Kristina A. Jahn

Correlative microscopy: providing new understanding in the biomedical and plant sciences.

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological, structural and chemical information that exceeds what is possible with any single technique. As a variety of complementary microscopy approaches rather than a specific type of instrument, correlative microscopy has blossomed in recent years as researchers have recognised that it is particularly suited to address the intricate questions of the modern biological sciences. Specialised technical developments in sample preparation, imaging methods, visualisation and data analysis have also accelerated the uptake of correlative approaches. In light of these advances, this critical review takes the reader on a journey through recent developments in, and applications of, correlative microscopy, examining its impact in biomedical research and in the field of plant science. This twin emphasis gives a unique perspective into use of correlative microscopy in fields that often advance independently, and highlights the lessons that can be learned from both fields for the future of this important area of research.

Three-dimensional organization of fenestrae labyrinths in liver sinusoidal endothelial cells.

Background/Aims: Liver sinusoidal endothelial cell (LSEC) fenestrae are membrane‐bound pores that are grouped in sieve plates and act as a bidirectional guardian in regulating transendothelial liver transport. The high permeability of the endothelial lining is explained by the presence of fenestrae and by various membrane‐bound transport vesicles. The question as to whether fenestrae relate to other transport compartments remains unclear and has been debated since their discovery almost 40 years ago.

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

To genuinely understand how complex biological structures function, we must integrate knowledge of their dynamic behavior and of their molecular machinery. The combined use of light or laser microscopy and electron microscopy has become increasingly important to our understanding of the structure and function of cells and tissues at the molecular level. Such a combination of two or more different microscopy techniques, preferably with different spatial- and temporal-resolution limits, is often referred to as ‘correlative microscopy’. Correlative imaging allows researchers to gain additional novel structure–function information, and such information provides a greater degree of confidence about the structures of interest because observations from one method can be compared to those from the other method(s). This is the strength of correlative (or ‘combined’) microscopy, especially when it is combined with combinatorial or non-combinatorial labeling approaches. In this topical review, we provide a brief historical perspective of correlative microscopy and an in-depth overview of correlative sample-preparation and imaging methods presently available, including future perspectives on the trend towards integrative microscopy and microanalysis.

GM1 expression in caco-2 cells: characterisation of a fundamental passage-dependent transformation of a cell line.

Caco-2 cells, which are known to spontaneously differentiate in cell culture, adopt typical epithelial characteristics and are widely used as a model to study cellular uptake, transport and metabolism processes. However, groups of flat and undifferentiated cells have been observed amid differentiating Caco-2 cell monolayers. In this study, we isolated and characterised these morphologically distinct, flat and island-forming Caco-2 cells. We visualised the undifferentiated cell islands with the aid of optical and electron microscopy and identified mono-sialo-ganglioside one (GM1) as their unique marker. Furthermore, two distinct subpopulations of morphology and GM1 expression were dilution cloned (Caco-2(GM1-) and Caco-2(GM1+) ), leading to the first documented Caco-2 clone that does not show differentiation characteristics. Caco-2(GM1+) cells were flat, non-polarising with extremely low transepithelial electrical resistance (TEER), whereas Caco-2(GM1-) cells showed typical epithelial features and high TEER. Importantly, the proportion of Caco-2(GM1+) cells in a culture increased with passage number and eventually dominated the cell culture. The novel GM1 passage-dependent cell transformation described here shows that careful monitoring is required when performing in vitro cell studies. Therefore, to guarantee consistent and valid experimental data, GM1 expression and the loss of differentiation characteristics should be carefully monitored and the use of fresh cultures should be standard practice.

The anticancer properties of iron core–gold shell nanoparticles in colorectal cancer cells

Previously, iron core–gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells’ relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the “active” component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core–shell nanoparticles are essential for the anticancer properties observed in CRC cells.

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin‐rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin‐rich structures and examine how they structurally may relate to detergent‐resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)‐ and electron microscopy (CFEM).

Monitoring membrane rafts in colorectal cancer cells by means of correlative fluorescence electron microscopy (CFEM)

Detergent-resistant membrane (DRM) rafts have been shown to play a pivotal role in regulating key cell biological processes, such as signal transduction, cellular transport and cell survival. The fine structure of membrane rafts are studied using various different imaging approaches and the outcomes are largely dependent on the detection methodology applied. All these microscopy techniques which employ light-, laser- and photon-optics, electrons as well as atomic force probing are characterized on their turn by their strengths and limitations for membrane raft identification. This explains in part the diversity of definitions available to describe these peculiar membrane structures. We present herewith an alternative and uncomplicated microscopy tool to study fluorescently labelled DRMs with information at the transmission electron microscopical level of the same cell, enabling us to obtain a snapshot of the morpho-functional relationships between the cells interior and DRMs. The proposed approach of correlative fluorescence electron microscopy (CFEM) can therefore be considered as an additional alternative imaging approach to unravel DRM structure-function relationships from micro- to nanometre length scales, from the cell to the molecule.

Caveolae and Caveolin-1 in Reptilian Liver

Caveolae are plasma-membrane invaginations that, by interacting with membrane-associated molecules such as endothelial nitric oxide synthase and tyrosine kinases, precisely regulate cell-signalling pathways responsible for cell structure and cell function. Indeed, there is widespread evidence that caveolae associate, structurally and functionally, with proteins, lipids and solutes to facilitate transcellular transport of these macromolecules. Caveolin-1, one of the family of membrane proteins that form caveolae, is most prominently expressed in endothelial cells of the vascular bed. Therefore, we have applied advanced electron microscopy as well as molecular biology techniques to study the presence of caveolae and caveolin-1 in the liver sinusoidal endothelium of reptiles. Reptiles are known to store excess lipid in the liver as an energy source for hibernation, and so offer a useful animal model in which to assess the structural and functional implications these subcellular compartments might have on liver sinusoidal endothelial transport. This study demonstrates that caveolae are indeed conserved across vertebrate species, whether mammalian or reptilian. It also presents as first novel data on the presence of caveolin-1-associated, tubular structures located within the cytoplasm of the lizard liver sinusoidal endothelium.

Visualization of detergent resistant membrane rafts in human colorectal cancer cells with correlative confocal and transmission electron microscopy

Detergent resistant membrane domains (DRM) can for the first time be visualised in the context of their native environment (i.e. attached to a cell) using correlative fluorescence and electron microscopy (CFEM). DRMs found on whole mounted colorectal cancer cells have a size range that is consistent with that widely accepted for lipid rafts (50–100nm). However, micron sized domains have also been observed. Furthermore, CFEM provides a tool to study DRMs on living cells yet still allows them to be prepared for high-resolution transmission electron microscopy (TEM) and electron tomographic analysis.