Table 3.

Basic techniques used for the characterization of magnetic nanoparticles

Technique	Advantages	Drawbacks	Refs.
Imaging techniques
Transmission Electron Microscopy (TEM)	- Detection of nanoparticles distributed in intracellular and extracellular thin fragments of tissues - Differentiation nanoparticles located in different structures - Information on biodistribution and MNPs degradation ways in the organism	- Costly preparation procedures - Information only from specific, very limited areas of tissue	[161, 162]
Magnetic Resonance Imaging (MRI)	- Noninvasive and repeatable method - Visualizing and distinguishing individual soft tissue - Used in examinations of practically the entire body -  Possibility of continuous imaging of moving objects in real time	- Necessity of application a very strong magnetic field - Quite expensive technique	[164−168, 171]
Magnetic Particle Imaging (MPI)	- Prominent contrast and signal-to-noise ratio - The selected region can be rapidly and continuously detected for real-time imaging of MNPs distribution	- Necessity to develop and apply the appropriate MPI tracers	[79, 169, 170, 173–177]
Spectroscopy techniques
Electron Spin Resonance (ESR)	- Characterization of physical properties of various nanomaterials - Observation the differences resulting from interaction between the material surface and environment - Differentiation between the endogenous and administered iron	- Results for only specific time points - The necessity to section the tissue samples in to 2 mm3 cubes to fit in the thin ESR glass tubes	[158–160]
Inductively Coupled Plasma (ICP) techniques coupled with Atomic Emission Spectroscopy (ICP-AES) or with Mass Spectroscopy (ICP-MS)	- Detection of iron present in tissues at very low concentrations	- Destructive methods - No differentiation between the endogenous and administered iron	[185–189]
Electron Paramagnetic Resonance (EPR)	- Sensitive and nondestructive method which results in a direct measurement of the MNPs not requiring further data analysis - Performed at low magnetic fields and frequencies, offering the advantage that a much larger sample volume can measured at room temperature - EPR can be combined with MRI which benefits among others in cell tracking studies	- Limitations of the method result from the instability of paramagnetic centers in the tested substances and the reduced sensitivity of their detection for samples containing water	[190, 191]
Ferromagnetic Resonance Spectroscopy (FRS)	- Powerful method for the quantitative determination of internal fields in ferro- or ferrimagnetic materials and nanostructures - Shape of the FMR spectrum contains valuable information about the internal fields in the sample	- Structural information cannot be obtained in a straight-forward way from spectra	[192]
Alternating Current (AC) Susceptibility (ACS)	- Non-invasive method - Tissue sample preparation is minimal and no separation or isolation procedures are needed for the simultaneous quantification of several iron-containing species - The large amounts of tissue can be characterized each time so that representative results are easily obtained	- The need to use ex vivo samples - Time, costs and the relatively low availability of these type of instruments	[181–184]
Magnetometry techniques
Magnetic Susceptibility Measurement (MSM)	- A fast and easy method to quantify MNPs in convenient and accurate way in different media - There is no need of any preliminary modification of the samples - MSM values are only influenced by the iron from magnetic particles and not by free iron in solution	- The same magnetic particles for the calibration and experiments must be used, magnetic susceptibility being sensitive to the size of the magnetic core	[25]
Technique with the use of Superconducting Quantum Interference Device (SQUID)	- Very sensitive technique - These instruments are used in MRI and magnetoencephalography (MEG) for recording the very weak fields, which are produced by electrical currents flowing in the brain’s neural networks	- The noise level is determined by environmental sources, except in those experiments where the SQUID and its signal source are enclosed in a superconducting shield	[193]
Magnetic Particle Quantification (MPQ)	- Method offers highly sensitive, room-temperature and rapid quantification of nanoparticle–cell interactions - The low invasiveness and high resolution - Possibility of measuring very low amounts of the nanoparticles without destruction of sample - Llow amplitude and frequencies used in MPQ protect the MNPs from heating and agglomeration	- Necessity to use only MNPs with nonlinear magnetization - MPQ method cannot distinguish the processes of particle dissolution, transformation of iron oxides to biological forms of iron, excretion of particles from the organism, etc.	[178–180]