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Technology Advancements Improve Both Clinical and Research-Based Imaging Needs

This editorial from JALA Editor-in-Chief Edward Kai-Hua Chow on the JALA Special Issue on High-Throughput Imaging Technologies is reprinted with permission of the Journal of Laboratory Automation.

On behalf of the Journal of Laboratory Automation (JALA), I am pleased to present this special issue on advancements in imaging technology that improve both clinical and research-based imaging needs, particularly focusing on advancements that integrate automation and high-throughput into traditional and emerging imaging applications.

Ever since Antonj van Leeuwenhoek and Robert Hooke began using microscopes to study biological samples, advancements in imaging technology have been fundamentally linked to the advancement of life sciences research and medical diagnosis and treatments. Scientific imaging technologies now cover a wide range of technologies and applications following a number of key advancements such as the invention of magnetic resonance imaging and the invention of monoclonal antibody hybridomas. In recent years, advances in micro/nano-technology and digital imaging quantification have led to the development of high-throughput, automated imaging applications in both the laboratory and the clinic. This issue features several reports that highlight these advancements and demonstrate how advances in imaging can improve life sciences research and drug-development as well as how clinicians diagnose and treat patients.

Advances in Imaging Technology in Life Sciences Research

Imaging technology has transformed the ability of life sciences researchers to interrogate cellular processes in ever increasing detail and efficiency. In particular, Förster or fluorescence resonance energy transfer (FRET) is an imaging technique that has allowed researchers to quantify and study changes in molecular structure and conformation as well as intermolecular interactions such as protein-protein, protein-nucleic acid or protein-small molecule interactions. FRET involves the transfer of energy from donors to acceptors to alter the fluorescence ratio between donors and acceptors that can be used to image and quantify molecular changes.

One of the most common methods of quantification of FRET activity involves the determination of fluorescence lifetime, such as fluorescence lifetime imaging microscopy (FLIM), which detects the decay of energy from the donor molecule as it continues to interact with the acceptor molecule. FLIM is useful in the study of protein-protein interactions in the context of live cells, particular in response to cellular stimuli such as toxin, drugs and activation of specific signaling pathways.

Advances in automated microscopy as well as integration of automated image capture and image analysis have allowed for the development of high-throughput FLIM-FRET microscopy to quickly quantify protein-protein interaction in cells. The addition of automation to FLIM-FRET microscopy allows researchers to quickly interrogate within a single slide or multi-well plate multiple samples or multiple cellular processes in a high-throughput manner. This will be useful in systems biology studies such as predictive chemogenomics that can correlate phenotypic drug response to key genomic alterations.

Advances in robotics and automation technology are integral to the development of high-throughput imaging tools. Just as important are advances in automated image analysis software to handle and process the large amounts of data generated by high-throughput imaging tools such as automated high-content microscopy systems. A major benefit of automated image analysis software is the ability to quickly identify and quantify multiple parameters in images that are difficult to quantify subjectively by the researcher. By providing a specific set of imaging analysis procedures, complex cellular processes such as autophagy can be quantitatively determine through imaging. One such software, IFDOTMETER, can be used to quickly automate the workflow needed to quantify autophagy, cell survival and other cellular processes by integrating both fluorescent protein expression signals as well as cellular morphology and cellular compartment determination into a single readout for quantifying desired cellular responses. As more researchers become exposed to high-content imaging analysis, automated imaging analysis software such as this will provide the wider research audience with the imaging tools needed to quickly analyze complex cellular processes in a large number of samples.

Marix-Assisted Laser Desorption Ionization (MALDI) imaging mass spectrometry (IMS) provides region-specific mass spectrometry to identify and measure individual molecular entities in tissue sections and provide an accurate and quantitative imaging approach to molecular measurements in tissue section samples. As such, unique images of tissue biology can be created based on highly sensitive measurements of proteins, lipids, drugs and even metabolites. Because of the versatility of MALDI-IMS in providing a comprehensive and informative image from tissue sections for such a wide variety of molecules, it is becoming an increasingly popular method for analyzing clinical samples when studying disease states or drug responses as well as analyzing biological samples when investigating molecular changes in preclinical models. In MALDI-IMS, a matrix solution is applied to the tissue section, followed by laser ablation to desorb and ionize analytes in specific regions to identify and quantify analytes.

Because of the complexity of these steps, much of this work has taken advantage of recent advances in automation and high-throughput mass spectrometry to achieve a stable and efficient MALDI-IMS system. Matrix application is critical to the success of MALDI-IMS as the matrix solution facilitates ionization and extraction of analytes. Thus, developing a reliable customizable automated matrix sprayer is critical to the success of any MALDI-IMS system. As much of the automated technology becomes more widely available, the ability to build custom matrix sprayers, such as the Langertech, is possible. These custom matrix sprayers can be programmed to minimize spot size and disperse matrix solution in a homogenous manner at a fraction of the cost of more traditional automated matrix sprayers. When used to analyze preclinical mouse kidney tissue sections by MALDI-IMS, images generated with the Langertech were only minimally less detailed than those generated following matrix spraying by more expensive commercial units. Furthermore, key peak intensities and peak counts were the same across both systems. By demonstrating that custom-built automated matrix sprayers can achieve research-grade quality MALDI-IMS results at a greatly reduced cost, this work brings MALDI-IMS to a wider audience of researchers who would otherwise not be able to afford to incorporate this powerful imaging method into their research.

Advances in Imaging Technology in Drug Development

Following the rise of biotechnology, molecular-target based drug screening replaced phenotypic-based drug screening as the preferred approach toward identifying new drugs, particularly after the mapping of the human genome and the development of molecular tools to understand the underlying genomic causes of diseases. Recent advances in quantitative high-throughput imaging, however, have led to a resurgence in phenotypic approaches to drug screening. For example, high-content analysis has allowed for multi-parameter quantitative phenotypic drug screening to identify candidate drugs against phenotypes and cellular functions that were previously difficult to quantify such as cellular differentiation or motility in combination with more traditional readouts such as cell survival or protein/gene expression.

In order to truly adapt high-content imaging towards drug-development, a number of support technologies are needed to further automate the process and increase throughput. These include integrated automated sample processing, cell seeding and high-content specific assays. Nowhere is this more true than the application of high-content screening toward 3D cell cultures, such as 3D tumor spheroids, that require more complex methods for sample preparation as well as imaging analysis compared to 2D surface adherent cell cultures. In order to facilitate high-throughput high-content 3D spheroid screening, 3D spheroid culture specific technology has to be developed to facilitate automated collection and analysis of 3D spheroids, such as specialized tissue culture plates that can handle transfer and imaging of 3D spheroids. Adaptation of automated liquid handling robotic technology and micropattern designs toward 3D spheroid culture as seen in the preparation of Oncospheres in 96-well plates are examples of key advances in high-throughput imaging technology that allow for reliable reproducibility of sample preparation and imaging that is critical to the implementation of automated high-throughput high-content screening into drug development.

Bioanalytical applications that allow for detailed detection and analysis of biomolecules and pharmaceutical compounds are needed at all phases of drug development and production to ensure consistent manufacturing and production of therapeutics. Enhanced automated bioanalytical assays can allow for lowered costs of drug development and production. One such bioanalytical application that shares crossover with imaging applications utilizes native fluorescence as well as fluorophore-mediated detection is capillary electrophoresis (CE). CE is capable of quickly detecting the fluorescence of protein, nucleic acids and small molecules in biological samples as well as in sample preparation for both drug development and clinical diagnostics.

One use of CE is in N-glycosylation analysis of therapeutic antibodies as glycosylation of antibodies and therapeutics with different glycans can affect safety and efficacy. Analysis of glycosylation by CE requires several sample processing steps including glycan release, fluorophore labeling and labeled sample isolation are all time consuming and previously not amenable to high-throughput analysis. The integration of all these processes into a fully automated laboratory workstation allows for the use of CE in antibody glycosylation analysis in a reliable high-throughput manner.

Advances in imaging technology related to all aspects of assay workflow are needed to fully realize automated high-throughput imaging capabilities. As shown in this special issue, different imaging techniques have unique hurdles that are being overcome through advances in everything from automated imaging analysis software and sample preparation to integration of multiple complex steps into a single automated workflow. In addition to improving how researchers interrogate cellular processes and develop better therapeutics, advances in imaging technology continue to improve clinical diagnostics and treatment as well.

Clinical Translation of Advances in Imaging Technology

Imaging technology has become critical to a wide variety of clinical diagnostic tests. Many advances in imaging technology that allow for automated high-throughput imaging also improve the implementation of complex diagnostic assays in the clinic by lowering the cost per assay and reducing the need for specialized training to perform diagnostic tests. Infectious diseases, such as influenza viral infections, represent a major global health issue that can benefit from advancements in imaging technology. Rapid diagnosis of influenza infections is critical to identifying and treating patients as well as for global surveillance of infection rates.

The most common assay to determine response to influenza infections is the hemagglutination inhibition (HAI) assay that is based on the ability of influenza viruses to agglutinate red blood cells (RBCs) and the fact that antibodies developed in response to influenza infections can inhibit this hemagglutination. This assay previously relied on subjective imaging analysis of RBC morphology that represented nonagglutinated and agglutinated RBCs. Automated imaging analysis of RBCs following an HAI assay reduces human error and decreases processing time of multiple samples through both automated image acquisition as well as automated image processing and analysis.25 Automation of the HAI assay can result in 400 samples being accurately processed within 30 minutes without human interference or aid.

Another imaging assay with implications in the clinical study of infection and cancer-related diseases is the optical analysis of hematological cells within pleural effusions or ascetic/peritoneal fluids. An increase in total nucleated cells (TNCs) as well as a prevalence of hematological cells is often representative of tuberculous peritonitis progression or metastatic cancer. Most commonly, a trained professional performs this assay manually by optical microscopy. Because of the reliance on manual preparation and subjective individual interpretations, this assay can lead to imprecise and variable results following time-consuming sample preparation.

Just as the integration of automated imaging workstations with user-friendly imaging analysis software has led to quantitative high-throughput imaging assays for life sciences research and drug development, the integration of automated imaging-based cell counters with image analysis software can be applied toward automated cell counting and differentiation in biological samples such as pleural effusions. TNC determination from automated hemocytometers, such as the Sysmex XE-5000, can outperform manual optical microscopy when paired with a set of rules for reflex testing to provide more precise TNC counts as well as improve cell identification in pleural effusions and peritoneal fluids, providing faster and more reliable diagnosis of disease statuses such as metastatic cancer.

Advances in imaging technology not only provide cheaper, faster and more reliable diagnosis. Advances in imaging technology through miniaturization, automation and ease of use can allow imaging to be part of interventional therapies as image-guided therapeutic techniques have begun to gain traction in the clinic. One of the most common image-guided interventional therapeutic techniques is arterial stent implantation. Using a variety of imaging techniques including ultrasound, fluoroscopic or magnetic resonance imaging for guided delivery and placement of stents, a wide variety of stents can be implanted in difficult to reach areas, including more advanced drug delivery stents.

More recently, renal denervation has been introduced as a method for treating hypertension in patients whose blood pressure cannot be therapeutically controlled by impairing sympathetic activity that was found to contribute to the pathogenesis of hypertension. A prime example of image-guided therapy, renal denervation commonly involves the use of a guided catheter that emits a radio frequency toward the intimal walls of the renal artery to ablate the renal nerve and reduce blood pressure. Because this technique is still in the early stages of adoption, there are a number of devices that are competing to become the standard in renal denervation. A key aspect of many of these devices lies in the miniaturization of various components into the form and function of catheters in order to deliver a radio frequency or ultrasonic wave suitable for renal nerve ablation. As such, renal denervation is a prime example of image-guided interventional therapy where recent advances in technology can greatly impact clinical practice.

The original research, technology briefs and informative review presented in this JALA special issue on high-throughput imaging cover a wide range of both basic life sciences research as well as clinical applications where advances in imaging technology surrounding these applications will greatly impact and improve how imaging is utilized to both better understand the intricacies of life as well as diagnose and treat patients in a more cost-efficient and less-invasive manner.

April 18, 2016