process using laser as the light source, two-photon or even multiphoton fluorescence phenomena may occur. At this time, the excitation light source used is of high intensity, and the photon density meets the requirement for fluorescent molecules to absorb two photons simultaneously. In the process of using a typical laser as the excitation light source, the photon density is still not sufficient to generate two-photon absorption phenomenon. Typically, femtosecond pulse lasers are used, and their instantaneous power can reach the level of megawatts. Therefore, the wavelength of two-photon fluorescence is shorter than that of excitation, which is equivalent to the effect produced by half excitation wavelength excitation.
