Focus On Virology

Movie courtesy of Rudolph Reimer, Heinrich Pette Institute, Leibniz Institute for Experimental Virology

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Technology for imaging and quantification of virus activity in real time

Due to the extremely small size of viruses (5-300 nm), their structure and function have always been a challenge to study. Over recent centuries, many viruses have been discovered, and vaccines against them developed, but it was the invention of the electron microscope in 1931 that enabled their complex structures to be visualized. Since then, Nikon has become an expert in providing advanced light microscopy systems to image virus structure and infectivity in real time with high resolution and high throughput. When rapid and reliable analysis of viruses is essential, the importance of such equipment has become even more apparent.

Live Cell

A wide range of Nikon’s capabilities, including high-speed widefield or confocal microscopy, Artificial Intelligent (AI) software technology and a real-time focus maintenance system (The Perfect Focus System, PFS) all contribute to long-term, time-lapse imaging of viral processes with minimal photobleaching.

The ECLIPSE Ti2 widefield inverted microscope is a powerful platform for live cell imaging in virology. Its unparalleled 25 mm Field of View (FOV) captures changes and reactions of cells and viruses over a wide area in one shot. The Perfect Focus System (PFS) prevents focal drift, keeping moving cells and viruses in constant focus and thus supporting high-resolution imaging during high-speed and long-term acquisitions.

The A1R HD25, CSU-W1 SoRa and Crest X-Light V3 confocal microscopes have a FOV (up to 25 mm) which is nearly twice that of conventional confocal microscopes. This allows more data to be collected within a single image, resulting in greater efficiency. Real-time observation of viral activities in live cells is also possible using Total Internal Reflection Fluorescence (TIRF) microscopy, modules for which can be installed on the Ti2 inverted microscope. TIRF enables the study of processes near the cell membrane with exceptionally high signal-to-noise, such as the mechanism of viral entry into cells and its impact on cell-cell interactions. Enhanced resolution is achieved on the CSU-W1 SoRa spinning disk confocal using the concept of optical photon reassignment. The Crest X-Light V3 spinning disk confocal provides homogeneous illumination over a large 25 mm FOV.

Phototoxicity and photobleaching of fluorescent probes used to stain viruses can be minimized using the high-speed resonant scanner of the A1R HD25. The power of the A1R HD25 confocal microscope lies in the combination of the high-definition resonant scanner and large 25 mm field of view, which provides high throughput. All of Nikon’s live-cell imaging solutions incorporate NIS-Elements, a unified acquisition and analysis software platform that now supports Nikon's NIS.ai deep learning-based modules, including Clarify.ai, Denoise.ai, and Enhance.ai.

Clarify.ai automatically removes blur from widefield fluorescence images using a pre-trained neural network. Denoise.ai removes Poisson shot noise from resonant confocal data, either in real-time or post-acquisition, and also using a pre-trained neural network. When combined with the A1R HD25 confocal microscope, Denoise.ai enables users to quickly acquire images of low signal-to-noise features that would normally require longer dwell times to sufficiently resolve. Enhance.ai is useful for low-light fluorescence imaging of viruses, which is necessary for light-sensitive detection markers present at low number. The neural network is trained using matched high and low signal-to-noise image data, allowing it to quickly predict high signal-to-noise versions of subsequent input images.

with Enhance.ai
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with Denoise.ai
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with Clarify.ai
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Case study:

The transport and spread of the alphaherpes virus in neuronal cultures has been studied by Dr. Lynn Enquist’s team at Princeton University, USA, utilizing a Nikon live-cell widefield imaging platform (the ECLIPSE Ti inverted microscope controlled by NIS-Elements software). Their work applies real-time and overnight time-lapse live-cell imaging of viruses expressing fluorescent protein fusions to illuminate viral assemblies during infection of primary neurons. Their approach also minimizes the impact of fluorescence imaging on the virus.

(Taylor, M.P. et al. (2013) J. Vis. Exp. (78), e50723, doi:10.3791/50723).

High-content screening

When large scale clinical screening, such as drug and virus testing, is required there is a considerable amount of multidimensional image data produced in a short time. Nikon provides a high-content cell screening platform (BioPipeline Live), a visual programming tool (JOBS) and a high-performance analysis tool (GA3) to streamline and automate your virus imaging workflow.

Nikon’s live-cell high-content imaging platform, BioPipeline Live, provides a great solution for fully automated live-cell imaging in multiple plates. Utilizing all the functionality of the ECLIPSE Ti2 inverted microscope, the BioPipeline Live includes robotic automation for rapid imaging and exchange of up to 44 plates!

The General Analysis 3 (GA3) tool enables simple implementation of complex post-processing analyses. Images include complete metadata, even for settings such as environmental condition, which can be crucial for virus studies. In addition to all the analysis and processing functions NIS-Elements provides, the JOBS module provides the ability to create customized experiment protocols, including conditional workflows. This allows for fully automated acquisition and analysis to be performed, including of multiple plates. This includes immediate viewing of measurement data, well-by-well, and via a heat map for trend observation and further analysis. Throughput with various tasks can be increased along with specimen viability using available NIS.ai deep learning-based modules for NIS-Elements. For example, Segment.ai applies a neural network to automatically segment your images (once trained). JOBS can streamline and automate the entire platform, integrating functions such as device control, image acquisition, processing, and analysis without the need for advanced programming knowledge.

Case study:

Dr. Vibor Laketa’s research group at Heidelberg University, Germany recently developed a semi-quantitative SARS-CoV-2 antibody test for use on human sera. This microscope-based test is demonstrated using a Nikon ECLIPSE Ti2 widefield microscope controlled by the JOBS module in the NIS-Elements software to automatically acquire images in 96-well plates. This new test demonstrates improved sensitivity and specificity compared to a standard ELISA-based diagnostic test, and with high throughput, which is necessary for large screening programs.

(Pape, C. et al. (2020) bioRxiv (preprint doi: https://doi.org/10.1101/2020.06.15.152587)).

Super-Resolution

Super-resolution imaging of viral structure and activity

Viral particles vary greatly in size, spanning approximately 5 to 300 nm in diameter, which ranges from beyond to close to the resolution limit of conventional fluorescence microscopy. For hundreds of years, optical microscopy could only resolve cellular details to approximately 200 nm in XY and 500 nm in Z, but new super-resolution techniques have been successful in resolving biological details to about 20 nm in XY and 50 nm in Z. This makes it possible to investigate small and, sometimes, rare microorganisms (including viruses) with near-molecular level detail. These super-resolution techniques help bridge the gap between conventional fluorescence microscopy and electron microscopy.

Nikon offers a range of super-resolution systems for different imaging applications. Structured Illumination Microscopy (SIM) provides twice the resolution of a typical widefield microscope in X, Y and Z while maintaining high acquisition speed and flexible sample preparation requirements. STochastic Optical Reconstruction Microscopy (STORM) provides the greatest level of detail - up to 10x greater resolution than conventional techniques.

SIM uses the concept of frequency mixing to identify super-resolution frequency information in the image. This works by illuminating the sample with various patterns of known spatial frequency and comparing how the image is modulated by each pattern, with subsequent analysis uncovering super-resolution frequency information that is not directly observable and using it to compute the final image. The acquisition speed for the N-SIM S is up to 15 frames per second, which is enabled by using a Spatial Light Modulator (SLM) for pattern modulation, and especially useful for visualizing the fast progression of viral activity in live cells. The N-SIM S can be implemented on the same microscope stand as a confocal system. Such a correlative approach allows fast confocal imaging of large fields of view, reserving super-resolution acquisition for regions of interest.

The N-STORM super-resolution microscope pushes the limits of optical resolution by only ‘switching on’ a small number of non-overlapping fluorophores in a sample. This allows the centroid position of single emission events to be identified with high precision. Repeating this process over many imaging frames allows for the complete image to be reconstructed at unprecedented resolution, to approximately 20 nm in the XY and 50 nm in the Z directions. Nikon’s 100x silicone immersion lens (NA = 1.35) enables superior 3D performance in live-cell cultures and thicker samples as it better corrects for spherical aberration in aqueous media.

As with all of Nikon’s microscopes, the NIS-Elements software is fundamental to successful imaging, greatly simplifying the acquisition and analysis of super-resolution images.

Case study:

The Nikon Center of Excellence (CoE) at the Heinrich Pette Institute (HPI), Hamburg, Germany, is a hub for research and education on the fundamentals of and advances in microscopy for investigating human pathogenic viruses. A study at HPI demonstrated the capabilities of multicolor 3D STORM technology for super-resolution imaging of hepatitis C virus (HCV) infection. STORM enabled the spatial distribution of the structural core and E2 envelope protein of HCV to be visualized in small lipid droplets on the membrane of the endoplasmic reticulum in infected cells. These areas of co-localization have a diameter of approximately 100 nm and are thought to represent viral assembly sites

(Eggert, D. et al. (2014) PloS ONE 9(7): e102511. Doi:10.1371/journal.pone.0102511).