RESUMO
Imaging is one of the key technologies underpinning discoveries in biomedical research. Each imaging technique however usually only provides a specific type of information. For instance, live-cell imaging using fluorescent tags can show us the dynamics of a system. On the other hand, electron microscopy (EM) gives us better resolution combined with the structural reference space. By applying a combination of light and electron microscopy modalities to a single sample one can exploit the advantages of both techniques in correlative light electron microscopy (CLEM). Although CLEM approaches can generate additional insights into the sample that cannot be gained by either technique in isolation, the visualization of the object of interest via markers or probes is still one of the bottlenecks in a Correlative Microscopy workflow. Whereas fluorescence is not directly visible in a standard electron microscope, gold particles, as the most common choice of probe for EM can also only be visualized using specialized light microscopes. In this review we will discuss some of the latest developments of probes for CLEM and some strategies how to choose a probe, discussing pros and cons of specific probes, and ensuring that they function as a dual modality marker.
Assuntos
Elétrons , Microscopia Eletrônica , Microscopia de Fluorescência/métodosRESUMO
Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.
RESUMO
Correlative Imaging (CI) visualizes a single sample/region of interest with two or more imaging modalities. The technique seeks to elucidate information that may not be discernible by using either of the constituent techniques in isolation. Correlative Light Electron Microscopy (CLEM) can be employed to streamline workflows, i.e., using fluorescent signals in the light microscope (LM) to inform the user of regions which should be imaged with electron microscopy (EM). The efficacy of correlative techniques requires high spatial resolution of signals from both imaging modalities. Ideally, a single point should originate from both the fluorescence and electron density. However, many of the ubiquitously used probes have a significant distance between their fluorescence and electron dense portions. Furthermore, electron dense metal nanoparticles used for EM visualization readily quench any proximal adjacent fluorophores. Therefore, accurate registration of both signals becomes difficult. Here we describe fluorescent nanoclusters in the context of a CLEM probe as they are composed of several atoms of a noble metal, in this case platinum, providing electron density. In addition, their structure confers them with fluorescence via a mechanism analogous to quantum dots. The electron dense core gives rise to the fluorescence which enables highly accurate signal registration between epifluorescence and electron imaging modalities. We provide a protocol for the synthesis of the nanoclusters with some additional techniques for their characterization and finally show how they can be used in a CLEM set up.
Assuntos
Elétrons , Platina , Corantes Fluorescentes , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field.