Abstract
Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold-particle tagged samples using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is presented and validated both on simulated data as well as experimentally.
Highlights
This paper presents a technique for 3D imaging of GNP-loaded cells using only two images
The proposed approach is based on the GS phase retrieval algorithm, and is generic and applicable to all wavelengths, given GNPs with an absorption peak that matches the laser’s wavelength
Scattered light from the GNPs is spatially coherent, which is a necessary condition for the GS algorithm
Summary
The solution was stirred for 5min, and the flask was removed from the hot oil and set aside until cooled. The solution was centrifuged to remove excess citrate. PEG7 solution was added to the GNP solution, stirred overnight and centrifuged in order to remove excess PEG. Excess EDC (N-ethyl-N -(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) (Thermo Fisher Scientific, Inc, Rockford, IL) were added to the solution, followed by addition of Glucose-2 (2GF) (D-(+ )-Glucosamine hydrochloride, Sigma-Aldrich, Israel Ltd.). Cells were centrifuged and a saline solution containing GNPs at concentration of 0.9% was added in excess. The concentration of the GNPs within the cells depends on the incubation time. GNP-labeled-cells were incubated in Formaldehyde solution at room temperature for fixation and were placed on a glass slide. The slides were covered with a #1.5 glass coverslip and sealed with nail-polish
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