To ensure different biochemical reactions take place simultaneously, eukaryotic cells have evolved several membrane-bounded organelles, which coordinate their functions through proteins involved in signal transduction, vesicle trafficking, and membrane interactions [1]. Therefore, determining the subcellular localization of a protein is an essential step toward understanding its functions and activities. To do this, people normally co-express genes of interest with a fluorescent organelle marker using the N. Benthamiana (Nicotiana benthamiana) transient expression system and image it with confocal microscopy[2]. This method is commonly used in horticulture research, where generating stable transgenic plants is still challenging for most species. Although there are a few common standards for this operation, such as signals should not be over-saturated, controls should be performed to eliminate the possibility of signal cross-talk between multiple channels (bleed-through), and the microscope settings (e.g. laser output, pinhole) should be kept identical when comparing two independent experiments, things may still go wrong in some circumstances. Here, we have highlighted a few examples of inappropriate image acquisition that happened in various previous studies and provide our reasonings and solutions to these issues.

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