The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients

Phenotypic analysis of extracellular vesicles:a review on the applications of fluorescence

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated

An automated fluorescence lifetime imaging multiwell plate reader: application to high content imaging of protein interactions and label free readouts of cellular metabolism

This thesis reports on work performed in the development and application of an automated plate reading microscope implementing wide field time gated fluorescence lifetime imaging technology. High content analysis (HCA) imaging assays enabled by automated microscopy platforms allow hundreds of conditions to be tested in a single experiment. Though fluorescence lifetime imaging (FLIM) is established in life sciences applications as a method whereby quantitative information may be extracted from time-resolved fluorescence signals, FLIM has not been widely adopted in an HCA context. The FLIM plate reader developed throughout this PhD has been designed to allow HCA-FLIM experiments to be performed and has been demonstrated to be capable of recording multispectral, FLIM and bright field data from 600 fields of view in less than four hours. FLIM is commonly used as a means of reading out Förster resonance energy transfer (FRET) between fluorescent fusion proteins in cells. Using the FLIM plate reader to investigate large populations of cells per experimental condition without significant user input has allowed statistically significant results to be obtained in FRET experiments that present relatively small changes in mean fluorescent lifetime. This capability has been applied to investigations of FOXM1 SUMOylation in response to anthracycline treatment, and to studies of the spatiotemporal activation profiles of small GTPases. Furthermore, the FLIM plate reader allows FLIM-FRET to be applied to protein-protein interaction screening. The application of the instrument to screening RASSF proteins for interaction with MST1 is discussed. The FLIM plate reader was also configured to utilise ultraviolet excitation radiation and optimised for the measurement of autofluorescence lifetime for label-free assays of biological samples. Experiments investigating the autofluorescence lifetime of live cells under the influence of metabolic modulators are presented alongside the design considerations necessary when using UV excitation for HCA-FLIM.Open Acces

High-Throughput Nonlinear Optical Microscopy

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.National Institutes of Health (U.S.) (9P41EB015871-26A1)National Institutes of Health (U.S.) (R01-EX017656)National Institutes of Health (U.S.) (5 R01 NS051320)National Institutes of Health (U.S.) (4R44EB012415-02)National Science Foundation (U.S.) (CBET-0939511)Singapore-MIT AllianceSkolkovo Institute of Science and TechnologySingapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)Wellcome Trust (London, England) (Massachusetts Institute of Technology. Postdoctoral Fellowship 093831/Z/10/Z

Fluorescence Lifetime Imaging of Quantum Dot Labeled DNA Microarrays

Quantum dot (QD) labeling combined with fluorescence lifetime imaging microscopy is proposed as a powerful transduction technique for the detection of DNA hybridization events. Fluorescence lifetime analysis of DNA microarray spots of hybridized QD labeled target indicated a characteristic lifetime value of 18.8 ns, compared to 13.3 ns obtained for spots of free QD solution, revealing that QD labels are sensitive to the spot microenvironment. Additionally, time gated detection was shown to improve the microarray image contrast ratio by 1.8, achieving femtomolar target sensitivity. Finally, lifetime multiplexing based on Qdot525 and Alexa430 was demonstrated using a single excitation-detection readout channel

Fluorescence Multiplexing with Combination Probes for Biological and Diagnostic Applications

Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts. Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures. I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells. Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes. I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer

Artificial Biosystems by Printing Biology

The continuous progress of printing technologies over the past 20 years has fueled the development of a plethora of applications in materials sciences, flexible electronics, and biotechnologies. More recently, printing methodologies have started up to explore the world of Artificial Biology, offering new paradigms in the direct assembly of Artificial Biosystems (small condensates, compartments, networks, tissues, and organs) by mimicking the result of the evolution of living systems and also by redesigning natural biological systems, taking inspiration from them. This recent progress is reported in terms of a new field here defined as Printing Biology, resulting from the intersection between the field of printing and the bottom up Synthetic Biology. Printing Biology explores new approaches for the reconfigurable assembly of designed life-like or life-inspired structures. This work presents this emerging field, highlighting its main features, i.e., printing methodologies (from 2D to 3D), molecular ink properties, deposition mechanisms, and finally the applications and future challenges. Printing Biology is expected to show a growing impact on the development of biotechnology and life-inspired fabrication