Photoelectric correlation microscopy based on small gold nanoparticle single probe

01 Introduction

Recently, Iestyn Pope et al. from the School of Biological Sciences at Cardiff University in the United Kingdom developed a photoelectric correlation microscopy (CLEM) based on a single probe of small gold nanoparticles, which uses a resonant four-wave mixed-frequency (FWM) optical microscope to locate single gold nanoparticles bound to epidermal growth factor proteins at nanoscale accuracy, and realizes high-precision mapping with correlated transmission electron microscopy images. When using nanoparticles with radii of 10 nm and 5 nm, relevant accuracy below 60 nm is obtained in the region of 10 μm without the need for additional fiducial markers; In addition, by further reducing the system error, the correlation accuracy can be improved to below 40 nanometers, and the positioning accuracy can be lower than 10 nanometers; FWM microscopes have polarization-resolution characteristics and are sensitive to the shape of nanoparticles, and are expected to be reused for detection by shape recognition in the future. Due to the good photostability of gold nanoparticles and the good applicability of FWM microscopy to living cells, this FWM-CLEM microscopy technique provides an effective alternative to fluorescence-based imaging.

02 Research Background

CLEM, which combines the advantages of light microscopy (LM) and electron microscopy (EM), is gaining traction in the life sciences. CLEM aims to combine LM’s live-cell imaging capabilities, large field of view, and molecular-specific advantages with EM’s high resolution and properties of providing ultrastructural information to determine specific events within cellular structures and visualize molecular components with nano- to atom-resolution precision. In order to determine the location of the biomolecule of interest with high accuracy in this case, probe labeling that is visible in both LM and EM is required, and it is critical to select and prepare suitable probes for different imaging modalities.

Commonly used commercial probes bind fluorophores to gold nanoparticles (AuNPs), such as Alexa594 fluorescent dye and AuNP 5 nm in diameter, to ligand transferrin, however, due to the non-radiative energy transfer of AuNP causing fluorescence quenching effects, the suitability of probes in CLEM is significantly reduced. Semiconductor nanocrystals, also called quantum dots (QDs), represent another type of CLEM probe. QDs often require a protective shell layer in biological applications to reduce their cytotoxicity, which increases their size; In addition, QDs have intermittent fluorescence scintillation, which limits their application. On the other hand, fixation and staining operations in sample preparation of EM make it difficult to preserve the fluorescence emission characteristics of a single fluorescent dye probe. Cryo-optical microscopy increases the photostability of fluorescence at low temperatures, but often presents technical challenges, such as the need to use a highly stable custom cryogenic stage and the need for high numerical aperture eyepieces to avoid sample crystallization. In addition, to achieve correlation accuracy between images in LM and EM modalities, it is often necessary to introduce labels, increasing the complexity of sample preparation.

Another approach is to use small gold nanoparticles (AuNPs) as a single probe. These nanoparticles are easily visible in electron microscopy and exhibit strong light scattering and absorption at their local surface plasmon resonance (LSPR) wavelengths. Compared to fluorescent dyes, they are photostable and offer significant advantages. However, conventional single-photon (i.e., linear) light microscopy has difficulty distinguishing small AuNPs from the autofluorescence background of cells.

Using the four-wave mixing (FWM) nonlinear effect of AuNP to create a triple resonance with LSPR wavelengths, it has been possible to detect a single small AuNP (radius less than 5 nanometers) in scattering cells and tissues, free from background limitations, and only affected by photon firing noise.

In this work, the researchers demonstrated a CLEM workflow using small AuNPs as a single probe to achieve simultaneous imaging of epidermal growth factor (EGF) proteins in mammalian cancer cells in LM and EM by leveraging the FWM effect of AuNP. The photostability of AuNP enables the position of a single AuNP to be located and observed with nanometer accuracy at room temperature, and provides high-precision correlation between EM and LM without additional reference markers.

03 Innovative research

Figure 1a shows the experimental schematic diagram of the implementation of FWM technology, using the pump light, reference light and detection light generated by the same laser source, their center frequency is the same, and it matches the local plasmon resonance frequency of the small AuNP, and the detected FWM can be understood as the change of the AuNP dielectric function caused by pumping, which is manifested as the scattering change of the detection beam. The authors used a high numerical aperture (NA) objective to focus pump light and probe light onto the sample and collect FWM signals in the reflected light path. To detect FWM by pump light and probe light, a mixed-frequency detection scheme is used, in which the pump light is modulated at νm, the probe light frequency is modulated at ν2, and interference between the FWM and the reference light is detected at frequency ν2±νm. Figure 1b shows the center position where a single AuNP can be positioned with high accuracy in an FWM image. Figure 1c shows that this detection protocol avoids the effects of incoherent background signals such as autofluorescence, and the spatial position of individual AuNP probes can be observed with high contrast by both FWM and EM techniques.

Figure 2 shows the results of the correlation accuracy analysis of the CLEM system. The image is formed by superimposing the transformed FWM field amplitude (yellow) and transmission electron microscopy (TEM) image (gray) to obtain a correlation accuracy of 54 nanometers.

Figure 3 shows an example of CLEM imaging using AuNP with a radius of 5 nm in HeLa cells. The results show that this small size of individual nanoparticles can be clearly distinguished, independent of noise and background, and the relevant accuracy obtained is 58 nanometers.

Figure 4 shows that considering that the position of the AuNP centroid of the AuNP is affected by defocusing aberrations when locating the AuNP centroid by FWM, the optimal focal plane coordinates of AuNP with a radius of 10 nm in HeLa cells are determined by fine axial three-dimensional (3D) scanning, thereby eliminating the influence of defocusing aberration, and the relevant accuracy of the CLEM system is 36 nm.

Figure 1 Correlation photoelectron microscopy based on FWM imaging. a) Schematic diagram of the FWM device. b) Example of volumetric FWM microscopic imaging with a single radius of 10 nm AuNP. c) Observation of AuNP within a radius of 10 nm binding to EGF protein in HeLa cells using the CLEM system.

Figure 2 Accuracy analysis of CLEM system. Overlay of FWM field amplitude (yellow) and TEM image (gray).

Figure 3 CLEM correlation accuracy using AuNP with a radius of 5 nm.

Figure 4 CLEM correlation accuracy obtained by 3D-FWM.

The article was published in the journal Light: Science & Applications under the title “Correlative light electron microscopy using small gold nanoparticles as single probes,” with Iestyn Pope as first authors, Paola Borri and Paul Verkade is the co-corresponding author. (Source: LightScience Applications WeChat public account)

Related paper information:‍-023-0‍1115-4

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