Table 2.
Toolkit of techniques to investigate nano/bio interactions in C. elegans: state-of-the-art techniques and proposed novel uses.
| Information of C. elegans | Information of NP status | Advantages | Limitations | Ref | |
|---|---|---|---|---|---|
| Microscopy | |||||
| Light Microscopy (LM) | NP biodistribution (organ level) | Visible color | Easy sample preparation. | Low spatial resolution (>200
nm). Only applicable when NP are coloured and accumulate significantly, or in combination with staining techniques. |
[67, 81] |
| Fluorescence Microscopy |
NP biodistribution (organ level) and uptake | Fluorescence | Easy sample preparation. | Low spatial resolution (>200
nm). Limited to fluorescent particles. Not quantitative. |
[12, 52] |
| Confocal microscopy | NP biodistribution (organ level) and status | Spectra | Easy sample preparation. Tomography capabilities. |
Limited to fluorescent
particles. High cost. Complex image processing. |
[51, 84] |
| HDFM | NP biodistribution (organ level) and status | Spectra | Easy sample preparation. | Lacks spatial resolution. | [33] |
| TEM | NP biodistribution (cellular level) | Size, Aggregation | High resolution (up to 1 nm) | Complex sample preparation of TEM
cross-sections. Not quantitative. Contrast between the cellular structures and the NPs is required. |
[53, 67, 69] |
| HAADF | NP biodistribution (cellular level) | Size, Aggregation | Higher contrast than TEM (the brightness
depends on the Z2 of the element). High resolution (up to 1 nm). |
Complex sample preparation of C. elegans cross-sections. | [67] |
| SEM | NP biodistribution (cuticle) | Aggregation | Allows investigation of the external surface of treated C. elegans | Low spatial resolution - single particle detection is not possible. | [34, 62] |
| TPLM | NP biodistribution (organ level) and uptake | Luminescence | Enhanced contrast compared to LM. No fluorescence required. Confocal in nature; offers tomography capabilities |
Limited to particles with UV-Vis-NIR
absorption. Lacks spatial resolution. Not quantitative. |
[63] |
| Light Sheet Microscopy | NP biodistribution (organ level) and uptake | Fluorescence | Enhanced contrast due to reduction of
background signal. Tomography capabilities. |
Limited to fluorescent
particles. Limited spatial resolution. |
[51] |
| μ-SRXRF | NP uptake | Chemical composition | High sensitivity. | Limited access to synchrotron-based
techniques. Limited to elemental identification (phase identification is not possible). |
[25, 43] |
| STORM | NP uptake, Elemental distribution | Switching fluorescence | High resolution (single particle detection). Quantitative. | Tissue autofluorescence hinders its
application. Limited to NPs with switching fluorescent signal. |
– |
| Spectroscopy | |||||
| EDX | NP biodistribution (i.e. endosomes) | Presence/Absence of elements | Multi-element detection. High sensitivity. |
Not quantitative. ot possible to discern the form of the element (NP / ionic) |
[34, 67] |
| EELS | NP biodistribution (i.e. endosomes) | Presence/ Absence of elements | Multi-element detection. High sensitivity. | Not quantitative. Not possible to discern the form of the element (NP / ionic) |
[67] |
| ICP-MS | NP uptake | Chemical composition | Multi-element detection. Quantitative. | High cost. Not possible to discern the form of the element (NP / ionic) |
[34, 85] |
| μ-FT-IR | Degree of tissue oxidation | – | Highly informative about lipid and protein status. | Limited access to synchrotron-based techniques. | [82] |
| Absorbance μ-spectroscopy | NP uptake | NP aggregation (by peak position and width) | Quantitative. | Limited to particles with UV-Vis-NIR absorption. | [63] |
| Raman μ-spectroscopy | Biomolecular phenotype | – | Quantitative. | Difficult interpretation. | [49] |
| μ -xanes | Ionic homeostasis | Redox status of elements | Informative of oxidation state. High sensitivity. |
Limited access to synchrotron-based techniques. | [25] |
| μ-pixe | NP uptake Ionic homeostasis | Chemical composition | Multi-element detection (also in
2D). High sensitivity. |
Limited access to microbeam line facilities. | [43] |
| Other techniques | |||||
| Magnetometry (i.e. SQUID) | NP composition | NP uptake and magnetic properties | Informative of NP size and magnetic
properties. High sensitivity. Quantitative. |
Limited to magnetic particles. | [34] |
| MRI | NP biodistribution | In vivo T1 / T2 | Safe imaging modality. | High sensitivity is
demanded. Difficult to make it quantitative. Limited to magnetic NPs. |
[66] |
Legend: TPLM: Two-Photon Luminescence Microscopy, MRI: Magnetic Resonance Imaging, SQUID: Superconducting Quantum Interference Devices; HAADF: High Angle Annular Dark Field, HDFM: Hyperspectral Dark Field Microscopy, SEM: Scanning Electron Microscopy, μ-SRXRF: Synchrotron Radiation X-ray Fluorescence; STORM: Stochastic optical reconstruction microscopy; XANES: X-ray Absorption Near Edge Spectroscopy; EDX: Energy-dispersive X-ray spectroscopy; EELS: Electron energy loss spectroscopy; ICP-MS: Inductively coupled plasma mass spectrometry; FT-IR: Fourier-Transform Infrared Spectroscopy; μ-PIXE: micro-proton-induced X-ray emission.