Practical Guide for Excellent GSDIM Super-Resolution Images

How to visualize cellular details with the Leica SR GSD 3D localization microscope

October 26, 2016

Do you know that most protists and bacteria lack one feature that each of our body cells has? Our cells  touch and communicate with each other. They send and receive a variety of signals which coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surfaces interact to organize tissues and body structures is of great interest.

The overall architecture of a tissue is determined by adhesion mechanisms that involve cell-cell and cell-matrix interactions formed by cytoskeletal structures, desmosomes and hemidesmosomes. The cytoskeleton is present in all cells forming a dynamic network of interlinking filaments and tubules that extend through the cytoplasm, giving the cell their particular shape and tension and maintaining the intracellular transport of vesicular proteins and signal cascades. Understanding the structural organization of cell interactions is of great interest, because defects in these connections are linked to a variety of diseases.

Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy.

In the following interview Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.

What is your main research subject? Why do you use a Leica SR GSD 3D super-resolution microscope?

Kees:I have always been interested in what makes a collection of dead chemicals ‘alive’, in other words, how does a cell work. Life is in the number and types of molecules, in their detailed localization and in their interaction.

Most molecules cannot be seen, not even with an ideal microscope. They have no color and are too small. Over the past years, tremendous advancements have been made in coloring the molecules: we have Fluorescent Proteins in all colors to tag proteins, we have probes to stain specific parts of the DNA and we have sensors that are able to see small-molecule messengers like Ca2+ and many more. With these, live-cell imaging allowed us to follow these invisible molecules in time and space. But only to the resolution of light microscopy, that is about 100 times lower resolution than what we would need to see molecules.

Functional imaging techniques like Fluorescence Resonance Energy Transfer (FRET), Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Lifetime Imaging (FLIM) allow us to see molecular interactions. But again, only with 0.25 µm resolution, and the experiments are labor-intensive. And one must also realize that the outcome of such experiments could be negative, even if the two molecules interact.

GSD and Super-Resolution are sort of the logical next step in obtaining sufficient resolution to study molecules and their interactions (Figure 1).

Fig. 1: Actin filaments, invadopodia and meshwork in N1E-115 cells, imaged in OxEA buffer with Alexa488. Scale bar: 500nm. Image Kees Jalink / Leila Nahidiazar, NKI Amsterdam

Which were the obstacles to overcome to adapt the GSD technique to your requirements? How did you solve the problems?

Kees: When we started, right out of the box, the Leica SR GSD 3D localization microscope worked fine. This was the time of single-color super-resolution, mostly in TIRF (Total Internal Reflection Fluorescence) mode.

We had anticipated that dye blinking needed optimization, and in fact this turned out to be the first major effort. Most dyes do not blink well enough, and the ones that do, often do not combine with each other. We optimized buffers extensively and found certain combinations that work together for two- and three-color imaging. The buffer system we developed is called OxEA and it has been tested in the labs of a number of colleagues worldwide before we published it (Figure 2). [1]

Fig. 2: Vimentin imaged at four different colors in OxEA buffer, scale bars: 500 nm. Images by Leila Nahidiazar, NKI Amsterdam.

Even very good dyes like Alexa Fluor®647 eventually run out, after maybe 20.000 frames or so. Leila Nahidiazar in our lab found, that if you are really careful you can keep the oxygen away perfectly with the help of a special designed Oxygen Tight Chamber (OTC). In these conditions, it is readily possible to get hundreds of thousands of images from a single cell. This is especially important for stitching. Still there are many things we do not understand. Did you know that the actin stain phalloidin, when coupled to Alexa Fluor®647, loses its affinity for actin if you illuminate it with strong laser light? Other colors are fine!

We also invested quite some time in 3D imaging. The Leica SR GSD 3D system has sectioning properties itself, so it is possible to combine different focal planes (routinely, with the help of the Oxygen Tight Chamber, Movie 1). We were the first to test the Leica astigmatic lens solution for 3D imaging (Movie 2). And it appeared possible to combine several astigmatic lens images to arrive at an extended 3D stack with ~50 nm resolution.

Finally, we started GSD imaging in tissue slices. Of course the results are not as crisp as those obtained in cells on cover slips, but a clear improvement in resolution is obtained in two-color images and these helped verifying that structures we observed in vitro are also discernible in tissue slices.

Movie 1: Extended 3D image of vimentin filaments constructed by stepping the focus through the cell. No cylinder lens was used. Axial resolution is approx. 500 nm. Image: Kees Jalink/ Leila Nahidiazar, NKI Amsterdam.
Movie 2: Detailed 3D image of a single focal plane (800 nm in thickness), acquired using the astigmatic lens. Axial resolution is approximately 50 nm. Image: Kees Jalink/ Leila Nahidiazar, NKI Amsterdam.

How did you analyze the data you acquired? What did you think about the image quality of initial experiments? Were there any adaptions you did? Any special analysis tools you created?

Kees: When we started working with the Leica SR GSD localization microscope we extensively studied sample drift using different commercially available dishes and mounting procedures. By analyzing long time-lapse imaging series taken from coverslips with sub-resolution fluorescent beads (Invitrogen) the Chamlide CMB magnetic chamber (Live Cell Instrument, Seoul, South Korea) was found to have minimal drift. In addition we place the sample on the microscope stage 20 min prior to imaging to let the sample settle down. If x/y drift is notable, we make a correction routine for that by using a home-built “de-jittering” routine or alternatively the analysis package ThunderSTORM.

We found that during image acquisition artefacts can be introduced in these already impressive images, especially when the background is very high. Existing background subtractions only partially solved that. So we worked together with Eelco Hoogendoorn and Marten Postma from the University of Amsterdam (UvA) who implemented a new background subtraction method: a running time-averaged median filter (Figure 3). This works great. [2]

Fig. 3: Clathrin-coated pits and plaques of HeLa cells imaged with Alexa Fluor®532 in Glucose-Oxidase (Gloxi) buffer. Note the improvement in image quality of this suboptimal dye/buffer combination by applying the temporal median filter. Images: Kees Jalink/ Daniela Leyton-Puig, NKI Amsterdam.

We then found that at such high magnification, small image errors that go unnoticed in the diffraction limited world can no longer be ignored. Chromatic aberrations are notably visible as was shown by Daniela Leyton in our lab, who was working on a protein that translocated to the plasma membrane after stimulation of cells. The question was whether it translocated to the plasma membrane lipid bilayer, or merely associated with the cortical actin just adjacent to that, a question that seems tailored for super-resolution.

Initially, Daniela found Clic4 almost always just next to the lipid bilayer, but depending on the orientation of the cell, they appeared sometimes in and sometimes just outside the cell. Suspecting chromatic aberrations to be the cause, together with physicist Bram van den Broek, 0.1 µm Tetraspec microspheres embedded in a matrix were imaged in the red, green and blue channel. With these data, we quantified chromatic aberrations and were able to precisely correct them with an affine transformation matrix using a home-built Image J macro (Figure 4). [3]

Please keep in mind that chromatic aberration is dependent on the objective in use and has to be corrected individually, but once characterized, the calibration remains valid throughout the years.

Fig. 4: Clathrin heavy chain (red) and Clathrin light chain in Clathrin coated pits stained with Alexa Fluor®647 and Alexa Fluor®532 imaged in Glucose-Oxidase buffer. In the right panel, chromatic aberrations have been corrected using an affine transformation implemented in Image J. Images: Kees Jalink / Daniela Leyton-Puig, NKI Amsterdam.

Daniela also spent much time to compare fixation protocols for super-resolution microscopy. The occurrence of fixation artifacts - and how to deal with them - is well documented in electron microscopy. Yet, these protocols cannot be simply copied for SR imaging because preparations are often imaged in aqueous buffers.

For the combined imaging of the actin cytoskeleton and associated actin-binding proteins, optimal results were obtained by the protocol in Leyton-Puig et. al, 2016. [4]

Fig. 5: Cos7 cells fixed with PFA in PEM (PIPES-EGTA-MgCl2) buffer and stained for actin with Phalloidin-Alexa Fluor®647. Imaging buffer: Glucose-Oxidase buffer. Images: Kees Jalink / Daniela Leyton-Puig, NKI Amsterdam.

What are the new insights the GSD has provided you for your research? How did this change the understanding of your research area?

Kees: Many! Too many to mention them all. For example, we observed that the molecular makeup of hemidesmosomes, the adhesion structures that make sure your skin doesn’t come off the rest of your body, are not quite as they were thought to be based on earlier electron microscopy and widefield fluorescence microscopy.

We found that stretches of DNA may enter empty spaces (a sort of pockets) in the lamin network at the inner leaflet of the nuclear membrane. And we are studying cytoskeletal components, cell surface receptors and vesicular proteins, all of which bring up new scientific insights, and also new scientific questions. A striking observation was also that the intermediate filament network seems to closely associate with microtubules, and this is seen predominantly in the periphery of the cell. The technique is so powerful that we receive requests constantly from groups that want to collaborate.

Another important insight is that quantitative image analysis is urgently needed – and more within reach than even before. With resolution far superior to fluorescence microscopy, and labeling density far superior to EM, we can start to count the number of receptors in a cluster, to quantitate the distance between filaments and the proteins that anchor them, we can quantitate spatial separation of two different molecular species and much more. But traditional tools like the Manders coefficient or Pearson correlation coefficient do no longer apply! Single-molecule localizations by definition do not overlap, so rather we have to ask the question: how close are the two molecular species, on average? That requires new analysis tools. [5,6,7,8,9]

Fig. 6: Three color Super Resolution image of human keratinocytes stained for actin (green), keratin (red), and integrin β4 (cyan). Imaging buffer: OxEA buffer. Scale bars: 500nm. Images: Kees Jalink / Leila Nahidiazar, NKI Amsterdam.

What are your current projects with the GSD technique? What possibilities do you see for using new camera techniques such as sCMOS?

Kees: The new sCMOS camera in our hands has several advantages. It delivers (almost) the same image quality at ~500 Hz, i.e. about 5 times faster than CCD. The camera can do 1500Hz but in that case we are not collecting enough photons for the highest quality images.

5 times faster means 5 times more experiments before your buffer runs out, much less drift and much less bleaching. It also works extremely well in low-power mode (40 x 40 µm field of view).

But the most exciting is that we start to do GSD in living cells. We found early on that mVenus blinks quite well when excited with 488 nm laser.

Fig. 7: Super-resolution live cell imaging of U2OS-cells with Venus-H2A in nucleoli. The cell has a somewhat atypical concentration of label in the nucleoli that is seen in about 1 - 2% of H2A-Venus cells. Scale bar: 500 nm. Image: Kees Jalink / Leila Nahidiazar, NKI Amsterdam.

Don’t expect to go to 5 nm localization precision, but certainly 20-30 nm is within reach. We are now also starting to do 2-color live cell imaging by imaging certain red fluorescent protein (FP) mutants that are still in development. We also got good results with mEOS.

Note that the excitation intensity is so high that cells are likely to be damaged within minutes. However, for resolution of fast events like vesicles that hop-on and hop-off microtubule highways, this has great potential.

Our current directions focus on various cell-biological problems, including internalization of receptors after stimulation, regulation of the actin cytoskeleton by formins, and interactions between the genome and the nuclear lamina. We also continue to work on cell adhesion structures and intermediate filaments: there is plenty to discover.

Could you describe a typical workflow for your GSD experiments?

Kees: Cells are grown and stained on a 1.5H cover slip and placed in a low drift Chamlide CMB magnetic chamber. For optimized blinking of two- and three-dye Super Resolution images the sample is mounted with 500 µL of Oxyrase buffer (OxEA). The sample is placed on the microscope stage 30 min before image acquisition to avoid initial sample movement.

After collection of 10 – 50k frames for each color we apply our temporal median background correction and our home-built software drift correction to the raw data. Finally the images are rendered using the Image J plugin ThunderSTORM and corrected for chromatic aberration with a home-built Image J macro.

Fig. 8: Experimental flow for excellent super-resolution images.
Movie 3: Super-resolution image of BP180 (red) and keratin-14 (green). Image: Kees Jalink/ Leila Nahidiazar, NKI Amsterdam.

References

  1. Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy, Nahidiazar L, Agronskaia AV, Broertjes J, van den Broek B, Jalink K, PLoS One (2016), 11(7), dx.doi.org/10.1371/journal.pone.0158884
  2. The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation, Hoogendoorn E, Crosby KC, Leyton-Puig D, Breedijk RM, Jalink K, Gadella TW, Postma M, Sci Rep. 2014 Jan 24;4:3854. doi:10.1038/srep03854
  3. CLIC4 regulates cell adhesion and β1 integrin trafficking, Elisabetta Argenzio et al., J Cell Sci (2015) 127, 5189-5203. doi:10.1242/jcs.150623
  4. PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins, Leyton-Puig D, Kedziora KM, Isogai T, van den Broek B, Jalink K, Innocenti M. Biol Open. (2016), doi:10.1242/bio.019570
  5. The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy, Leila Nahidiazar et al., Journal of Cell Science (2015) 128, 3714-3719. doi:10.1242/jcs.171892
  6. Genome-wide Maps of Nuclear Lamina Interactions in single Human Cells, Jop Kind et al. Cell (2015) 163 (1), 134-147. doi:10.1016/j.cell.2015.08.040
  7. The rod domain is not essential for the function of plectin in maintaining tissue integrity, Mirjam Ketema et al., Mol Biol Cell (2015) 26 (13), 2402-2417. doi:10.1091/mbc.E15-01-0043
  8. Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy, Robert P.J. Nieuwenhuizen and Leila Nahidiazar et al., PLoS One (2015), 10 (7). doi:10.1371/journal.pone.0131756
  9. Cholesterol and ORP1L-mediated ER contact sites control Autophagosome transport and fusion with endocytic pathway, Ruud H. Wijdeven, Hans Janssen, Leila Nahidiazar, Kees Jalink, Ilana Berlin, Jacques Neefjes, Nature Communications (2016), 11808. doi:10.1038/ncomms11808

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