Fluorescence-based Method Research Articles

Polymer-mediated tuning of the monomer-aggregate equilibrium of a coumarin derivative for ratiometric sensing of protamine.

Amongst the various existing methods of analyte quantification, fluorescent-based methods, especially the ratiometric methods, continue to gain significant attention due to their high reproducibility, low environmental influence, and self-calibrating behavior. This paper presents the modulation in a monomer-aggregate equilibrium of coumarin-7 (C7) dye at pH ∼ 3, under the influence of a multi-anionic polymer, poly(styrene sulfonate) (PSS), leading to a significant modification in the ratiometric optical signal of the dye. At pH ∼ 3, cationic C7 formed aggregates in the presence of PSS via a strong electrostatic interaction, resulting in the development of a new emission peak at 650 nm at the expense of the monomer emission at 513 nm. Such contradicting changes in fluorescence intensities at two different wavelengths gave rise to a ratiometric signal, which was found to be highly sensitive towards external stimuli such as pH, and ionic strength. The stability of the C7-PSS complex was found to decrease as the pH of the solution was increased beyond 5, which indicated the decline in the electrostatic attraction between C7 and PSS due to the deprotonation of the C7 dye. Furthermore, an increase in the monomeric peak and a concomitant decrease of the aggregate peak with added salt in the solution (at pH ∼ 3) clearly justified the presence of an electrostatic attraction between C7 and PSS for the complex formation. This was further validated by the excited-state lifetime measurement of the C7-PSS complex, which showed a systematic increase in lifetime contribution from the monomeric species at the expense of aggregated species, as the concentration of NaCl increased in the solution. Thus, protamine (Pr), being a highly positively charged polypeptide, largely affected the monomer-aggregate equilibrium of the C7-PSS system, leading to a phenomenal change in the ratiometric signal, which was utilized to quantify with LOD as low as ∼2.8 nM in buffer for the bio-analyte Pr. Moreover, the ratiometric response of the C7-PSS assembly demonstrated excellent selectivity towards Pr, facilitating its practical relevance for the quantification of Pr in a 1% human serum matrix. Therefore, the studied C7-PSS can be utilized as a potential candidate for the quantification of the protamine even in complex biological media.

Quantitative Spatio-temporal Analysis of Phagosome Maturation in Live Cells.

Phagocytosis and phagosome maturation are central processes to the development of the innate and adaptive immune response. Phagosome maturation is a continuous and dynamic process that occurs rapidly. In this chapter we describe fluorescence-based live cell imaging methods for the quantitative and temporal analysis of phagosome maturation of beads and M. tuberculosis as two phagocytic targets. We also describe simple protocols for monitoring phagosome maturation: the use of the acidotropic probe LysoTracker and analyzing the recruitment of EGFP-tagged host proteins by phagosomes.

Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

Detection of Azo Dyes Using Carbon Dots from Olive Mill Wastes

Azo dyes are widely spread in our day life, being heavily used in cosmetics, healthcare products, textile industries, and as artificial food colorants. This intense industrial activity, which inherently includes their own production, inexorably leads to uncontrolled release of dyes into the environment. As emerging pollutants, their detection, particularly in water systems, is a priority. Herein, a fluorescence-based method was employed for the sensitive and selective detection of anionic and neutral azo dyes. Carbon dots (CDs) synthesized from wet pomace (WP), an abundant semi-solid waste of olive mills, were used as probes. An outstanding capability for detection of azo dyes methyl orange (MO) and methyl red (MR) in aqueous solutions was disclosed, which reached a limit of detection (LOD) of 151 ppb for MO. The selectivity of WP-CDs for the anionic azo dye (MO) was established through competitive experiments with other dyes, either anionic (indigo carmine) or cationic (fuchsin, methylene blue, and rhodamine 6G); perchlorate salts of transition metal cations (Cu(II), Co(II), Fe(II), Fe(III), Hg(II), and Pb(II)); and sodium salts of common anions (NO3−, CO32−, Cl−, and SO42−). Evidence has been collected that supports static quenching as the main transduction event underlying the observed quenching of the probe’s fluorescence, combined with a dynamic resonance energy transfer (RET) mechanism at high MO concentrations.

Metabolite quantification: A fluorescence-based method for urine sample normalization prior to 1H-NMR analysis.

Metabolomics is a multi-discipline approach to systems biology that provides a snapshot of the metabolic status of a cell, tissue, or organism. Metabolomics uses mass spectroscopy (MS) and nuclear magnetic resonance (NMR) to analyze biological samples for low molecular weight metabolites. Normalize urine sample pre-acquisition to perform a targeted quantitative analysis of selected metabolites in rat urine. Urine samples were provided from rats on a control diet (n = 10) and moderate sucrose diet (n = 8) collected in a metabolic cage during an eight hour fast. Urine from each sample was prepared by two different methods. One sample was a non-normalized sample of 1200µL and the second sample was a variable volume-normalized to the concentration of urobilin in a standard sample of urine. The urobilin concentration in all samples was determined by fluorescence. Ten metabolites for each non-normalized and normalized urine sample were quantified by integration to an internal standard of DSS. Both groups showed an improvement in pH range going from non-normalized to normalized samples. In the group on the control diet, eight metabolites had significant improvement in range, while the remaining two metabolites had insignificant improvement in range comparing the non-normalized sample to the normalized sample. In the group on the moderate sucrose diet all ten metabolites showed significant improvement in range going from non-normalized to normalized samples. These findings describe a pre-acquisition method of urine normalization to adjust for differences in hydration state of each organism. This results in a narrower concentration range in a targeted analysis.

Investigation of facile spectroscopic approaches for rapid calycosin determination in invitro biological samples and pharmaceuticals; application to the content uniformity of capsules

Calycosin, the major bioactive isoflavonoid inAstragali Radix and an important anti-viral drug with a variety of pharmacological actions, is being determined by five different spectroscopic methods. Two spectrophotometric methods have been investigated including measuring the absorption spectra at λmax = 270 nm and the first derivative spectra at λ = 288 nm for methods I and II, respectively. For the first time; the native fluorescence of calycosin is measured without adding any reagents. The fluorescence intensity was measured at 340 nm after excitation at 282 nm in method III. The fourth method involves the direct measuring of a first derivative spectrofluorimetric emission peak at 292 nm. In method V synchronous fluorescence spectra were recorded in methanol at Δλ = 70 nm. The linear range for the fluorescence-based methods was 0.05–1.0 µg/mL and for the UV-based methods was 0.5–10.0 µg/mL. The methods were validated per International Council of Harmonization (ICHQ2R1) guidelines. The limits of detection were found to be down to 0.11 and 0.12 µg/mL for the spectrophotometric methods, and 15.0, 18.0,16.0 ng/ mL, for the spectrofluorimetric approaches respectively, representing the high sensitivity. Accordingly, this permitted the quantitation of calycosin in spiked human plasma samples with satisfactory percentage recoveries (94.50.–102.50 %). The methods were utilized for calycosin analysis in different matrices including plasma and capsules with high precision and accuracy.

Accurate identification of cell doublet profiles: Comparison of light scattering with fluorescence measurement techniques.

Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.

Evaluating mitophagy in embryonic stem cells by using fluorescence-based imaging.

Embryonic stem cells (ESCs), which are characterized by the capacity for self-renewal and pluripotency, hold great promise for regenerative medicine. Increasing evidence points to the essential role of mitophagy in pluripotency regulation. Our recent work showed that PINK1/OPTN take part in guarding ESC mitochondrial homeostasis and pluripotency. Evaluating mitophagy in ESCs is important for exploring the relationships between mitochondrial homeostasis and pluripotency. ESCs are smaller in size than adult somatic cells and the mitophagosomes in ESCs are difficult to observe. Many methods have been employed—for example, detecting colocalization of LC3-II and mitochondria—to evaluate mitophagy in ESCs. However, it is important to define an objective way to detect mitophagy in ESCs. Here, we evaluated two commonly used fluorescence-based imaging methods to detect mitophagy in ESCs. By using autophagy- or mitophagy-defective ESC lines, we showed that the mito-Keima (mt-Keima) system is a suitable and effective way for detecting and quantifying mitophagy in ESCs. Our study provides evidence that mt-Keima is an effective tool to study mitophagy function in ESCs.

Phytochemical Profiling of Tupistra nutans Wall. ex Lindl. Inflorescence Extract and Evaluation of Its Antioxidant Activity and Toxicity in Hepatocarcinoma (HepG2) and Fibroblast (F111) Cells.

Tupistra nutans Wall. ex Lindl. is a medicinal plant found in the Eastern Himalayan region. Besides being used as a folk medicine for pain and high blood sugar, its inflorescence is consumed as a vegetable. However, its medicinal properties have not been proven in vitro and in vivo till now. Therefore, in this study, we reported the phytochemicals present in the methanolic extract of Tupistra nutans Wall. ex Lindl. inflorescence (METNI) and its comparative effect in liver carcinoma HepG2 cells against non-cancerous murine fibroblast F111 cells. Phytochemical profiling by gas chromatography-mass spectrometry (GC-MS) analysis showed that METNI was rich in unsaturated fatty acids, vitamin E, and anticancer compounds like diosgenin, linoleic acid, and palmitoleic acid. METNI was found to have in vitro antioxidant property as determined by DPPH and pyrogallol methods, and UV protection property as investigated by fluorescence-based and spectrophotometric methods. MTT assay revealed METNI caused significantly more cell proliferation inhibition in HepG2 (IC50 = 138µg/ml) compared to F111 (IC50 = 347µg/ml) cells. Although in both HepG2 and F111 cells METNI showed significant antioxidant activity, it led to intracellular ROS generation and cell cycle alteration at higher exposure. The obtained results suggest that Tupistra nutans can be a promising application for anticancer drug and skin care product development, but can be harmful if overconsumed.

Design of graphene quantum dots decorated MnO2 nanosheet based fluorescence turn “On-Off-On” nanoprobe for highly sensitive detection of lactoferrin

Lactoferrin estimation is increasingly acquiring prominence as a novel biomarker for the diagnosis of periodontal disease. To date, diverse lactoferrin detection methods which include electrochemical, surface-enhanced Raman scattering, colorimetric, and others have been extensively portrayed. Unfortunately, these systems have significant shortcomings including low sensitivity, selectivity, high cost, arduous and time-consuming technique, and so forth. Recently, the fluorescence-based method shows remarkable uniqueness that overcomes the demerits of traditionally reported techniques. Therefore, graphene quantum dots (GQDs) and manganese dioxide nanosheets (MnO2-NS) based simplistic, highly sensitive, and selective fluorescent turn ‘Off-On’ mediated [email protected]2-NS nanoprobe was designed. Herein, MnO2-NS addition demonstrated the quenching of GQDs containing fluorescence through inner filter effects (IFE) and strong interaction between GQDs and MnO2-NS. The lactoferrin addition destroyed the MnO2-NS and fluorescence emission of GQDs reappeared which may be because of redox reaction between lactoferrin and prepared MnO2-NS. Herein, nanoprobe offers a wide concentration range and low limit of detection of 5 to 1600 ng/mL and 1.69 ng/mL, respectively. As fabricated [email protected]2-NS nanoprobe sensor demonstrated high selectivity, good stability, and reproducibility towards lactoferrin that assuring applicability of biosensor. Therefore, the [email protected]2-NS nanoprobe will offer a simplistic sensor with adequate sensitivity to achieve highly responsive and selective detection of lactoferrin.

Fluorometric setup and method for studying the functional activity of corneal endotheliocytes

Subject of study. Experimental methods for investigating the state of preservation and functionality of the corneal endothelium, which is determined by the water permeability of the cells and activity of ion pumps, mainly by sodium–potassium adenosine triphosphatase, were considered. Aim of study. The aim of this study was to identify significant measurable parameters such as the fluorescence intensity integrated over the entire cornea preparation and the temporal dynamic of this fluorescence. Additionally, a specialized experimental fluorometric setup was designed based on fiber optics. Method. Modifications were introduced to the composition and design of a universal experimental setup for the measurement of cell functions that enabled implementation of a fluorescence-based method for determination of the intracellular sodium concentration without processing the digital image of the cornea preparation. Main results. The specialized experimental setup and method were developed for investigating functions of the corneal endothelial cells and the intracellular signaling mechanisms whose activation or disfunction under conditions of a traumatic inflammation and after a hypothermic preservation can be related to the development of a corneal edema and a corneal graft disease. Practical significance. The specialized experimental setup and methods for functional testing of the corneal endothelial cells developed in this study can be applied to determine the degree of suitability of the cornea as a transplantation material. They can also be used as a system of tests for determining the effectiveness of the cornea preservation methods.

Exploring effects of MC-LR to lysosome localization by a unique fluorescence-based method

Maintaining lysosomal pH homeostasis is crucial in regulating physiological processes, such as macromolecule degradation, vesicle trafficking, and plasma membrane repair. However, simultaneous investigation of distribution and interrelation of lysosomes and Microcystin-LR (MC-LR) in living systems is restricted by lack of functional molecular tools. To address this critical challenge, herein, we describe a novel probe Bptp-MR can concurrently report MC-LR and lysosomes for the first time. This probe can realize lysosomal localization and imaging before MC-LR enters the cell. However, its fluorescence signals are no longer limited to lysosomes in the presence of MC-LR in cells because of their pH similarity. This result directly indicates that MC-LR interferes with the intracellular acid region in cells. Thus, this work first reported that MC-LR could disrupt the intracellular acidic regions as indicated by its fluorescence interference with lysosomes in cells. The occurrence and development of major diseases related to MC-LR are possibly associated with the interference of MC-LR with the intracellular acid region. The results provide important insights into studying the pathogenesis of major MC-LR-related diseases.

Synthesis of new fluorescent metal-organic framework (4-sulfo-1,8-naphthalimide-UiO66) for detection of Cr(III)/Cr(VI) ions in aqueous media and its antimicrobial studies

Detection of toxic metals both rapidly and at low concentrations is of great importance. Researchers put a lot of effort to detect toxic metals and develop various detection systems. In recent years, fluorescent-based methods have been used for the detection of toxic metals such as chromium. Metal-organic frameworks (MOFs) are organic materials with unique properties such as excellent chemical-thermal stability, designable structure, well-defined pore structure, and high surface area. MOFs are used in the development of fluorescent-based sensor systems thanks to these advantages. In this study, 4-sulfo-1,8-naphthalimide immobilized UiO66-NH2 (UiO66-Nap) with fluorescent properties was synthesized and used for the detection of Cr(III) and Cr(VI). The structures of the synthesized compounds were elucidated using 1H-NMR, FTIR, PXRD, and SEM techniques. The MOF-based fluorescent sensor shows good sensing performances for Cr(III) and Cr(VI) ions including high selectivity, and great sensitivity (18.3 μM and 3.42 μM, respectively). Also, the antimicrobial potentials of these compounds were elucidated by a broth microdilution test against standard bacteria and yeast. Results showed that Ag-associated UiO66 had great antimicrobial potential with a MIC value of 0.019 mg/mL against Gram-positive and Gram-negative bacteria. In addition, this compound revealed very good antifungal activity against Candida at a dose of 0.039 mg/mL.

Multi-functional carbon dots from simple precursors: An excellent heavy metal ions sensor with photocatalytic activity in aqueous environment

Carbon dots (CDs) with core and surface structures were prepared from simple precursors (ethylenediamine and ethylene glycol) by using house-hold microwave oven. The structural and optical properties were exclusively studied with HR-TEM, XRD, UV-Vis and PL techniques. The fluorescence emission of the CDs was predominantly dependent and/or independent on excitation-wavelength are owed to the existence of assorted functional groups on surface and uniform arrangement in core structure. Hence, the CDs shows better stability against photo bleaching and ionic strength. In addition, CDs are successfully used to detect heavy metal ions such as Cr6+ and Hg2+ selectively by fluorescence-based detection method through different quenching mechanisms with lower detection limit of 0.09 µM for Cr6+ and 0.16 µM for Hg2+. Similarly, we attempted rapid, simple and low cost on-field application by using two different cellulose-based filter papers for visual detection of Cr6+ and Hg2+ metal ions for quick and cheap method. Moreover, CDs were successfully employed for detoxification/degradation of Rhodamine B (RhB) dye and also 87 % degradation were achieved over the time period of 140 min under direct sunlight irradiation. At finally, the innovative CDs are superior multi-behavior material for the several environmental applications.

CELL CO-CULTURE MICROFLUIDICS PLATFORM WITH AN INTEGRATED HYDRAULIC VALVE FOR INVESTIGATION OF SIGNAL-MEDIATED INTERACTIONS IN THE BLOOD-BRAIN BARRIER

Lab-on-a-chip systems for real-time analysis of neural cell communication is an emerging topic of neuroscience research that can provide a better understanding of brain functionality. Astrocyte and HBEC5i co-culture provide in vitro model of the blood-brain barrier. The successful employment of lab-on-achip cell co-culture devices in research settings requires fabricating materials that are not cytotoxic to the cells. Controlled and reversible separation of cell culture chambers is crucial for real-time studies of extracellular-mediated cell-to-cell communications. This study demonstrated a 3D printed cell co-culture microfluidic platform that enables controlled separation of the chambers and provides the long-term viability of HEBC-5i cells. The platform consists of two 27.5 mm × 35 mm × 10 mm cell culture chambers separated by an Elastic Resin 3D stereolithography printed valve (10 mm × 35 mm × 9.5 mm). The actuation of the valve is controlled using hydraulic pressure exerted by the chamber positioned directly above the valve. The deflection of the valve barrier provides separation of the cell chambers and the individual microenvironments. Upon the release of the pressure, the valve returns to its original position and allows the exchange of signaling molecules between the cells. The lower glass channel wall of the microfluidic device was coated with gelatin, polydopamine (PDA), and poly-L-lysine (PLL) to provide cellular attachment for HBEC-5i cells and astrocytes. The polyelectrolyte immobilization efficacy was assessed via atomic force microscopy while the viability of the HBEC-5i cell was assessed using fluorescent-based methods.

Defocusing μPTV With Increased Measurement Depth Using Shadowgraphy

In this study we can show that shadowgraphy is a feasible alternative to fluorescence for microscopic particle imaging. Furthermore, using the particle shadow image geometry (diameter for defocusing methods or axis lengths for astigmatism methods), the particle location along the optical axis can be determined, enabling a three-dimensional flow measurement with a single camera. However, the rate of change of the particle shadow image geometry as a function of the distance to the focal plane is one order of magnitude smaller as compared to fluorescence/scattering imaging approaches, such that the measurement depths is effectively increased. This increase is due to the fact that the signal to noise ratio of the relatively small particle shadow images is large, even if the particle is located far away from the focal plane, as the intensity is distributed over a small sensor area. In addition, due to their smaller dimensions, more particle shadow images can be captured on the camera sensor, yielding a larger information density for microscopic particle imaging. A measurement of a micro-channel (channel height: 650 µm) flow showed a good agreement with the ideal parabolic flow profile, proving the viability of this microscopic particle shadow imaging approach as a suitable addition to the existing fluorescence-based methods.

Application of fluorescence-based analysis methods

Fluorescence has been discovered over decades and enormous fluorescence technology appear and been applied to different field. This article mainly focuses on the fluorescence technologies with good function or potential that has relationship with health. The field of the health is extremely broad, so we just summarized it into two parts, one is the cause of unhealthy, one is the restore health. Apart from those physical injuries, food safety is a major risk of un-wellbeing, so to effectively improve the accuracy and efficiency of the detection technology of contaminants is very important, where metal-organic frameworks (MOFs) can improve this. Fluorescent imaging and other fluorescent biosensors have great abilities and potential in clinical technology and biomedical field to help to restore healthy. While not just these fluorescence technologies themselves need to discuss, fluorophore is also an essential part. As most fluorescence technologies need to use it. As a result, this research shows the application of fluorescence-based analysis methods in different fields, including food safety and biomedicine.

Non-destructive insights into photosynthetic and photoprotective mechanisms in Arabidopsis thaliana grown under two light regimes

Probing insights into understanding photosynthetic processes via non-invasive means has an added advantage when used in phenotyping or precision agriculture. We employed Raman spectroscopy and fluorescence-based methods to investigate both the changes in the photosynthetic processes and the underlying protective mechanisms on Arabidopsis thaliana wild-type (WT), and ros1, which is a mutant of a repressor of transcriptional gene silencing, both grown under low light (LL: 100 μmol m−2s−1) and high light (HL: 400 μmol m−2s−1) regimes. Raman imaging detected a lower carotenoid intensity after two weeks in those plants grown under HL, compared to those grown under the LL regime; we interpret this as the result of oxidative damage of β-carotene molecules. Further, the data revealed a significant depletion in carotenoids with enhanced phenolics around the midrib and tip of the WT leaves, but not in the ros1. On the contrary, small necrotic zones appeared after two weeks of HL in the ros1 mutant, pointing to the starting oxidative damage. The lower maximum quantum yield of the photochemistry (Fv/Fm) in the WT as well as in the ros1 mutant grown in HL (compared to those in the LL two weeks post-exposure), indicates the HL partially inactivated photosystems. Chlorophyll a fluorescence imaging further showed high non-photochemical quenching (NPQ) in the plants grown under the HL regime for both the WT and the ros1 mutant, but the spatial heterogeneity of NPQ images was much higher in the HL-grown ros1 mutant. Fluorescence screening methods revealed significantly high values of chlorophyll proxies in the WT as well as in the ros1 mutant two weeks after in the HL compared to those under LL. The data generally revealed an increased accumulation of phenolics under HL in both the WT and ros1 mutant plants, but the proxies of anthocyanin and flavonols were significantly lower in the ros1 mutant than in the WT. The comparatively low accumulation of anthocyanin in the ros1 mutant compared to the WT supports the Raman data. We conclude that integrated use of these techniques can be efficiently applied for a better understanding of insights into photosynthetic mechanisms.

Abstract 639: Rapidly evaluating cancer dependencies by label-free imaging of zero-passage primary cells

Abstract Successful mapping of cancer dependencies requires conducting genetic and pharmacological screens in a diversity of cell models. However, existing model development approaches require long periods of culture time during which evolutionary pressures reduce heterogeneity. It also remains difficult to create long-term models of many cancers, greatly limiting the share of patient samples that can be studied. These limitations make it challenging to create large and diverse datasets for the discovery and validation of biomarker-vulnerability relationships. To enable high-throughput genetic and pharmacological screens in primary, including nondividing, cells without an intermediate model generation step, we are developing a label-free imaging-based platform of early living tissue perturbation. Here, we describe our efforts to establish an ex vivo cell-preservation and imaging system for malignant ascites from patients with advanced gastroesophageal (GE) cancer, whose prognosis remains poor and there is an urgent need for rapid evidence-based therapeutic discovery. First, we optimized a workflow to acquire and perturb cells within 24 hours of sample collection. We observed that incorporating microenvironmental factors by mixing ascites fluid with organoid media extends the preservation of cellular composition and viability. We next hypothesized that label-free microscopy can be a substitute for fluorescence-based labels which fade over time in live-cell imaging. To test this, we created a dataset consisting of over 1.0M cells from 14 samples (10 unique patients). Before seeding, we added fluorescence labels to annotate cell type and viability during imaging. We then trained predictive models to infer cell annotations based on brightfield morphological features only and observed an overall accuracy of 92% and 82% for cell identity and viability, respectively. Prediction of tumor cell fraction from label-free images alone showed strong correlation with tumor fractions estimated by flow cytometry. We also integrated single-cell RNA sequencing data to generate a candidate panel of 28 compounds which are predicted to exhibit antitumor activity via different mechanisms that are of relevance to our study cohort. We observe that label-free inference of compound activity showed strong correlation (R2 > 0.8) with fluorescent-based methods. We are now expanding the scale of our rapid screens and utilizing transcriptomics to link the molecular profile with response. Since our approach couples the timing of drug or genetic perturbation with the preservation of subcellular heterogeneity, it will serve as a strong foundation for preclinical studies. Importantly, our method substantially expands the fraction of samples that can be interrogated and will have application in other areas of need where material is limited or long-term model generation remains unsuccessful as is the case in many rare diseases. Citation Format: Mushriq Al-Jazrawe, Csaba Molnar, Niklas Rindtorff, Steven Blum, William Colgan, Maria Alimova, Sean Misek, Carmen Rios, Moony Tseng, James M. McFarland, Aviv Regev, Beth A. Cimini, Anne E. Carpenter, Adam Bass, Samuel J. Klempner, Jesse Boehm. Rapidly evaluating cancer dependencies by label-free imaging of zero-passage primary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 639.

Abstract 2748: High-speed and high-precision FL-based cell count and viability assays using the Cellaca™ MX high-throughput cell counter

Abstract With multiple FDA-approved cell therapy products available and others in pre-clinical and clinical trials, now, more than ever, it is critical to provide fast and accurate measurements of cell concentration and viability. For certain clinical products, cell concentration is synonymous with dosage, and proper cell viability is crucial to avoidance of potential harmful side effects. The complex nature of patient-derived samples makes legacy cell analysis methods such as the trypan blue exclusion assay difficult or impossible to perform. Nonspecific objects such as RBCs and cellular debris can significantly increase cell counting variation. Meanwhile, the need for cell-based products that are custom-tailored to each patient has resulted in the analysis of smaller and more numerous batches, leading to cell counting bottlenecks. With the challenges of messy samples and counting bottlenecks in mind, we investigate the use of the Cellaca™ MX, a cell counter that can increase throughput and employ fluorescence-based cell counting methods. The high-throughput cell counter can utilize 5 fluorescence imaging channels for cell count and viability analysis in less than 3 min for 24 samples. In addition, fluorescent cell-based assays such as apoptosis (Annexin V or Caspase 3/7), cell cycle, reactive oxygen species measurement can be performed to better characterize the target cell samples. In this work, we demonstrate the capabilities of Cellaca™ MX for high-throughput fluorescence-based cell counting. Comparison among multiple instruments using Jurkat cells and fluorescent beads reveals high consistency between replicate counts, between Cellaca consumable plates, and between instruments. We also compare cell counts made on the Cellaca™ MX with manual counts performed using a hemocytometer, as well as counts performed using other fluorescence-based automated cell counting methods. In addition, we illustrate the use of the ISO 20391-II cell counting standard to evaluate and compare the performance of cell counting methods. Finally, we include real-world primary T cell linearity results over a 4-log range of concentration. The results of the experiments confirm the suitability of the Cellaca™ MX for general fluorescence-based assays applicable to cell therapy-related workflows. Citation Format: Jordan Bell, Yongyang Huang, Sun Yung, Henry Qazi, Charles Hernandez, Bo Lin, Jean Qiu, Leo Chan. High-speed and high-precision FL-based cell count and viability assays using the Cellaca™ MX high-throughput cell counter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2748.