Multicolor Fluorescence Imaging Research Articles

Bio-inspired green fluorescent gold-naringin nanoclusters as a dual-functional optical probe for bio-imaging and intracellular sensing applications

Human exposure to mercury (Hg2+) happens in everyday life via exposure to aquatic animals like fishes, shrimps, and prawns. We developed bio-inspired gold-naringin nanoclusters (GNNCs) as a novel fluorescent probe for sensitive and selective detection of Hg2+ ions. GNNCs exhibit green-color emission under UV illumination with excellent selectivity for Hg2+ over other metal ions. GNNCs demonstrated excellent photostability and excitation-dependent emission behaviors. As-synthesized GNNCs facilitate Hg2+ detection in the range of 0–5 µM with a detection limit of 26.42 nM. GNNCs also provide effective multi-color fluorescence imaging of shrimp as an animal model. GNNCs displayed excellent intracellular fluorescence sensing of Hg2+ and in vitro cytotoxicity in cancer (A549) cells. The practicality and feasibility of GNNCs were examined using real water samples from streams and tap water.

One-step synthesis of carbon dots for selective bacterial inactivation and bacterial differentiation

Novel carbon dots (CDs) were synthesized by a one-pot hydrothermal approach using ampicillin as a precursor, and the as-prepared CDs exhibited a high quantum yield (23%). The CDs were found to possess abundant surface functional groups, thus providing good permeability to the cell, and the antibacterial activity of CDs was evaluated. S. aureus and L. monocytogenes were selected as model bacteria, and our results showed that the CDs exhibited antibacterial activity against S. aureus and L. monocytogenes under visible light illumination, even at low concentrations. The antibacterial mechanism is believed to be the production of reactive oxygen species (ROS) from CDs under visible light irradiation, which attacked the bacterial cell membranes, resulting in the death of the bacteria. In addition, because of the multicolor fluorescence properties of CDs, staining of S. aureus and L. monocytogenes obtained multicolor fluorescence images at different excitation wavelengths. Based on these results, CDs are a promising candidate material for biological applications. Graphical abstract.

Additive-color multi-harmonic generation microscopy for simultaneous label-free differentiation of plaques, tangles, and neuronal axons.

Multicolor fluorescence imaging has been widely used by neuroscientists to simultaneously observe different neuropathological features of the brain. However, these optical modalities rely on exogenous labeling. Here, we demonstrate, for the first time, a label-free additive-color multi-harmonic generation microscopy to elucidate, concurrently with different hues, Alzheimer's disease (AD) neuropathological hallmarks: amyloid β (Aβ) plaques and neurofibrillary tangles (NFT). By treating third harmonic generation (THG) and second harmonic generation (SHG) as two primary colors, our study can simultaneously label-free differentiate AD hallmarks by providing different additive colors between Aβ plaques, NFT, and neuronal axons, with weaker THG presentation from NFT in most places of the brain. Interestingly our pixel-based quantification and Pearson's correlation results further corroborated these findings. Our proposed label-free technique fulfills the unmet challenge in the clinical histopathology for stain-free slide-free differential visualization of neurodegenerative disease pathologies, with a sub-femtoliter resolution in a single image field-of-view.

A hydrogen peroxide activatable nanoprobe for light-controlled "double-check" multi-colour fluorescence imaging.

A new probe for precise and accurate bioimaging contributes significantly to advancing biomedical research for early disease diagnosis and treatment monitoring. Through wrapping a photochromic molecule (SP-Np-B) within a polymer nanoparticle, a new light-controlled multicolour fluorescence nanoprobe (Poly-SP-Np-B) is developed for precise fluorescence subcellular bioimaging. Poly-SP-Np-B shows an "OFF-ON" red-emitting fluorescence response upon alternate UV/Vis light irradiation. After activation by hydrogen peroxide (H2O2), a green-emitting Poly-SP-Np nanoparticle is generated, thus allowing light-controlled fluorescence response simultaneously, i.e., green and yellow switch upon alternate UV/Vis light irradiation for 10 and 20 s, respectively. Such a "blinking" fluorescence signal change is not possible by only using a photochromic molecule probe (SP-Np-B) with alternate UV/vis light irradiation for over 5 min. Poly-SP-Np-B has large isomerization kinetic constants (kSP-MR = 0.4543 s-1 and kMR-SP = 0.0809 s-1), excellent biocompatibility and lysosome distribution capability, enabling multicolour fluorescence imaging in live cells. With exo-/endogenous H2O2 activation in lysosomes, light-controlled "double-check" fluorescence imaging at the subcellular level is successfully achieved. More specifically, the change in fluorescence imaging is reversible in green, red and yellow channels in live cells upon excitation under alternate UV and visible light. This work thus provides a new strategy to develop switchable photochromic probes for precise fluorescence bioassay and bioimaging.

Eco-friendly synthesis of tunable fluorescent carbon nanodots from Malus floribunda for sensors and multicolor bioimaging

In this work, green and economical synthesis of highly fluorescent nitrogen-doped carbon nanodots (N-CNDs) is reported using Malus floribunda (M. floribunda) fruit and aqueous ammonia as carbon and nitrogen source, respectively by a simple hydrothermal method. The synthesized N-CNDs show exceptional advantages including acceptable fluorescent quantum yield (18 %) and N-CNDs sizes are around 3.5 nm. The fluorescence of N-CNDs based nanosensor can be selectively and sensitively quenched by ferric ions in the aqueous medium. This phenomenon was used to develop a fluorescent method for facile detection of ferric ions within a linear range of 10–50 μM with a detection limit of 2.5 μM (S/N = 5) having linear regression value (R2) of 0.994. The synthesized N-CNDs incubated with human colon cancer (HCT-116) cells which did not stimulate cell death, indicating that the N-CNDs have good biocompatibility. Hence, the N-CNDs was used as a fluorescent probe for in vitro imaging of HCT-116.TheN-CNDswereuniformly internalized into the whole body of cells and displays the multicolor fluorescence imaging. To best of the authors' knowledge, this is the earliest report on the selective sensing of ferric ions and live-cell imaging using M. floribunda fruit-derived N-CNDs by fluorescent quenching mechanism.

Noninvasive determination of toxic stress biomarkers by high-throughput screening of photoautotrophic cell suspension cultures with multicolor fluorescence imaging

BackgroundWith increasing pollution, herbicide application and interest in plant phenotyping, sensors capturing early responses to toxic stress are demanded for screening susceptible or resistant plant varieties. Standard toxicity tests on plants are laborious, demanding in terms of space and material, and the measurement of growth-inhibition based endpoints takes relatively long time. The aim of this work was to explore the potential of photoautotrophic cell suspension cultures for high-throughput early toxicity screening based on imaging techniques. The investigation of the universal potential of fluorescence imaging methods involved testing of three toxicants with different modes of action (DCMU, glyphosate and chromium).ResultsThe increased pace of testing was achieved by using non-destructive imaging methods—multicolor fluorescence (MCF) and chlorophyll fluorescence (ChlF). These methods detected the negative effects of the toxicants earlier than it was reflected in plant growth inhibition (decrease in leaf area and final dry weight). Moreover, more subtle and transient effects not resulting in growth inhibition could be detected by fluorescence. The pace and sensitivity of stress detection was further enhanced by using photoautotrophic cell suspension cultures. These reacted sooner, more pronouncedly and to lower concentrations of the tested toxicants than the plants. Toxicant-specific stress signatures were observed as a combination of MCF and ChlF parameters and timing of the response. Principal component analysis was found to be useful for reduction of the collected multidimensional data sets to a few informative parameters allowing comparison of the toxicant signatures.ConclusionsPhotoautotrophic cell suspension cultures have proved to be useful for rapid high-throughput screening of toxic stress and display a potential for employment as an alternative to tests on whole plants. The MCF and ChlF methods are capable of distinguishing early stress signatures of at least three different modes of action.

A platform for primary tumor origin identification of circulating tumor cells via antibody cocktail-based in vivo capture and specific aptamer-based multicolor fluorescence imaging strategy

Circulating tumor cells (CTCs) are expected to serve as a blood-based biomarker in the diagnosis of cancers at an early stage, providing an opportunity to increase the survival of cancer patients. Current techniques for CTC detection were designed for some particular types of cancer with confirmed primary tumor origin. In this work, a platform for the detection of two cancer types and the identification of the primary tumor origin of CTCs was established to meet the requirement of cancer diagnosis and clinical application. A combined strategy based on in vivo capture method using antibody cocktail and multicolor fluorescence imaging using aptamer was designed to achieve the high-efficiency capture of CTCs and the accurate location of the primary tumor. An antibody cocktail of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) was applied to capture breast cancer CTCs and hepatocellular CTCs in vivo. The capture efficiency of hepatocellular CTCs was significantly increased from 3.17% to 26.67% and the capture efficiency of breast cancer CTCs slightly increased from 27.00% to 29.84% compared with EpCAM-based capture of CTCs. Meanwhile, the primary tumor origins of breast cancer CTCs and hepatocellular CTCs were simultaneously distinguished by specific aptamer-based fluorescence probes without any signal crosstalk. The results of in vivo experiments using the dual tumor-bearing mouse model confirmed the feasibility of this method to capture CTCs and identify primary tumor origins. This simple and efficient approach has potential for future applications in cancer diagnosis and prognosis.

A practical guide to intelligent image-activated cell sorting.

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in ~3 months.

Abstract LB-137: Three-dimensional cytotoxicity assay using magnetic 3D bioprinting for measuring CAR T cell function in heterogeneous solid tumor microenvironments

Abstract Chromium release assays are routinely utilized to assess cytotoxic activity and function of tumor targeting T cells expressing chimeric antigen receptors (CAR T cells). Although these assays can predict the activity of CAR T cells targeting hematological malignancies, results of such assays do not accurately reflect the limited efficacy of T cell therapy observed against solid tumors. This limitation may be due to the disparity between the in vitro cell culture setting and the complex nature of solid tumor microenvironments and their vascular structure. To this end, we have developed a cytotoxicity assay based on a three-dimensional (3D) cell culture model, facilitated by magnetic 3D bioprinting, to more precisely mimic the complexity of solid tumor microenvironments in vitro. This model can be customized to reflect the heterogeneity of solid tumors by incorporating multiple cell types and immune cells. In these studies, we have demonstrated the feasibility of this assay using CD19-specific CAR T cells specifically targeting CD19+ leukemia cells in a heterogeneous 3D cell culture. We also show the CAR T cells infiltrating and targeting glioblastoma (U87) 3D cultures in real-time with fluorescence microscopy. Incorporation of this assay as a routine cytotoxicity assay will provide new dimensions to rapid immune function assessments by revealing the kinetics of cell killing through time-lapse microscopy, the persistence and targeting patterns of CAR T cells, and cell-specific killing by multicolor fluorescence imaging. This assay is being adapted into a 96-, 384- and 1,536-well formats for high-throughput applications. Citation Format: Glauco R. Souza, Amer Najjar. Three-dimensional cytotoxicity assay using magnetic 3D bioprinting for measuring CAR T cell function in heterogeneous solid tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-137.

Deep eutectic solvents-derived carbon dots for detection of mercury (II), photocatalytic antifungal activity and fluorescent labeling for C. albicans.

A novel nitrogen and chloride co-doped carbon dots (N/Cl-CDs) based choline chloride-urea deep eutectic solvents (DESs) were synthesized by one-step hydrothermal method with high quantum yield of 37% and excellent photoluminescent properties. This N/Cl-CDs fluorescent probe had been successfully applied to sensitively and selectively detect the concentration of Hg2+ with a linear range of 0.2-40 μM and a detection limit of 0.05 μM. Moreover, the N/Cl-CDs displayed a strong photocatalytic antifungal activity against C. albicans and their photoinduced antifungal functions were evaluated under conditions of varying other experimental parameters. The antifungal efficiency of N/Cl-CDs (7 mg/mL) against C. albicans is upon 100% when extending the visible light irradiation time to 80 min. In addition, the excellent luminescence properties of N/Cl-CDs can also label C. albicans and displayed multicolour fluorescence imaging at different excitation wavelengths. Based on their functions of fluorescence probe, antifungal and fluorescence imaging, N/Cl-CDs would provide potentials for a wide range of applications in the detection and microbe in the near future.

Multifunctional iron oxide-carbon hybrid nanoparticles for targeted fluorescent/MR dual-modal imaging and detection of breast cancer cells

An efficient method for the highly sensitive and specific detection of cancer cells is crucial for the early diagnosis of cancer. In this work, we propose a one-pot approach to fabricating magnetic-fluorescent iron oxide-carbon hybrid nanomaterials (MCNP) with excellent stable, high quantum yield and excellent magnetic properties for breast cancer cells recognition and detection via magnetic resonance and multicolour fluorescence imaging. MCNPs were efficiently synthesised via one-pot, multi-component reactions of FeCl3, FeCl2•4H2O, citric acid and ethylenediamine in diethylene glycol. The MCNPs showed strong excitation wavelength-dependent fluorescence in the blue-red region with a high quantum yield of 58.4%, and they presented higher stability and T2 relaxivity than pure iron oxide nanoparticles. After conjugating with CD44 monoclonal antibodies, the fabricated targeting nanoprobe, MCNPs-CD44, demonstrated a specific fluorescence/MRI dual imaging contrast effect in 4T1 breast cancer cells. Biological transmission electron microscope imaging showed a significant preferential uptake of the nanoparticle conjugates by the 4T1 cells. By taking advantage of the high binding affinity and specificity of the CD44 antibodies to the overexpressed CD44 on the cancer cell surface, the developed MCNPs-CD44 probe distinguished 4T1 breast cancer cells from normal cells and detected as low as a few hundred cancer cells, thus indicating the potential application of multifunctional nanocomposites in the MR diagnosis and fluorescence positioning of breast cancer at cellular-level resolution.

Multicolor fluorescent imaging by space-constrained computational spectral imaging.

Spectral imaging is a powerful technique used to simultaneously study multiple fluorophore labels with overlapping emissions. Here, we present a computational spectral imaging method, which uses sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive spectral imaging and the low throughput issue of scanning spectral imaging. With simulated and experimental data, we have demonstrated the reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method for differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further improve multiplexing capability.

Triarylmethane Fluorophores Resistant to Oxidative Photobluing.

Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.

Covalently Assembled Dipeptide Nanoparticles with Adjustable Fluorescence Emission for Multicolor Bioimaging.

Peptide self-assembly, inspired by the naturally occurring fabrication principle, remains the most attractive in constructing fluorescent nanoagents towards bioimaging. However, the noncovalent interactions that drive peptide self-assembly are usually susceptible to the complex physiological environment; thus leading to disassembly and dysfunction of fluorescent nanoagents. Herein, a covalently crosslinked assembly strategy for fabrication of stable peptide-based nanoparticles with adjustable emission is introduced. In the process of cationic diphenylalanine peptide (H-Phe-Phe-NH2 ⋅HCl) self-assembly, glutaraldehyde is used as a crosslinker and the resulting product of the Schiff base reaction can be fluorescent. More importantly, the emission wavelength can be readily tuned by controlling the covalent reaction time. It has been demonstrated that the nanoparticles are stable, even after intracellular uptake, and can be used for sustainable multicolor fluorescent imaging. The strategy with integrating peptide self-assembly and covalent crosslinking could be promising for the design and engineering of functional fluorescent nanoparticles with robust physiological stability and adjustable emission towards improved bioimaging applications.

Frequency-Encoded Multicolor Fluorescence Imaging with Single-Photon-Counting Color-Blind Detection

Standard fluorescence microscopy relies on filter-based detection of emitted photons after fluorophore excitation at the appropriate wavelength. Although of enormous utility to the biological community, the implementation of approaches for simultaneous multicolor fluorescence imaging is commonly challenged by the large spectral overlap between different fluorophores. Here, we describe an alternative multicolor fluorescence imaging methodology that exclusively relies on the absorption spectra of the fluorophores instead of their fluorescence emissions. The method is based on multiplexing optical excitation signals in the frequency domain and using single color-blind detection. Because the spectral information is fully encoded during excitation, the method requires minimal spectral filtering on detection. This enables the simultaneous identification of multiple color channels in a single measurement with only one color-blind detector. We demonstrate simultaneous three-color confocal imaging of individual molecules and of four-target imaging on cells with excellent discrimination. Moreover, we have implemented a non-negative matrix factorization algorithm for spectral unmixing to extend the number of color targets that can be discriminated in a single measurement. Using this algorithm, we resolve six spectrally and spatially overlapping fluorophores on fixed cells using four excitation wavelengths. The methodology is fully compatible with live imaging of biological samples and can be easily extended to other imaging modalities, including super-resolution microscopy, making simultaneous multicolor imaging more accessible to the biological research community.

Abstract 5651: Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging

Abstract With very high spatial and biological complexity, tumor micro-environments present a significant characterization challenge, which is only heightened by the need to image entire sections of cancer tissue on many different samples. A variety of techniques offer very high levels of multiplexing, but always fail on either speed requirements (incredibly long times required to image a whole slide) or on compatibility issues (expensive, hard-to-operate CapEx not suitable for high-throughput CLIA operation). Ultivue has developed a platform reagent technology (InSituPlex™) that simultaneously enables whole- slide higher multiplexing by immunofluorescence AND complete compatibility with existing instrumentation. The InSituPlex technology comprises five key foundational elements. First, short unique DNA barcodes are covalently attached to each primary antibody (Ab). These conjugates are purified and functionally characterized in tissue for optimal staining specificity. Importantly, all of this work is done in advance by Ultivue. Second, tissue samples are stained in a single step. Third, all the DNA sequences attached to targets (via primary Ab binding) are simultaneously amplified, allowing for detection of less abundant markers e.g. PD-1, PDL-1 etc. Fourth, complementary DNA sequences each with different, covalently- attached fluorescent labels (“imager strands”) are simultaneously hybridized to the amplified DNA on the targets. All these wet chemistry steps are carried out in walkaway mode using commercially available auto-stainers. The samples are then imaged using a standard, whole-slide multi-color fluorescence imaging system. Finally, in an optional fifth step, the imager strands are gently removed via an enzymatic dehybridzation- with complete retention of antigenicity- to enable higher multiplexing via additional rounds of hybridization/imaging. The InSituPlex reagent technology offers many advantages for cancer research over current approaches: • the ability to detect 5, 10, or more targets simultaneously via whole-slide imaging on an each and every sample, with no loss in antigenicity; • the ability to leverage existing auto-stainers and existing fluorescent microscopes with Ultivue's reagent kits to carry out high-throughput analyses of multiple cancer biomarkers; and • the ability to design custom biomarker panels that leverage Ultivue's unique barcoding capability and infrastructure. This poster will elaborate on each step of the technology, and provide multiple examples of data acquired using different biomarker panels and different cancer tissue types. Citation Format: Michael Natan. Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5651.

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.

PO-025 Antiproliferative effects of rosemary (rosmarinus officinalis, L.) extract in colon cancer cells lines

IntroductionInfections by oncoviruses are an important risk factor for the development of neoplasms. Particularly, anogenital cancers are strongly associated with persistent infections by high-risk human papillomaviruses (HPV). In the case of cervical cancer the physical state of the virus seems to be an important prognostic marker. Especially the integration of viral DNA into the host cell genome bears the risk of following degenerations. Therefore, the early and sensitive detection is of high interest. Besides Pap-test and PCR, fluorescence in situ hybridization (FISH) is used for a quantitative and qualitative detection of HPV infections and risk assessment.Material and methodsFor analysis high-risk HPV FISH probes should be developed for standardised, type-specific and quantitative detection of HPV nucleic acid in host cells. Due to the short viral DNA sequence, present with just few copies, conventional FISH probes have little sensitivity. Therefore, we tested specific FISH probes targeting different regions of the HPV genome in combination with signal amplification. PCR-produced FISH probes were biotin labelled by direct incorporation of biotinylated nucleotides or nick translation. Our biotinylated HPV probes were tested on HPV-positive and –negative cells and detected using fluorescence labelled streptavidin or subsequent tyramide signal amplification. In addition to FISH, probes were used for chromogenic in situ hybridization. All analysis were performed with our fully automatized multicolor fluorescence imaging platform VideoScan.Results and discussionsThe presence of HPV DNA in cell lines was confirmed by (digital) PCR. The protocol for synthesis of biotinylated probes using HPV sequence specific primers and genomic DNA as template was successful. Biotin incorporation was checked with agarose gel electrophoresis and modified dot blot hybridization. In in situ hybridization experiments, our probes showed high signals in HPV high-copy number CaSki cells and a tissue model, compared to HPV negative samples.ConclusionHighly sensitive and specific FISH probes offer a great potential for the detection of HPV DNA in patient samples and thereby for the assessment of tumour risks. Using our probes, we got positive results in hybridization experiments using chromogenic and fluorescent detection methods with or without further amplification steps. Next, comparative FISH experiments will be performed to check probe sensitivity in cells containing few HPV copies and their practicability in biopsy derived tissue samples.

Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy

Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.

Phenotyping of Arabidopsis Drought Stress Response Using Kinetic Chlorophyll Fluorescence and Multicolor Fluorescence Imaging.

Plant responses to drought stress are complex due to various mechanisms of drought avoidance and tolerance to maintain growth. Traditional plant phenotyping methods are labor-intensive, time-consuming, and subjective. Plant phenotyping by integrating kinetic chlorophyll fluorescence with multicolor fluorescence imaging can acquire plant morphological, physiological, and pathological traits related to photosynthesis as well as its secondary metabolites, which will provide a new means to promote the progress of breeding for drought tolerant accessions and gain economic benefit for global agriculture production. Combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging proved to be efficient for the early detection of drought stress responses in the Arabidopsis ecotype Col-0 and one of its most affected mutants called reduced hyperosmolality-induced [Ca2+]i increase 1. Kinetic chlorophyll fluorescence curves were useful for understanding the drought tolerance mechanism of Arabidopsis. Conventional fluorescence parameters provided qualitative information related to drought stress responses in different genotypes, and the corresponding images showed spatial heterogeneities of drought stress responses within the leaf and the canopy levels. Fluorescence parameters selected by sequential forward selection presented high correlations with physiological traits but not morphological traits. The optimal fluorescence traits combined with the support vector machine resulted in good classification accuracies of 93.3 and 99.1% for classifying the control plants from the drought-stressed ones with 3 and 7 days treatments, respectively. The results demonstrated that the combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging with the machine learning technique was capable of providing comprehensive information of drought stress effects on the photosynthesis and the secondary metabolisms. It is a promising phenotyping technique that allows early detection of plant drought stress.