Measuring the Interactions between Peroxisomes and Chloroplasts by in situ Laser Analysis

Abstract

Quantitative analysis has been necessary for deeply understanding characteristic of organelles function. This is the detailed protocol for the quantification of the physical interaction between peroxisomes and chloroplasts taken by laser scanning microscopy described by Oikawa et al. (2015). To clarify the morphological interactions between both organelles, we measured the contact length between two organelles (interaction length) in the fluorescent microscope image by using image analysis software ImageJ. The result clearly revealed that the contact length in light condition is much longer than that in dark condition. In addition, the force of the morphological interaction was quantified utilizing intersection technology of femtosecond laser and atomic force microscope (AFM). When an intense femtosecond laser is focused near the interface of two organelles, the adhesion is broken by a force due to the laser. The adhesion strength in light and dark conditions was estimated from the force calibrated by AFM. The detailed procedure is described in Bio-protocol as another protocol entitled “Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology” (Hosokawa et al., 2016). These methods can be applied to other physical interaction between different types of organelles such as nuclei, mitochondria, Golgi, and chloroplasts.