Three-Dimensional Super-Resolution GSDIM Microscopy

Polar epithelial cells are the prerequisite for a functional organ. They build a monolayer on the outer surface of the organ, providing a barrier and exchange system between the organ and the environment. The polarized structure of epithelial cells is characterized by two distinct domains of their plasma membrane, the apical domain facing the organ lumen and the basolateral domain where cell-cell and cell-extracellular-matrix contacts occur. The two membrane domains are separated by tight junctions and exhibit different protein and lipid compositions. To maintain this polarity, epithelial cells have evolved a highly specific polarized sorting of proteins and lipids to their target membrane.

Apical protein sorting requires various sorting signals for a variety of different pathways to the cell surface. GPI (Glycosylphosphatidyl inositol) anchors as well as the involvement of N- and O-glycans are identified as apical sorting signals. The sorting pathways can be subdivided by the affinity of the transported protein to lipid rafts. Therefore, there is a lipid raft-dependent and a non-dependent pathway. Consequently, proteins are separated into distinct vesicle populations at the trans-Golgi network (TGN) or at a post-Golgi compartment [1].

The sorting process involves many proteins that are essential for the correct delivery of cargo proteins. One important sorting receptor is galectin-3. It can bind specific cargos due to their glycosylation and leads to a transport into the correct plasma membrane [2]. Moreover, cytoskeletal tracks formed by actin microfilaments and microtubules are involved in this process by the movement of secretory vesicles and organelles. Microtubules in particular are required for long passages inside the cell. They consist of α- and β-tubulin subunits that polymerize to dimers. Posttranslational modification like the tyrosination-detyrosination of tubulin, where a tyrosine residue is added or removed from the C-terminal domain of α-tubulin, is required for correct delivery of cargo to the apical membrane [3].

Highly resolved GSDIM images depict the distribution of these modifications along single molecules and can resolve cellular components that are involved in polarized protein trafficking in detail.

The diffraction barrier limits the imaging resolution of conventional light microscopy to 200–300 nm in the lateral dimension and 500–800 nm in the axial dimension, leaving many intracellular organelles and structures unresolvable. Many cellular structures are much smaller, e.g. the cisternae of the Golgi are arranged like pancakes spaced <100 nm apart, the ATP synthases are spaced 10 nm apart in the inner mitochondrial membrane, and microtubules are composed of dimerized subunits forming hollow rods 25 nm in outer diameter. Several approaches have been used to overcome this diffraction limit in two dimensions by super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy, photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and ground state depletion followed by individual molecule return (GSDIM) microscopy. The third dimension of a cellular compartment can be resolved by direct stochastic optical reconstruction microscopy (dSTORM) and recently by 3D GSDIM microscopy (Leica SR GSD 3D).

In dSTORM and GSDIM microscopy, the determination of the exact position of a fluorochrome is based on the principle of temporal separation. This requires switching of fluorochromes between on and off states followed by a sequential readout. At the beginning of an experiment, the majority of fluorochromes are transferred into a long-living dark state (OFF state) by applying a high intensity laser. Only single molecules will return to the ON state stochastically and emit fluorescence for a short time before switching back to the dark state. The fluorescence signals from these molecules that cycle from dark to bright state and back to dark state are recorded over time. Finally, a super-resolution image is reconstructed from thousands of images [4].

A fluorescence signal will be depicted as a point spread function (PSF) several hundreds of nanometer in size. But the PSF can be adapted/adjusted by a two-dimensional Gaussian function to localize the exact position of the molecule. The precision of the molecule position depends on the signal intensity, i.e. the number of collected photons [5]. An increase in resolution is achieved by detection of single fluorescence spots temporally separated from each other.

To enable the localization of single fluorophores in z direction, the astigmatism effect is used by introducing a cylindrical lens into the imaging path of the microscope. As a result the ellipticity and orientation of the fluorescence spot vary depending on its position in z dimension, thereby enabling 3D reconstruction. When the fluorophore is in the focal plane, the image appears round. When the fluorophore is below the focus, the image spot shape is elongated in the vertical direction, conversely when it is higher than the focus position the spot is elongated in the horizontal direction [6].

The high power laser of the Leica SR GSD 3D system can switch a wide range of fluorochromes and can collect many photons in a short time, allowing the researcher to use the set of fluorescent antibodies and Alexa dyes that were also used for classical epifluorescence. The high power laser objective with a 160x magnification additionally contributes to a precise localization of single molecules in high resolution. For this accuracy an extended period of time is required. The prerequisite of a non-drifting sample over the imaging period is achieved by the SuMo (Suppressed Motion) stage technology that minimizes lateral drift by less than 20 nm/10 min.

The Leica SR GSD 3D system is a new approach in localization microscopy for showing cellular compartments with high resolution in the third dimension. To understand transport processes in polarized epithelial cells the exact localization of different cellular organelles involved in protein trafficking is necessary. Using high-resolution GSD microscopy it was possible  to analyze the Golgi matrix protein gm130, the mitochondrial ATP synthase ATP-2B and detyrosinated microtubules (detyr-tubulin). MDCK wild-type cells cultured on coverslips for one day were fixed with ice-cold methanol or 4 % PFA. The cells were blocked with 1 % milk powder or 1 % BSA (bovine serum albumin) in PBS++ (PBS with 1 mM CaCl₂ and 1 mM MgCl₂) and immunostaining was performed by using antibodies directed against gm130, ATP-2B or detyr-tubulin. Primary antibodies were then labeled with Alexa647-coupled antibodies, using 100 mM MEA (β-Mercaptoethylamine) in PBS as embedding medium. The Golgi is labeled by round structures of gm130 facing the Golgi lumen in super-resolution (Figure 2A). In addition to the detailed information of the 2D image compared to the epifluorescence image, the third dimension clearly defines the cellular localization of this compartment using a color gradient that indicates the z-axis from 0–800 nm. Although mitochondria and microtubules are already well resolved in the 2D GSD image (Figure 2B), the 3D GSD super-resolution image displays the positioning of mitochondria in close proximity to microtubules, indicating a movement of these organelles along cytoskeletal tracks.

Fig. 1: Comparison of widefield microscopy, 2D GSDIM and 3D GSDIM microscopy.
A) MDCK cells were cultivated on coverslips, fixed and immunostained for the Golgi matrix protein gm130/Alexa647. Scale bar 2 µm.
B) MDCK cells grown on coverslips were fixed and stained for mitochondrial ATP synthase using an anti-ATP-2B antibody and microtubules using an anti-detyr-tubulin antibody. Both were labeled with an Alexa647 coupled secondary antibody. Scale bar 2 µm. The color gradient indicates the x-axis 0–800 nm.

Super-resolution GSDIM helps to elucidate the localization of components that are involved in polarized protein trafficking in greater detail and will therefore shed light on previously unresolved transport mechanisms. The 3D GSD image shows an improvement in resolution over the conventional epifluorescence image and, moreover, it provides the 3D information that was not provided by 2D images. Using this super-resolution technique in 3D, it will be possible to monitor more components of the protein trafficking pathway, like the transport vesicles themselves, in future. Leica SR GSD 3D is a new super-resolution microscope with a multi-application platform including TIRF, widefield and super-resolution that enables a deeper insight into fundamental processes inside the cell.

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