Table 1 The problems and solution methods during the protocol
Number | Problem | Possible reason | Solution |
|---|---|---|---|
1 | A lack of good contiguous microsections | An inappropriate microtome was used | A suitable microtome should be selected based on the characteristics of the sample |
2 | Intensity is too low | Laser intensity is too low | Make sure the intensity of the laser at the sample position is correct; increase laser power accordingly |
| Â | Exposure time is insufficient | Increase the exposure time | |
| Â | Samples are overtreated and not suitable for imaging | Prepare the samples carefully according to step 1 | |
3 | Poor spectral resolution of the CRS spectrum | The sections of the sample are too thick, or the slit is too wide | Make sure the sections are ≤ 10 μm thick and that the slit opening is ≥ 50 μm |
4 | Too much fluorescence of background | Coverslips are dirty, there are bubbles, or the samples are damaged | Use new coverslips or prepare a new sample for imaging |
5 | Other fluorescence interference | Chloroplast auto-fluorescence | The sections of the sample can be immersed in 75% ethanol before spreading onto the slide |
6 | Sample burns | The sections of the sample were embedded in LR White resin or laser intensity is too high | Both herbaceous and woody samples should be prepared from fresh or dry plant material but not embedded The laser intensity should be sufficiently high to obtain good counts per molecule (CPM), to ensure a good signal-to-noise ratio, but also sufficiently low to avoid photobleaching |