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Po-Kang Lin, Chieh-Hsiung Kuan; Focused ion beam sectioning retinal pigment epithelium cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1485.
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© ARVO (1962-2015); The Authors (2016-present)
In the biological field, transmission electronic microscopy (TEM) plays an important role on exploring the ultrafine structure of the cross-section of biological samples. Instead, the role of SEM is limited to observe the sample surface only. In the semiconductor industry, both focused ion beam (FIB) and scanning electron microscopy (SEM) have been widely used in the semiconductor industry. According the previous literatures, the attempts to apply the FIB/SEM dual system to mill/section and observe biological samples, however, usually resulted in poor cross section image. We plan to overcome the problem, to improve the image quality in terms of resolution and contrast for further biological investigation.
We developed a set of sample holders, each with different semiconductor substrate and pattern, through microprocessing such as e-beam lithography and PECVD. We utilized retinal pigment epithelium cells ARPE-19 as the milling targets. The cultured ARPE’s were fixed with glutaraldehyde and post-fixed with OsO4 (O-T-O-T-O method). Then the specimen underwent CPD procedure separately. The ARPE-19 cells were subsequently milled with FEI nova-600i FIB/SEM system with Gallium ion beam as the source in different parameters. The cross-section of the ARPE-19 cells was viewed by SEM and photographed.
With the sample holders and the milling methods we developed, the ultrafine structures of ARPE-19 cells could be observed in detail. The SEM image quality SEM from the cross-section of the milled samples approximated the TEM image of other biological samples.
For the first time, FIB/SEM system plays a role in the biological field, as exploring the ultrafine structure of the cross-section of ARPE-19 cells by FIB milling.
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