Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Summary

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 μm and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

Abstract

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1 to 24 hr after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 μm. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response: elemental, molecular, or cellular.

Introduction

The wide development of nanoparticles for biological applications urged the parallel improvement of analytical techniques for their quantification and imaging in biological samples. Usually the detection and the mapping of the nanoparticles in organs are made by fluorescence or confocal microscopy. Unfortunately these methods require the labeling of the nanoparticles by a near infrared dye that can modify the biodistribution of the nanoparticles, especially for very small nanoparticles due to its hydrophobic properties. The detection of labeled nanoparticles, and especially the very small nanoparticles (size < 10 nm), might thus interfere with their biodistributio....

Protocol

1. Biological Sample Preparation

All the experiments described in this study were approved by the Animal Care and Use Committee of the CECCAPP (Lyon, France) (authorization #LYONSUD_2012_004), and the experiments were carried out under the supervision of authorized individuals (L. Sancey, DDPP authorization #38 05 32).

1. Add 1 ml of H₂O to 100 µmol of Gadolinium (Gd)-based nanoparticles, wait 15 min, and add 20 µl of HEPES 50 mM, NaCl 1.325 M, CaCl₂ 20 mM to 100 µl of H₂O and 80 µl of the primary solution of Gd-based nanoparticles to obtain a 200 ....

Results

As shown in Figure 1, the beam of a Nd:YAG laser in the fundamental wavelength of 1,064 nm was focused vertically down on the tissue slice by a quartz lens of 50 mm focal distance. The pulse energy was 4 mJ and the repetition rate 10 Hz. In order to avoid the generation of plasma in air, the laser beam was focused around 100 µm under the surface of the sample. No air plasma was observed in this condition. During the experiments, the sample was moved by a step motor in order to generate one plasma in only.......

Discussion

Applied to biological sample, this technique allows the chemical imaging, i.e. the mapping and quantification, of Gd and Si from injected Gd-based nanoparticles in different organs. From the main critical settings, the control of laser properties (wavelength, pulse energy, focusing, and stability) is critical for a precise and fine tissue ablation (i.e. mapping resolution) as well as for sensitivity. Working at high energy provides a better sensitivity but unfortunately generates degraded spatial resolu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Laser nanosecond Nd:YAG	Quantel	Brillant	5 nsec pulse width, wavelength 1,064 nm
Spectrometer	Andor Technology	Shamrock 303	with 1,200 lines/mm blazed at 300 nm grating
Detector ICCD	Andor Technology	Istar	2 nsec temporal resolution
LIBS Unit	ILM		Homemade Instrumentation
Gd-based nanoparticles	Nano-H		particles
HEPES	Sigma-Aldrich	H4034	for particle's dilution
CaCl2	Sigma-Aldrich	21108	for particle's dilution
NaCl	Sigma-Aldrich	S5886	for particle's dilution
Mice	Charles River	depending of animal breeding
Isoflurane	Coveto / Virbac		for anaesthesia - Isofluranum
Isopentane	Sigma-Aldrich	59060	to froze the sample  slowly
Liquid nitrogen	Air Liquide		to cool down the isopentane
Cryostat	Leica	CM-3050S	to slide the samples
Petri dishes	Dutscher	353004	to stick the sample