Endothelial-like nitric oxide synthase immunolocalization by using gold nanoparticles and dyes.

Ramla Gary,Riccardo Barberi,Daniela Amelio,Maria Penelope De Santo,Gia Petriashvili,Filippo Garofalo,Yuen Kwong Ip

doi:10.1364/boe.6.004738

Abstract

Immunofluorescence is a biological technique that allows displaying the localization of the target molecule through a fluorescent microscope. We used a combination of gold nanoparticles and the fluorescein isothiocianate, FITC, as optical contrast agents for laser scanning confocal microscopy imaging to localize the endothelial-like nitric oxide synthase in skeletal muscle cells in a three-dimensional tissue phantom at the depth of 4µm. The FITC detected fluorescence intensity from gold-nanoparticles-labelled cells was brighter than the emission intensity from unlabelled cells.

Highlights

Nitric oxide (NO), produced by the different nitric oxide synthases (NOSs) isoforms including endothelial-like NOS have ubiquitous tissue locations, including skeletal muscles
We used a combination of AuNPs and the fluorescence dye (FITC) for a cellular imaging of laser scanning confocal microscopy (LSCM), where the skeletal muscle cells from lungfish Protopterus annectens were tested for the endothelial NOS localization
The higher optical contrast between the autofluorescence and endothelial-like NOS (eNOS) emitted signal is obtained when the AuNPs treated sample is irradiated with the laser beam at 496 nm and the emitted signal is detected in the 506-750 nm range, since this wavelength is within the surface plasmon resonance (SPR) band of AuNPs and it is the closer one to the excitation peak of FITC (494nm)

Summary

Introduction

Nitric oxide (NO), produced by the different nitric oxide synthases (NOSs) isoforms including endothelial-like NOS (eNOS) have ubiquitous tissue locations, including skeletal muscles. It exerts an universal multi-faceted regulatory role, including modulation of the aerobic biome (redox and energy balance) and cardio-circulatory homeostasis and muscle contractile efficiency [1,2,3,4]. All cells have some intrinsic level of autofluorescence, which is most commonly caused by NADH, riboflavins, and flavin coenzymes [7,8] These molecules excite over a broad range of wavelengths including the blue region of the spectra. The peak autofluorescence emission after 488 nm excitation is in the green region of the spectra [9], heavily overlapping with the FITC fluorescence detection region

Methods

Results

Conclusion