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[Observation of the free calcium of lens epithelial cell using laser scanning confocal microscopy]
Objective:
Using a new fluorescent Ca++ indicator (fluo-3) and laser scanning confocal microscopy (LSCM) to monitor the intracellular Ca++ of rat lens epithelial cell (LEC).
Methods:
The LEC was loaded with fluo-3, then the distribution of free Ca++ in the LEC was observed by using LSCM monitoring the distribution of fluorescence that is given due to the chelation between fluo-3 and Ca++. At last, ionophore (A23187) and heavy-metal ions (Mn++) were applied to calibrate fluo-3 intensities to obtain [Ca++]i value.
Results:
The free calcium in LEC is mainly located in the nucleus. Free calcium concentration of rat LEC is 259.79 +/- 49.24 nmol/L.
Conclusion:
Fluo-3 can be loaded into LEC by incubation with acetoxymethyl (AM) ester of the dye, and LSCM can be used to observe the cellular free calcium directly by surveying the fluorescence. This method will pave a way technically for the further study of intracellular free Ca++ of the LEC.