Microscopy Techniques

In our group we use a variety of imaging techniques, with a main focus on nanomaterials and the interaction of nanomaterials with living cells. We use super-resolution microscopy, in particular single molecule localization microscopy techniques, such as dSTORM and PAINT, as well as transmission electron microscopy (TEM) and the combination of them in Correlative light and electron microscopy (CLEM). 

STORM

Stochastic Optical Reconstruction Microscopy (STORM) is one of the most used super-resolution microscopy techniques, allowing us to resolve structures beyond the diffraction limit and study structures as small as 20 nm.  

Since each fluorophore emits a diffraction-limited fluorescence spot and these spots overlap forming a blurred image, conventional fluorescence microscopy cannot resolve structural features than are closer than 200 nm. To overcome this limitation, the mechanism that STORM uses is to reconstruct a super-resolution image by the localization of the centre of emission of each fluorophore. This can be done using photoswitchable fluorophores than can be turned on and off in a light-controlled fashion. When switching enough fluorophores off individual emitters can be observed and their position localized with nanometric precision using a Gaussian fitting. By the superimposition of the centroid of all the fluorophores localized a super-resolution image can be obtained. Key aspects for this method are the choice of the correct blinking fluorophore and the sample preparation, as labeling modality and density, buffer and environmental conditions determine the ability of the fluorophore to undergo optimal photoswitching. 

Our group successfully applied STORM for imaging of nanoparticles in vitro and in the biological environment.   

Pujals, S., Albertazzi, L., 2019. Super-resolution Microscopy for Nanomedicine Research. ACS Nano 13, 9707–9712. https://doi.org/10.1021/acsnano.9b05289

PAINT

PAINT is an implementation of single molecule localization microscopy where localization events are associated to the stochastic interaction between the target and a fluorescent probe diffusing in solution at low concentration. In this way, “blinking” is decoupled from the specific photophysical properties of fluorophores and it’s determined by the binding parameters of the target-probe pair, such as concentrations, affinity and association/dissociation rates. Thanks to the continuous influx of fresh probe binding to the target, photobleaching can be overcame. Because the amount of photons collected during a binding event is often higher if compared to a on/off switching event (like in STORM microscopy), PAINT is also endowed with the highest spatial resolution among single-molecule localization techniques. Moreover, multiple targets can be imaged by exchanging sequentially the type of binder probe in solution, without need of introducing multiple types of fluorophoresHowever, the control of binding properties, providing a reversible but still specific interaction, is a crucial requirement. It’s therefore not surprising that this approach gained substantial popularity when DNA hybridization was introduced as binding modality for PAINT. The predictable and well-controllable interaction between two short single strands of DNA, a docking placed on the target and a fluorophore-conjugated imager diffusing in solution, is particularly convenient for quantification, i.e. target counting, and high multiplexing. Indeed, different DNA docking strands are easily conjugated to popular binders, such as antibodies, that can be imaged by exchanging sequentially the complementary DNA imager strands exploiting the DNA- barcoding. 

Our group successfully applied DNA-PAINT for imaging of complex cellular components and nanomaterials. Additionally, we explored alternative approaches relying on different typologies of interaction. 

PAINT streptavidin

Delcanale, P., Miret-Ontiveros, B., Arista-Romero, M., Pujals, S., Albertazzi, L., 2018. Nanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT). ACS Nano 12, 7629–7637. https://doi.org/10.1021/acsnano.7b09063

TEM

The transmission electron microscope (TEM) is one of the most powerful tools available for characterisation in material sciences as it provides direct structural information about nano-scaled materials with exquisite resolution. In contrast to light microscopy, TEM uses an electron beam instead of light. Since electrons have a wavelength smaller than that of light, TEM can achieve a much higher resolution than that from a light microscope. The common voltage used for biological samples is 80 kV, which leads to wavelengths enough to reach a resolution of few nanometers.  

In TEM, a beam of electrons is transmitted through an ultra-thin specimen of maximum a few hundreds of nanometers thickness mounted on a metal grid, interacting with the specimen as it passes through. Sample preparation forms a very crucial part for imaging, and it is very specific to the material analysed and the kind of information required from it.  

Sample staining is often required, especially for soft materials. Normally heavy metals such as osmium, uranium or gold are used for negative staining. For nanomaterials, sample sectioning is not required as they can be fixated on the TEM grid using either a negative staining material such as uranyl acetate or by plastic embedding. Biological samples must be trimmed to a few hundred nanometers thickness by passing the sample over the glass or diamond knife of a microtome before staining and finally imaging.  

Our group employs TEM alone or in correlation with fluorescence techniques to characterize size and morphology of materials in vitro as well as to obtain the cell ultrastructure in biological samples.  

 

CLEM

Correlative light and electron microscopy (CLEM) combines the strengths of fluorescence and electron microscopy (EM). This is achieved by imaging the same sample and same field of view with both techniques and by overlapping the two resulting images gaining information from both methods. 

The recent development of super-resolution microscopy (SRM) pushes the limit of light diffraction of the fluorescence microscope down to 20nm, allowing the localisation of biological targets and their interactions with high sensitivity and specificity as well as with nano-meter spatial resolution. Multi-colour imaging using different fluorophores permits the simultaneous imaging of different biological targets and their interactions. EM possesses a resolution even greater than that of SRM, and hence can complement fluorescence microscopy with exquisite cellular ultrastructure detail, finding rare cellular and subcellular events in their cellular context. Although this powerful technique has been used in biology in various studies, it has not yet been applied to the understanding of nanomaterials. In our group we focus on the correlation between Single Molecule Localisation Microscopy (SMLM) techniques including dSTORM (direct Stochastic Optical Reconstruction Microscopy) and DNA-PAINT (DNA Points Accumulation for Imaging in Nanoscale Topography) with Transmission Electron Microscopy (TEM). Our aim is to study the structure-activity relationship between nanomaterials and cells for the development of novel nanotechnology-based therapies for the treatment of cancer and infectious diseases.  

CLEM STORM

van Elsland, D.M., Pujals, S., Bakkum, T., Bos, E., Oikonomeas‐Koppasis, N., Berlin, I., Neefjes, J., Meijer, A.H., Koster, A.J., Albertazzi, L., van Kasteren, S.I., 2018. Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens. Chembiochem 19, 1766–1770. https://doi.org/10.1002/cbic.201800230