Analysis of Acanthamoeba cysts isolated from contact lenses with the Raman spectroscopy microscope

Abstract

Purpose: : The Raman spectroscopy confirms molecular identity and allows analysis of geometric arrangements of molecules distinguishing between crystalline structures. The Microscope associating Raman spectra analysis is able to align automatically the images with the laser beams and makes possible time and space integration of Raman emission and visual beam paths. The goal of this study was to assess the Raman spectra obtained from clinical isolates of Acanthamoeba cysts.

Methods: : Acanthamoeba cysts isolated from the cornea of contact lens users (genotype T4) were suspended in saline, smeared on routine microbiology diagnosis glass-slides and air dried. Spectrum analysis was performed with the DXR Raman Microscope with alignment of the z-axis resolution of 2µm (ThermoFisher, Cortaboeuf, France). The signal was emitted by a calibrated laser (532 nm; 10 mW) and detected with a Silicon camera device integrated to the microscope (2 exposures; resolution:1.9285; 900 lines/mm; 3475 points; X Raman shift cm-1 from 50.2753 to 3400.0222; exposure time 57 sec; 512 background exposures;). Automatic fluorescence correction did compensate for fluorescence prior to data analysis and autofocus optimized Raman signal from sample while moving the z-axis.

Results: : The molecular Raman spectra analysis of the double wall of Acanthamoeba cultured in saline consistently shows Silicate complexes. When the z axis of the microscope was fixed on the surface, this pattern was found for all the cyst-walls (20 tested). The spectra were significantly different for signal obtained 2µm deeper or for Acanthamoeba suspended for 48 hours (and washed 3 times) in distilled water. The inorganic signals obtained from Acanthamoeba cyst walls correspond to ortho-silicates from clay and are consistently different from silicate spectra obtained from the glass-slide controls.

Conclusions: : During encystment, the cytoskeleton is rearranged and actin and cellulose synthesis is enhanced. According to these results it appears that Acanthamoeba trap (or synthesize) clay-related silicate compounds. These siliceous crystalline complexes assessed by Raman spectroscopy may be involved in the resistance of cysts to osmotic stress, disinfectants, chemicals and inappropriate environment.

Keywords: Acanthamoeba • keratitis • imaging/image analysis: non-clinical