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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 biodistribution at the whole body scale but also at the tissue and cell levels. The development of new devices able to detect nanoparticles without any labeling offers new possibilities for the study of their behavior and kinetics. Moreover, the role of trace elements such as iron and copper in brain illnesses and neurodegenerative diseases such as Alzheimer¹, Menkes^2,3, or Wilson⁴ suggest the interest to study and localize these elements in tissues.

Various techniques have been used to provide elemental mapping or microanalysis of different materials. In their review paper published in 2006, R. Lobinski et al. provided an overview of available standard techniques for elemental microanalysis in biological environment, one of the most challenging environments for analytical sciences⁵. The electron microprobe, which consists of energy dispersive X-ray microanalysis in a transmission electron microscope, can be applied to numerous studies if the element concentration is sufficient (>100–1,000 μg/g). To reach lower detection limits, the following techniques have been used:

• ion beam microprobe using particle induced X-ray emission μ-PIXE (1–10 μg/g)⁶
• synchrotron radiation microanalysis μ-SXRF (0.1–1 μg/g)⁷
• secondary ion mass spectrometry SIMS (0.1 μg/g)⁸
• laser ablation inductively coupled mass spectrometry LA-ICP-MS (down to 0.01 μg/g)^9,10

The above-mentioned techniques provide micrometric resolution as shown in the Table 1 extracted from Lobinski et al.

3D reconstruction of serial 2D investigations could also be proposed for the reconstruction of deeper tissues¹¹. However, all devices and systems require both qualified professionals, moderate to highly expensive equipment and long-lasting experiments (typically more than 4 hr for a 100 µm x 100 µm for µ-SXRF and 10 mm x 10 mm for LA-ICP-MS)¹². Altogether, these requirements make elemental microanalysis very restricting and incompatible with conventional optical imaging systems, fluorescence microscopy or nonlinear microscopy. Another point that we can mention here is that the quantitative measurement capability is still quite limited and depends on the availability of matrix-matched laboratory standards. The further generalization of the use of elemental microanalysis in industry processes, geology, biology and other domains of applications will generate significant conceptual and technological breakthroughs.

The purpose of the present manuscript is to propose solutions for quantitative elemental mapping (or elemental microanalysis) in biological tissues with a tabletop instrumentation fully compatible with conventional optical microscopy. Our approach is based on the laser-induced breakdown spectroscopy (LIBS technology). In LIBS, a laser pulse is focused on the sample of interest to create the breakdown and spark of the material. The atomic radiation emitted in the plasma is subsequently analyzed by a spectrometer and the elemental concentrations can be retrieved with calibration measurements performed beforehand^13,14. The advantages of LIBS include sensitivity (µg/g for almost all the elements), compactness, very basic sample preparation, absence of contact with the sample, instantaneous response and precisely localized (micro) surface analysis. However, the application of tissue chemical imaging remains challenging since the laser ablation of tissue must be finely controlled to perform maps with high spatial resolution together with sensitivity in the µg/g range^15,16.

With such solution, the adjunction of tracers or labeling agents is not needed, which allows detecting inorganic elements directly in their native environment in biological tissues. The LIBS instrument developed in our laboratory offers a current resolution inferior to 100 µm with an estimated sensitivity for Gd below 35 µg/g, equivalent to 0.1 mM ¹⁶, which allows the mapping of large samples (>1 cm²) within 30 min. In addition, homemade software facilitates the acquisition and exploitation of the data. This instrument is used to detect, map, and quantify the tissue distribution of gadolinium (Gd)-based nanoparticles^17-18 in kidneys and tumor samples from small animals, 1 to 24 hr after intravenous injection of the particles (size <5 nm). Inorganic elements, which are intrinsically contained in a biological tissue, such as Fe, Ca, Na, and P, have also been detected and imaged.

Protokół

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 µl solution at 40 mM ready-to-inject (City: Villeurbanne, at lab).
2. Inject the 200 µl Gd-based nanoparticles solution intravenously into anesthetized tumor-bearing rodents (City: Lyon Sud (Oullins), 15 km from the lab).
3. 1-24 hr after injection, sacrifice the mice and put the biological samples into isopentane cooled by liquid nitrogen. Store the samples at - 80 °C (City: Lyon Sud (Oullins), 15 km from the lab).
4. Slice the sample (City: usually Grenoble, 100 km from the lab; I will try to have an access in Lyon Sud) in 100 µm-thick slides and put the biological slides on specific plastic dishes (Petri dish). Store at -80 °C.
   Note: Plastic dishes are basically a very pure polymer sample. They are used to avoid interference with elements contained in the tissue.

2. Sample Preparation for Calibration

1. Prepare vials with increasing doses of Gd-based nanoparticle into water (0 nM, 100 nM, 500 nM, 1 µM, 5 µM, 10 µM, 50 µM, 100 µM, 500 µM, 1 mM, and 5 mM).
2. Put a 5 µl-drop of each solution regularly spaced by 3 mm on the Petri dish.
3. Dry at room temperature for 20 min.

3. LIBS Experiment

1. Initializing the LIBS Setup
   1. Laser Settings. After switching on the instruments, wait 10-20 min for laser pulse energy stabilization and cooling down of the ICCD camera to -20 °C. Adjust the pulse energy with the attenuator.
      Note: The optimum laser parameters for mapping tissues are 5 nsec pulse duration, 1,064 nm wavelength, and pulse energy of about 4 mJ. The laser uses is a typical Nd:YAG nanosecond laser.
   2. LIBS Settings. Set the laser focusing position (with respect to the sample surface) to obtain the smaller crater diameter (about 50 µm or less).
      Note: This corresponds to a laser pulse focalization 100 µm below the sample surface.
   3. Spectrometer Settings. Use the Czerny-Turner spectrometer combined with a 1,200 lines/mm grating and a high temporal resolution ICCD camera. Control all these devices by computer. Set the input slit value to 40 µm. Set the spectral range regarding the element to be analyzed. Use the spectral range covering 325 to 355 nm to detect Gd with high sensitivity, as well as Na, Cu and Ca. Set the ICCD parameters with a delay of 300 nsec, a gate of 2 µsec, and a gain of 200.
2. Mapping Measurement
   1. Place the biological sample on the LIBS motorized sample holder.
   2. Adjust the height of the sample according to the laser focus position.
   3. Take a high resolution photograph of the sample slice.
   4. Set the mapping module of the LIBS acquisition software to perform a map with typically 100 x 100 measurement points spaced by a resolution of about 100 µm.
   5. Start the acquisition. From this point automate everything; the moving sequence as well as the spectrum recording and saving. 40 min are required for a mapping of 10,000 points (equivalent to 1 cm² for 100 µm resolution). Regroup all the recorded spectra into a same file.
   6. When finished, take a second photograph of the sample slice
3. Calibration Measurement

With the same experimental parameters, measure the calibration samples (for preparation details, see section 2). Perform a map or record 25 spectra (obtained from measurement sites in the center part of the drop) in each of the calibration drops.

4. LIBS Spectrum Analysis: Constructing of Chemical Images

1. Record all LIBS spectra from the tissue mapping and load them in the LIBS software analysis. Subtract the baseline for each spectrum and construct the chemical images with relative intensity scale using a false color.
   Note: An algorithm retrieves specific line intensities, such as Gd, Cu, Na, or Ca.
2. Perform the same operation on the spectra measured from the calibrated sample to allow the calibration curves calculation (relation between intensity and concentration) and build a quantitative map or image for Gd (or other element of interest).
3. Apply the adequate treatments to the chemical images, such as interpolation or smoothing. Save the intensity or concentration maps in image format (bitmap).

Wyniki

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...

Dyskusje

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...

Materiały

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