Characteristics and Methods of Fluorescence Detection


Fluorescence Detection

Fluorescence detection is a natural luminescence reaction. Through the reaction of luciferase and ATP, it can detect human cells, bacteria, molds, and food residues, and get the reaction results within 15 seconds. Illuminance is measured by special equipment and expressed in digital form. It was first used in the food industry in 1975 and in the cosmetics manufacturing industry in 1985.

Main disadvantages:

The main disadvantage of fluorescent detection is that only a few compounds fluoresce. Most molecules do not fluoresce, but contain derivatizable functional groups for the synthesis of fluorescent derivatives. For example, o-phthalaldehyde is a commonly used fluorescent group for post-column derivatization of amino acids. Although fluorescence detection is sensitive, such high sensitivity is not required for common sample analysis. Because the response of ELSD is fluorophore-independent, derivatization is not required, thus greatly reducing sample pretreatment and analysis time.

Basic Features:

Fluorescence quantification: The international mainstream fluorescence quantitative PCR technology is adopted to rapidly improve the technical level.
The results are stable: the CV value of the test results is close to that of the imported automatic PCR instrument, and it has the function of self-checking to avoid the output of wrong data.
Closed operation: All detection processes are closed-tube operation. Fluorescence is detected at the reaction tube, which can effectively prevent contamination and solve the most difficult problem in PCR technology.
Accurate quantification: meet the clinical needs for quantification, with a wide quantitative range, covering most pathogenic microorganisms, and automatic curve fitting.
High sensitivity: Take advantage of spectral signal sensitivity to effectively improve detection sensitivity.
Safety: The whole process of non-toxic testing.
Easy to operate: save the tedious process of post-processing.
Direct Print: Print the results directly without recording a large amount of data.
Upgraded version: FS1000 has a data transmission port connected to the computer, which can be connected to the computer, and provides advanced analysis software according to user needs to comprehensively analyze experimental data. (Computer and printer options)
Low failure rate: maintenance is very simple.
Saving resources: The existing PCR amplification instrument can be used to save resources to the greatest extent.


The advantage is that the speed is fast, which is very suitable for large-scale statistics, and the sample is only tested once, so there is no need to worry about the problem of fluorescence quenching. The disadvantage is that it can only detect the presence or absence and intensity of fluorescence, cannot provide spatial positioning information, and cannot do experiments on changes in fluorescence positioning; since the sample is only detected once, it is impossible to continuously observe the same sample. For example, when doing membrane protein localization, you will get the result that you can detect the signal with flow cytometry, but you can’t see anything with the microscope, excluding the problem of instrument and filter selection, it is likely to be nuclear autofluorescence or non-specific Flagging causes false positive results for flow.

fluorescence microscope
It is just complementary to flow cytometry, and can be used for spatial observation to determine the localization of target proteins, but it is not suitable for large flow detection, and it is estimated that no one can stand the test of time. There are objective lenses with different magnifications to choose from. You can use a high-magnification objective lens to see fine structures, or use a small-magnification objective lens to do a small amount of statistics. The CCD and software matched with it are the key to the performance of the system. Especially CCD, it is not easy to choose a suitable one from so many products, you need to know a lot of knowledge, otherwise you can only listen to others.

The upgraded product of fluorescence microscope has good optical slice effect and can obtain good three-dimensional positioning information. However, it would be a shame to use confocals for the sole purpose of taking a nice photo of your sample and getting a nice picture. Confocals are basically fully automatic systems, which can freely define illumination areas, select multiple fluorescence channels, and set timing acquisitions. Therefore, doing Time-laps, FRAP and FRET has its own unique advantages. In addition, 4Pi and STED are the best among them, with ultra-high spatial resolution, but they are inconvenient to use and may not be suitable for biological experiments.

total internal reflection
Another upgraded product of fluorescence microscope, which belongs to the category of near-field optics, is only suitable and most suitable for membrane research, and cannot see intracellular information. CCD is also used for imaging, but the requirements for CCD are higher – I even feel that there is no upper limit to the requirements for CCD.

Another type of confocal, because it uses long-wavelength excitation fluorescence, can perform deep detection (breaking the 100-micron limit of confocal), and the most typical application is to observe living brain tissue. It is extremely expensive, there are only a few in China, and there are not many people who can use it – I won’t say it.

High Content Drug Screening System
The hybrid of flow and microscope, using a motorized stage, can automatically complete the experiment according to the predetermined program, can do large flow detection (although the speed is slower), and has the imaging ability, which can determine the positioning.