Blog: Frequently Asked Questions (FAQs) for Microscopy

20th May 2024

In our latest blog, we asked our Bioassay expert, Robert Francis, to shed some light on the frequently asked questions that drug developers may have in regards to Microscopy.

1. What are the advantages of microscopy assays? 

Live cell microscopy is the cornerstone of biology, allowing observation of events in real-time. For us, being able to assay living cells is key to understanding the dynamics, rather than the endpoint of the assay. We can also observe the biology happening, providing far more information than just the measured parameter. An example where this can be effective is observing cell death, where we cannot just see the increase in fluorescence relating to the breakdown of the plasma membrane, but also the proliferation of the cells to ensure drugs that induce stasis are captured. Equally, a key advantage is that it is the least invasive technique in our repertoire. 

Even in end-point assays, microscopy can provide extremely valuable information that cannot be measured by any other technique. For example, super-resolution microscopy can be used to see the co-localisation of two proteins or receptors, close to single molecule resolution (down to 10 nm). Equally, with the advent of high content imaging and associated image analysis pipelines, applications such as trafficking of antibodies through the cells can be measured in a multi-well format, moving microscopy to a “high throughput” technique.  

One of the growing areas of in vitro biology is the use of models that more closely resemble in vivo testing to reduce the use of animals. With this, 3D spheroid models are now routinely used, for example, to test solid tumour penetrating drugs / ADCs. The best technique to interrogate these is imaging, and especially, 3D confocal imaging.   

At Abzena, we have a broad range of microscopy capabilities. For an overview of the key applications at Abzena, see the questions below or our webpage. 

 

2. Why use confocal over widefield microscopy? 

Widefield is the more “traditional” type of microscopy where a 2D image is taken of a 3D object (such as a cell). This is still a very useful type of microscopy, with the Incucyte we run many of our longer microscopy assays and is essentially a widefield microscope sitting in an incubator (see question 9 below). This can be used with fluorescent labelling, as mentioned below. However, one of the key issues of widefield microscopy can be out-of-focus light.  

Out-of-focus light can cause a blur in the images in certain circumstances, and this can make discerning where the fluorescent signal is, very difficult in these cases. To alleviate this, confocal microscopy was invented whereby pinhole apertures are placed in front of the excitation source and detectors. These pinholes remove the out-of-focus light, but also reduce the light available. This means that much more powerful excitation sources such as lasers, and highly sensitive detectors such as photomultiplier tubes or CCD cameras, are required.  

On thin samples, such as fixed cells, widefield and confocal microscopy can both be used. For thick samples, or where out-of-focus light may be a problem, such as fluorescence in the media, confocal microscopy may be a better option. For example, for the addition of a fluorescently labelled antibody, without washing steps, to see uptake dynamics in cells, confocal is recommended. Also, in any 3D applications, such as spheroids confocal microscopy will produce better images. 

Our experts at Abzena can work with you to find the best readout for your assays. 

 

3. What are the key applications of microscopy at Abzena? 

At Abzena, we can use microscopy to interrogate the functionality of your biologics, as well as screening applications to find lead candidates for further development.  

One of the key questions in the development of an ADC / bioconjugate is whether the biologic is internalizing and reaching the correct compartment in the cells. For example, we use internalization imaging assays and pH sensitive dyes to ensure that an ADC or antibody backbone is reaching the acidic lysosomes, as this will be essential to ensure drug release into the cell, enabling the cytotoxic effect. 

To investigate the mode of action of cell killing by ADCs, we routinely use microscopy to observe kinetics of cell death pathways, such as apoptosis and/or necrosis. To capture the bystander effect, we are perfecting co-culture models to see death sequentially between antigen positive and antigen negative cells. 

An application that is a bit more specialized is the use of 3D spheroids and co-cultures, which allows us to get closer to an in vitro model of a solid tumour microenvironment in vivo 

We can also use imaging to investigate the Fc-mediated function of a biologic, such as assessing antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). In ADCC assays, we measure cancer cell killing by NK cells, while in ADCP assays, we quantify cancer cell phagocytosis by macrophages. In both assays, we use peripheral blood monocyte (PBMC) derived primary effector cells from our large pre-screened PBMC cell bank. Further assays that can be performed using PBMCs as effectors include assessment of bispecific T cell engagers in co-cultures with cancer target cells.  

 

 4. How do you select the optimal fluorophores and labelling strategy? 

Fluorescence is a photo-chemical phenomena that is vital for many applications of microscopy. In essence, a photon is absorbed by a “fluorochrome, which then produces a photon in turn. The released photon will always be of a longer wavelength, known as “Stoke’s shift”. For example, fluorescein (FITC) will absorb blue light (approximately 488 nanometers (nm)) and release a green photon (approximately 550 nm).  

In fluorescence microscopy, as the photon released can be dim, we need sensitive monochrome detectors with fixed wavelength filters to measure the photons. For example, a 525-575 nm filter to pick up fluorescence from FITC. However, bleed-through from different fluorochromes can happen within a sample, so careful fluorochrome selection is key when wanting to interrogate multiple parameters. 

As well as the excitation and emission fluorescence properties, select fluorescent dyes can vary in intensity and this can be used to capture many different dynamic cellular processes. For example, at Abzena we use pH-sensitive dyes which increase in their fluorescence intensity as they enter the lysosomal acidic environment. This allows us to assess whether a mAb or biologic is reaching this compartment, a key prerequisite for ADC development. The same fluorophores can also be used for different techniques (e.g. flow cytometry) to provide an orthogonal readout for the same function. If this is the case, both instrument’s configurations need to be taken into consideration when choosing the fluorophore. 

Besides selecting fluorophores, the labelling strategy also needs to be decided. There are two key ways to label a biologic, such as a mAb, for use in fluorescence microscopy: either through direct chemical conjugation such as esterification; or through secondary labelling such as a fluorescently labelled secondary antibody or Fab fragment. Generally, we use polyclonal Fab fragments wherever possible, as this “off-the-shelf add and use” method allows us to assess a large number of biologics at a reduced cost. When a biologic doesn’t have the Fc region, for example nanobodies, then we gravitate towards direct conjugation.  

As explained above, designing optimal microscopy assays can be a complex task. Our scientists at Abzena have the expertise to help design and execute such experiments to maximize the chance of success.  

 

5. How many parameters can we image? 

For the Incucyte, we have two fluorescent channels available: excitation of 440-480 nm and emission of 504-544 nm for the green channel; and excitation of 565-605 nm and emission of 625-705 nm for the red channel. In addition, we have the phase contrast channel to image all the cells present and can measure parameters such as proliferation.  

The confocal microscope instruments are designed to be as versatile as possible. Generally, we only go to 5 colors maximum across the visible light spectrum, right from near-ultra-violet to near-infrared, plus the phase contrast image. Add time to these experiments and this adds a total of 7 parameters.  

However, with the use of advanced image processing or spectral imaging, we can now push this to 6 fluorescent parameters or more. One of the recent developments in high content imaging, “cell painting”, uses 6 fluorescent parameters to capture the complete cell health by using fluorescent dyes to label different cellular compartments including the nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, Golgi apparatus, and RNA. 

Our scientists have the experience to provide the best advice in the design of your experiments. So again, if you have a research question that you think could be addressed by the instruments mentioned above, please speak to us.  

 

6. Does microscopy provide qualitative or quantitative data? 

It is a common myth that microscopy is solely qualitative! Using machine learning algorithms, we can use the phase contrast channel to measure the size of the cells and the total cell area within an image. This allows us to produce a parameter such as area change (in µm2) over time to illustrate proliferation of the cells. 

In fluorescence microscopy, the intensity can be measured on a cell, and even on a per-pixel basis. We can then use this information to quantify the fluorescence overlap or intensity changes. With the use of single cell analysis, such as the “cell-by-cell” analysis on the Incucyte, we can produce flow cytometry style intensity plots using the median fluorescence intensity (MFI) in the individual cells. In fact, there is so much quantitative data you can get from a high-throughput real-time microscopy experiment, that choosing the right parameters to plot, and how to present them to best inform both you and your stakeholders, can be a tricky task. We can support your program by making sure we assemble the most suitable data package for your specific purposes. 

However, with all the benefits of quantitative data, the impact of qualitative data should not be underestimated. Nothing makes a bigger impact on non-scientific audiences, such as investors or the public, than having a movie of your lead candidate doing what they are supposed to be doing, such as cancer cells popping when necrosis is induced from an ADC! 

 

7. What instruments do we have available? 

We have the Sartorius Incucyte® widefield imaging platform that sits within an incubator, allowing us to complete long-term timelapse microscopy of cultured cells. Compared to traditional microscopes, the way the Incucyte images is novel, as it moves the microscope underneath the stationary samples, rather than traditional high throughput systems that move the samples, and the microscope is stationary. By moving the microscope under the stationary samples there is minimal disruption to the samples, allowing for further long-term tracking. Many of our “off-the-shelf” assays are performed on this system, including internalisation of antibodies to the lysosome, ADCP using donor PBMC derived macrophages, ADCC using isolated NK cell or PBMC effectors, co-culture assays for bispecific natural killer cells, bispecifics using T cell engagers, bystander assays, 2D viability assays using monolayer culture, and 3D viability assays using spheroids.  

For confocal microscopy, we have access to the Babraham Institute confocal microscopy facilities. This includes spinning disk confocal microscopy, confocal laser scanning microscopy, and super-resolution microscopy (PALM and SIM). As the microscope systems have heated chambers and CO2, it is possible to image live cells under cell culture conditions (5% CO2 and 37OC). Some of the applications we run on these microscopes include trafficking of biologics through cellular pathways (in 2D and 3D), and Ca2+ signaling in response to agonists.  

 

8. Why use Abzena for your microscopy assays? 

Microscopy can be used at various stages of the discovery and development process, providing critical value by generating data we can trust to make high quality decisions, such as selecting the best candidate for development. At Abzena, we have some of the most capable imaging scientists around. We have produced microscopy data to support our customers with Investigative New Drug (IND) submissions and securing funding for their programs.  

We have various ‘off-the shelf’ assays such as internalization, ADCP, ADCC, 2D viability using monolayer culture, and 3D viability assays using spheroids, which we routinely use to assess biologics, even as part of high throughput screening. However, if you have a biologic with a novel, complex mode of action, we can collaboratively work with you to create bespoke microscopy assays tailored to your biologic and function of interest. Microscopy assays can be run as standalone projects or complement other assays available at Abzena.  

We can even multiplex assays combining techniques in a single run, for example, combining apoptosis and necrosis measurements on the Incucyte with the industry standard CellTiter-Glo® assay for viability; or fluorescence measurements using both imaging and flow cytometry.  

Taken together, at Abzena, we have the depth of expertise to provide comprehensive support for the imaging needs of your biologic or ADC development program. Click here, to learn more about our Bioassay capabilities and how they can help your program start smart and finish fast.