Describe in detail the selection steps and methods of fluorescent probes for confocal microscopy
In experiments with confocal microscopes, some experimental indicators can be detected using the autofluorescence of the sample itself or exogenous substances, but most of the substances to be tested do not have measurable fluorescence and need to be labeled with fluorescent probes. It has a measurable fluorescent signal. So, how to choose fluorescent probes for confocal microscopy?
1. Determine the indicators to be tested according to the purpose of the experiment
Once the detection index is determined, the selection range of fluorescent probes is also determined. It can be seen that the choice of which fluorescent probe to use in the experiment is determined by the purpose of the experiment. For example, if the purpose of the experiment is to study the changes of calcium ions in cardiomyocytes when atrial fibrillation occurs, it can be determined that the substance to be tested is calcium ions in cardiomyocytes, and the selection range of fluorescent probes is limited to calcium ion probes that can label living cells. Needle.
2. Determine the range of available fluorescent probes
For new experimental substances or indicators, if the purpose of the experiment is not to explore new fluorescent probes, you can choose to apply mature probes and labeling methods. The type or range of fluorescent probes that can label the substance to be tested can be determined by searching authoritative literature or product catalogs of well-known reagent companies at home and abroad.
3. Investigate whether the characteristics of the fluorescent probe meet the requirements for the preparation of fluorescent samples
The characteristics of fluorescent probes directly affect the labeling of samples and the experimental results. Therefore, before selecting fluorescent probes and labeling samples in the experiment, it is necessary to fully understand their fluorescence characteristics. The characteristics of fluorescent probes to be investigated include:
1. Reaction characteristics of light probes and samples
2. Sensitivity and fluorescence intensity of fluorescent probes
3. Spectral properties of samples labeled with fluorescent probes
4. Stability of fluorescence
5. Interaction between multiple fluorescence in the sample
