Fluorescent layers for characterization of sectioning microscopy with coverslipuncorrected and water immersion objectives
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Label-Free Optical Microscopy for Biology Lab
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AbstractWe describe a new method to generate thin (thickness > 200 nm) and ultrathin (thickness < 200 nm) fluorescent layers to be used for microscope optical characterization. These layers are obtained by ultramicrotomy sectioning of fluorescent acrylic slides. This technique generates sub-resolution sheets with high fluorescence emission and uniform thickness, permitting to determine the z-response of different optical sectioning systems. Compared to the state of the art, the here proposed technique allows shorter and easier manufacturing procedure. Moreover, these fluorescent layers can be employed without protective coverslips, allowing the use of the Sectioned Imaging Property (SIP)-chart characterization method with coverslip-uncorrected objectives, water immersion objectives and micro-endoscopes. © 2014 Optical Society of America.
SponsorsWe thank Roberta Ruffilli for her contribution to the preparation of the fluorescent layers, Mattia Pesce for his assistance in confocal measurements, Claudio Moretti for his help with two- photon measurements and Giuseppe Vicidomini for useful discussion. This work was supported by the Italian Institute of Technology "Interdisciplinary/Interdepartmental project" to TF and CL and grants from San Paolo "Programma in Neuroscienze", MIUR FIRB (RBAP11X42L), Telethon-Italy (GGP10138) and a FP7 DESIRE project to TF.
PublisherThe Optical Society