This is the detailed protocol to quantify adhesion strength between peroxisomes and chloroplasts in plant leaf palisade mesophyll cells described by Oikawa et al. (2015). The quantification was performed by utilizing local explosion induced by focusing femtosecond laser pulses into a mesophyll cell under a confocal microscope. When an impulsive force generated by an explosion is loaded on the interface between a peroxisome and a chloroplast, the peroxisome is frequently detached from the chloroplast. The probability of a peroxisome detaching from a chloroplast was estimated (left-top of Figure 1). Next, the magnitude of the impulsive force was quantified by an atomic force microscope (AFM) cantilever (right-top of Figure 1). On the basis of these results, the pressure to break adhesion between a peroxisome and a chloroplast was quantified as an index of the adhesion strength (bottom of Figure 1). In this protocol, these procedures are summarized. As the local explosion is induced not only in the medium of the mesophyll cells but also in aqueous medium generally, this method could be applied to various adhesions between organelles and between cells around 1 to 100 μm in diameter (e.g., adhesions between mitochondria and chloroplasts, between nucleus and cell membrane, and between two cells with weak physical interaction). Additionally, we have evaluated the interaction between peroxisomes and chloroplasts from the interaction length between two organelles. This protocol has been presented in Bio-protocol as “Measuring the interactions between peroxisomes and chloroplasts by in situ laser analysis” (Oikawa et al., 2015).
Figure 1. Flow chart estimating adhesion strength between a peroxisome and a chloroplast by utilizing femtosecond laser and atomic force microscope
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