Designing Microscopy Experiments Related To Infectious Diseases And Antivirals

Designing microscopy experiments related to infectious diseases and antivirals can be challenging, but there’s never been a more vital time than right now to design adequate microscopy experiments. The novel coronavirus (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and spread across the globe becoming the pandemic that the world is reeling with today. Currently, COVID-19  has no targeted therapies approved by the FDA, so the best coronavirus prevention happens through social distancing and good hygiene practices. However, companies are rapidly testing candidate molecules and vaccines as fast as they can. Initial tests suggest there may be some drugs that could be repurposed for COVID-19 treatment. Repurposed drugs have already shown their safety, so clinical trials need to be conducted for effectiveness only. 

SARS-CoV2 belongs to a family of viruses that we’ve encountered before, so there is hope that we can apply the knowledge we have about SARS and MERS for treatment. Looking towards the future, since 75% of emerging infections have come from animals (zoonosis) we will continue to see new pathogens in our lifetimes. What does the research & development pipeline look like for combating these new infectious diseases?  

In 2013, I worked for a company called Evrys that works on host-directed antivirals. Drugs like these are special because the therapeutic target is our cells instead of the virus directly. There are different pathways towards a successful drug, but this outline should be applicable to many research and development (R&D) workflows. Here, we will go over the steps that R&D takes to develop new treatments and how microscopy assists with the research.

Taken from Hughes et al. 2010

1. Determine a druggable target.

Viruses can be grouped into a major class even if they are new.  All coronaviruses (including SARS-CoV & MERS-CoV) are enveloped positive-sense RNA viruses that have club-like spikes on their surface and similar life cycle steps, allowing for prior knowledge of similar viruses to be used. If we encounter a new virus, sequencing the genome and obtaining electron microscopy images is the best place to start to determine what type of virus we are dealing with. Common antiviral targets interfere with receptor binding, membrane fusion/entry, replication, protein translation, and viral release. While writing this, Clinicaltrails.gov listed 112 active clinical trials for COVID-19 treatment. Current targets include RNA-dependent RNA polymerase (remdesivir, favipiravir) and viral entry (Baricitinib, ACE inhibitors). Knowing the target can help narrow down the compounds used in the primary screen, but it is not necessary due to the simple readout of “virus” or “no virus.”

2. Primary Screens

Primary screens are when scientists test compounds (numbering from 1K to 10K) for antiviral activity. The “effective concentration” is the concentration of product at which virus replication is inhibited by 50%, e.g. EC50 for cell-based assays and IC50 for biochemical or subcellular assays.

The most common microscopy technique used in this stage of drug development is high throughput screening (HTS). There are a variety of microscopes that are capable of HTS, but they all share similar features.  These microscopes accept 96-well to 1536-well plates and automatically image all the wells for the users. 

Ideally, specific antibodies have been produced at this point to allow for fluorescence microscopy.    

This entails using widefield or confocal microscopes to observe a decrease in viral load, as well as cytopathic effects (structural cell changes). Cytopathic effects can often be seen using phase contrast or DIC microscopy.  For more information on determining the health of your cells, check out our 6 Microscopy Assays To Determine Cell Health and Improve Your Experimental Results blog.

3. Secondary Screens

Once a potential drug has been identified, it is important to ensure that there are not any unintended side effects on healthy cells. The goal is to stop viral replication without killing the uninfected cells. This work again uses a HCS microscope. It tests a series of increasing concentrations of the possible drugs to determine the concentration at which the compound kills 50% of the cells (known as CC50 value). The concentration for which the compound kills 50% of the cell in culture is divided by the concentration needed to kill 50% of the virus, giving us a therapeutic index. The therapeutic index is important because it provides the difference in concentration between when it is helpful and when it could be toxic to the patient. Many over the counter drugs such as Claritin (loratadine) have a high therapeutic index and thus it’s difficult for a patient to overdose. The drugs that have the best therapeutic index will undergo further optimization with chemists making many small changes. And you guessed it, more cellular assays on HCS microscope to ensure safety.

4. Antiviral Activity in Vivo 

A lot of good information can come from in vitro studies, but they can’t replicate the effects observed in a whole organism. This is where in vivo studies become useful. In vivo studies generally rely on developing an animal model of the infection and treating it with possible drugs. Work on coronavirus is currently being performed using a specialized mouse that has the human ACE2 receptor.  

Animal studies study the effect of the drug on a whole-body level. The main goal is to determine important information for regulatory agencies such as dosage, toxicity, and/or carcinogenicity. For a more complete list of parameters check here. During the course of the in vivo study, researchers measure parameters such as weight and temperature regularly.  After the animal dies, researchers use brightfield microscopy and histological staining of tissues and blood to determine any toxic or carcinogenic damage that has occurred due to treatment.  

The most basic of the assays is staining tissue sections from different organs with hematoxylin and eosin (H&E). Animal studies for coronavirus would obviously examine the lungs, but it is also important to examine any histopathological changes in the gastrointestinal tract, liver, kidneys, heart, thyroid, and brain. Drug side effects occur when there are off-target effects in the cells. Although anyone can perform the histology sample preparation and imaging, someone with proper pathology training should analyze the samples. Imaging can be done using any brightfield microscope, but drug development companies have moved to use slide scanners. Slide scanners are digital microscopes that image the entire slide (or specified region) and can image 100s of slides each day. Not only do slide scanners allow high throughput imaging, but they can achieve incredible reproducibility.  

Other microscope assays that are important in the in vivo stage are blood smears and apoptosis assays. Blood smears are paired with histological dyes to examine changes in red blood cells, white blood cells, and platelets. Changes in these blood components can be an indicator of more serious systemic changes.  Finally, apoptosis assays are important to determine any cellular death caused by the antiviral.  There are a variety of different ways to measure apoptosis, but in tissue sections, TUNEL assays are the preferred method due to the simplicity and the ability to perform the assay in a high throughput manner. 

Microscopes are necessary at several stages of the research and development of antivirals. Time is very valuable, especially with the current pandemic, so learning to use high throughput machines is key to being successful. Although many of the features are automated, it is important to know enough about the microscope and the biology to ensure biologically relevant data are obtained. Just like in normal microscopy, don’t forget the proper controls! You don’t want thousands of pictures that are of no use to you or even worse, follow an incorrect lead during development.

To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class
Heather Brown-Harding
Heather Brown-Harding

Heather Brown-Harding, PhD, is currently the assistant director of Wake Forest Microscopy and graduate teaching faculty.She also maintains a small research group that works on imaging of host-pathogen interactions. Heather is passionate about making science accessible to everyone.High-quality research shouldn’t be exclusive to elite institutions or made incomprehensible by unnecessary jargon. She created the modules for Excite Microscopy with this mission.

In her free time, she enjoys playing with her cat & dog, trying out new craft ciders and painting.You can find her on twitter (@microscopyEd) a few times of day discussing new imaging techniques with peers.

Similar Articles

How To Profile DNA And RNA Expression Using Next Generation Sequencing

How To Profile DNA And RNA Expression Using Next Generation Sequencing

By: Deepak Kumar, PhD

Why is Next Generation Sequencing so powerful to explore and answer both clinical and research questions. With the ability to sequence whole genomes, identifying novel changes between individuals, to exploring what RNA sequences are being expressed, or to examine DNA modifications and protein-DNA interactions occurring that can help researchers better understand the complex regulation of transcription. This, in turn, allows them to characterize changes during different disease states, which can suggest a way to treat said disease.  Over the next two blogs, I will highlight these different methods along with illustrating how these can help clinical diagnostics as well as…

Optimizing Flow Cytometry Experiments - Part 2         How To Block Samples (Sample Blocking)

Optimizing Flow Cytometry Experiments - Part 2 How To Block Samples (Sample Blocking)

By: Tim Bushnell, PhD

In my previous blog on  experimental optimization, we discussed the idea of identifying the best antibody concentration for staining the cells. We did this through a process called titration, which  focuses on finding the best signal-to-noise ratio at the lowest antibody concentration. In this blog we will deal with sample blocking As a reminder, there are two other major binding concerns with antibodies. The first is the specific binding of the Fc fragment of the antibody to the Fc Receptor expressed on some cells. This protein is critical for the process of destroying microbes or other cells that have been…

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

By: Heather Brown-Harding, PhD

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

What Is Next Generation Sequencing (NGS) And How Is It Used In Drug Development

What Is Next Generation Sequencing (NGS) And How Is It Used In Drug Development

By: Deepak Kumar, PhD

NGS methodologies have been used to produce high-throughput sequence data. These data with appropriate computational analyses facilitate variant identification and prove to be extremely valuable in pharmaceutical industries and clinical practice for developing drug molecules inhibiting disease progression. Thus, by providing a comprehensive profile of an individual’s variome — particularly that of clinical relevance consisting of pathogenic variants — NGS helps in determining new disease genes. The information thus obtained on genetic variations and the target disease genes can be used by the Pharma companies to develop drugs impeding these variants and their disease-causing effect. However simple this may allude…

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

By: Tim Bushnell, PhD

Over the next series of blog posts, we will explore the different aspects of optimizing a polychromatic flow cytometry panel. These steps range from figuring out the best voltage to use, which controls are critical for data interpretation, what quality control tools can be integrated into the assay; how to block cells, and more. This blog will focus on determining the optimal antibody concentration.  As a reminder about the antibody structure, a schematic of an antibody is shown below.  Figure 1: Schematic of an antibody. Figure from Wikipedia. The antibody is composed of two heavy chains and two light chains that…

5 Drool Worthy Imaging Advances Of 2020

5 Drool Worthy Imaging Advances Of 2020

By: Heather Brown-Harding, PhD

2020 was a difficult year for many, with their own research being interrupted- either by lab shutdowns or recruitment into the race against COVID-19. Despite the challenges, scientists have continued to be creative and have pushed the boundaries of what is possible. These are the techniques and technologies that every microscopist was envious of in 2020. Spatially Resolved Transcriptomics Nature Methods declared that spatially resolved transcriptomics was the 2020 method of the year. These are a  group of methods that combine gene expression with their physical location. Single-cell RNA sequencing (scRNAseq) was originally developed for cells that had been dissociated…

Picking The Right Functional Imaging Probe

Picking The Right Functional Imaging Probe

By: Heather Brown-Harding, PhD

As biologists, we study the process of life, however, it’s intricacies cannot be captured by a snapshot in time. Generally, the easiest imaging experiments are those where the samples are stained, fixed, and imaged within a few days of procurement, but that too doesn’t capture the dynamic processes common in cells and organisms. Live cell imaging when combined with reporters serves as a powerful tool to provide solid imaging data. Cameleon —one of the first reporters— was developed in 1997 in Roger Tsien’s lab.  Cameleon is a green fluorescent protein (GFP) that undergoes a conformational change in the presence of…

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

By: Tim Bushnell, PhD

The heart and soul of the flow cytometry experiment is the ‘panel.’ The unique combinations of antibodies, antigens, fluorochromes, and other reagents are central to identifying the cells of interest and extracting the data necessary to answer the question at hand. Designing the right panel for flow cytometry is essential for detecting different modalities. The more parameters that can be interrogated will yield more information about the target cells. Current instruments can measure as many as 40 different parameters simultaneously. This is exciting, as it allows for more complex questions to be studied. Panel design is also valuable for precious samples,…

7 Key Image Analysis Terms For New Microscopist

7 Key Image Analysis Terms For New Microscopist

By: Heather Brown-Harding, PhD

As scientists, we need to perform image analysis after we’ve acquired images in the microscope, otherwise, we have just a pretty picture and not data. The vocabulary for image processing and analysis can be a little intimidating to those new to the field. Therefore, in this blog, I’m going to break down 7 terms that are key when post-processing of images. 1. RGB Image Images acquired during microscopy can be grouped into two main categories. Either monochrome (that can be multichannel) or “RGB.” RGB stands for red, green, blue – the primary colors of light. The cameras in our phones…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.