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Friday, 06 January 2017 16:10

Super “Resolution”: Reflections on Microscopy in the New Year

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As we shake off the holidays, 2017 brings a new opportunity for personal reflection. For many trainees, it is also a great time to focus one’s professional goals, including those related to microbiology and microscopy. In celebration of the new year, I’d like to reflect on some of the advanced microscopy work of 2016, and offer some inspiration for the year ahead. As we welcome 2017, I encourage you to embrace complexity, mystery, and the small picture.

Resolution: Go on a blind date.

Inspiration: “Super-resolution Microscopy for Microbiology.”

Super-resolution imaging can aid in the discovery of cellular structures. For details that are not resolvable with phase contrast or fluorescence microscopy, several super-resolution methods are suitable for use in microbiological research. Three major categories of super-res imaging exist: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illustration microscopy (SIM), and stimulated emission-depletion (STED) microscopy. These techniques take advantage of the fluorescent labeling methods use in fluorescence light microscopy, as well as its live-cell compatibility—with the added benefit of a 10-50 nm resolution. In that way, it approaches the resolutions of electron microscopy. Photoactivatable and photoswitchable fluorophores allow discreet fluorescence to take place (e.g. one fluorophore at a time), and the positions of these fluorophores are localized with nanometer accuracy using computer algorithms. Key to the localization capacity of super-resolution systems is the Gaussian function that approximates the microscope’s point spread function. Three important concepts for the various super-res techniques are photoactivation and photoswitching (PALM/STORM), the moiré effect (SIM), and stimulated depletion (STED). Followed by additional New Year’s Resolution ideas, these concepts are summarized in the image below (Fig. 1).

fig1a-reolutionFigure 1. Summary of super-resolution techniques in microbiology (Coltharp and Xiao 2012). Source

Resolution: Be more of a team player.

Inspiration: “Two-Photon Excitation STED Microscopy with Time-Gated Detection

The numerous molecular interactions occurring each second within living organisms are a microbiologist’s playground. Before the advent of super-resolution microscopy, however, clunky modifications for visualization with the microscope often irreversibly changed natural mechanisms and environments. Super-resolution microscopy improves resolution several-fold up to an order of magnitude, creating images that are much more realistic when compared with fluorescence microscopy. For example, two-photon excitation stimulation emission depletion implementation, or “2PE-STED implementation,” is a combination of two super-resolution techniques that allows scientists to image subcellular structures within thick specimens  (Fig. 3). 2PE-STED implementation demonstrates synergy between two existing techniques: 2PE-CW-STED and deconvolution algorithms. In general, super-resolution imaging is limited by the labeling density and the size of fluorophore particles. Additional combination microscopy techniques as well as advanced computer algorithms may solve these problems (now that’s a New Year’s Resolution).

Resolution: Learn to make better predictions.

Inspiration: “Architecture and Function of Mechanosensitive Membrane Protein Lattices

Super-resolution technologies are also helpful for creating membrane protein lattice models. By drawing from structural data and cooperative membrane protein interactions elucidated by super-resolution microscopy, Kahraman et al. predicted bilayer-mediated protein clustering. In the world of microbiology, super-resolution may also assist in the analysis of protein lattice models in HIV.  We are reminded of the HIV-1 protein-protein interactions analyzed by Barros et al. in the Journal of Virology this year. Advanced imaging may help bring the plasma membrane to life, in alignment with well-described electrostatic, hydrophobic, and lipid-specific interactions.

Resolution: Organize all of your important documents.

Inspiration: “Spatial organization shapes the turnover of the bacterial transcriptome

fig2-reolutionFigure 2. Phase contrast (grey) and STORM (color) images of transcriptional organization in E. coli (Moffitt et al.). Source

Microbes can teach us a lot about organization. For example, the spatial organization of the transcriptome in E. coli plays a major role in the post-transcriptional fate of RNA. The genome-wide spatial organization of the E. coli transcriptome can be observed through 3D-STORM imaging (a FISH-based RNA imaging approach). After imaging 75% of the E. coli transcriptome, Moffitt et al. demonstrated that mRNAs that encode inner-membrane proteins are associated with the membrane, while mRNAs that encode cytoplasmic, periplasmic, and outer-membrane proteins were diffusely distributed within the cytosol. The organizational patterns in E. coli also had post-transcriptional effects: if mRNAs were associated with the membrane, they were more rapidly degraded when compared with cytosolic mRNAs. These are nicely visualized in Fig. 2, below.

Resolution: Spend less time on social media.

Inspiration:  “Abnormal intermediate filament organization alters mitochondrial motility in giant axonal neuropathy fibroblasts

Super-resolution imaging has also allowed scientists to describe the proximity between cytoskeletal elements like fibroblasts and vimentin intermediate filaments. Below, we see indirect immunofluorescence and mitochondria (anti-vimentin and MitoTracker Red, respectively), showing the details of their interaction. These imaging techniques are well-aligned with recent molecular biology work by Azariyas Challa and Branko Stefanovic, who analyzed vimentin in 2011. Via transduction of lung fibroblasts with adenoviruses, the scientists examined the association of vimentin filaments with various types of collagen. Overall, its role in tissue fibrosis may be better understood by the conjunction of molecular biology and imaging techniques, perhaps helping to create the first effective therapy for fibroproliferative disorder. Imagine if these scientists had spent all their time on Facebook instead.

Resolution: Donate more--even if it’s something you want for yourself.
Inspiration:  “The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum

Super-resolution imaging can also help discern membrane dynamics within the endoplasmic reticulum. Differentiating the borders of the ER and its vesicles can help scientists determine how bacteria hijack the cell’s normal machinery to facilitate its own membrane traffic. The success of bacterial infections often depends on the ability to overcome multiple host barriers and utilized host machinery for transmission. In one example, A. anaplasmosis (a human and veterinary pathogen) was studied in order to characterize debilitating infections by tick-transmitted obligate intracellular bacteria. These bacteria live inside “pathogen activated vacuoles (POVs),” and imaging of ER lumen markers and related proteins showed bacterial recruitment to these POVs. Other Anaplasma species are known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Super-resolution imaging can be used to make quantitative measurements, such as structural dimensions, while characterizing these interactions. The new year is a time to find new uses for old things. Perhaps these highly resolved cytoskeletal elements will inspire you to clarify your own personal goals and aspirations in 2017.

Resolution: Post more realistic images on Instagram, with fewer filters (X-pro II won’t solve everything).

Inspiration: “Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells

fig3-reolutionFigure 3. Super-resolution analysis of replication compartment fractions in adenovirus-infected cells (Hidalgo et al. 2016). Source

In the ASM Journal of Virology, Hidalgo et al. published work describing the morphology of isolated replication compartment particles and nuclei. In order to establish the adenovirus replication microenvironment, subnuclear fractions were isolated and imaged with an Olympus IX-81 inverted microscope configured for total internal reflection fluorescence excitation. Adenoviruses have unique nuclear microenvironments, termed “replication compartments (RCs),” where the viral genome is replicated. Proteins involved in virus-host interactions are often centered within the replication compartments, and imaging of these activities require advanced morphological analyses. The result of these techniques show that RC-containing fractions are a powerful system for studying replication activities (Fig. 3). Dazzling, well in advance of the holidays.

Resolution: Learn to play a musical instrument.
: “Multiple signal classification algorithm for super-resolution fluorescence microscopy

A discussion of super-resolution microscopy would be incomplete without the inclusion of statistical algorithms, which are necessary to crunch large amounts of imaging data and compile the final image result. In order to shorten lengthy super resolution computational times, resolve fuzzy fluorophores, or work around biologically toxic photochemical environments, a new statistical algorithm has been described called “MUSICAL.” Short for MUltiple SIgnal Classification ALgorithm, this algorithm exploits the “eigenimages of the image stacks and applying point-spread functions. It has been thought to exceed the quality of some STORM imaging results that are muddled by high-density fluorophores. MUSICAL can be used for live cell fast imaging (around 49 frames in 250 milliseconds).

The year ahead

The past 8,760 hours have been an excellent time in the world of super-resolution microscopy. From wavelength-dependent super-resolution images of dye molecules coupled to plasmonic nanotriangles to mRNA transcriptome spatial organization, super-res advances have truly run the gamut. I hope I have inspired the reader toward veracity, brilliance, and resolve—but if not, there’s always next year…


Further Reading

Last modified on Wednesday, 25 January 2017 13:38
Jennifer Brubaker

Jennifer Brubaker is a medical student at Ohio University Heritage College of Osteopathic Medicine. She received her graduate degree from Johns Hopkins University in Biotechnology, where she studied Human Molecular Genetics and Emerging Infectious Diseases. She graduated from Boston College with a degree in Biology in 2012.

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