Fluorescence Microscopy Technologies Research Articles

Development and Application of Light Sheet Fluorescence Microscopy Technology (Invited)

Development and Application of Light Sheet Fluorescence Microscopy Technology (Invited)

Super-resolution fluorescence microscopy for investigating bacterial cell biology.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.

Label-free fluorescence microscopy: revisiting the opportunities with autofluorescent molecules and harmonic generations as biosensors and biomarkers for quantitative biology.

Over the past decade, the utilization of advanced fluorescence microscopy technologies has presented numerous opportunities to study or re-investigate autofluorescent molecules and harmonic generation signals as molecular biomarkers and biosensors for in vivo cell and tissue studies. The label-free approaches benefit from the endogenous fluorescent molecules within the cell and take advantage of their spectroscopy properties to address biological questions. Harmonic generation can be used as a tool to identify the occurrence of fibrillar or lipid deposits in tissues, by using second and third-harmonic generation microscopy. Combining autofluorescence with novel techniques and tools such as fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSI) with model-free analysis of phasor plots has revolutionized the understanding of molecular processes such as cellular metabolism. These tools provide quantitative information that is often hidden under classical intensity-based microscopy. In this short review, we aim to illustrate how some of these technologies and techniques may enable investigation without the need to add a foreign fluorescence molecule that can modify or affect the results. We address some of the most important autofluorescence molecules and their spectroscopic properties to illustrate the potential of these combined tools. We discuss using them as biomarkers and biosensors and, under the lens of this new technology, identify some of the challenges and potentials for future advances in the field.

Effect of M-resin-based Stabilizer on the Performance of SBS Modified Asphalt

Industrial solid waste M-resin (MR) contains high sulfur content which was difficult to be comprehensively utilized. According to the vulcanization reaction mechanism, MR was used as vulcanizing agent, nano zinc oxide (ZnO) as an activator, tetramethyl thiuram disulfide (TMTD) as an accelerator to produce M-resin-based stabilizer. MR (0.5, 1, 1.5, 2, 2.5), ZnO (0.1, 0.2, 0.3, 0.4, 0.5) and TMTD (0.06, 0.08, 0.1, 0.12, 0.14) were mixed with different contents, a three-factor and five-level orthogonal experiment was designed to test the penetration, ductility, softening point and 48 h softening point difference of SBS modified asphalt. Based on the four indexes, the basic formula of the stabilizer was obtained. The influence of kaolin, an auxiliary material, on the thermal stability was compared, and the formula were optimized, the appropriate dosage was determined. The microscopic morphology of the modified asphalt and SBS developmental fineness was analyzed with the fluorescent microscopy technology, and the SBS developmental fineness was studied with the generated binary graph and skeleton graph. DSR test was carried out to detect rheological properties of modified asphalt with different dosages of MR-based stabilizes. The results show that MR can significantly improve the thermal storage stability of modified asphalt. The ratio of MR:TMTD:ZnO:Kaolin is 1.5:0.5:0.08:12, and the appropriate dosage is 1.02 %. The stabilizer can reduce the 48 h softening point difference 33.6 ℃, increase the softening point 13.6 ℃, decrease the penetration degree 9.7 mm, but it has little effect on 5 ℃ ductility index. The microscopic images show that a cavity two-phase continuous network structure is formed inside the modified asphalt under the appropriate dosage of M-resin-based stabilizer. The SBS developmental fineness is 2.36 μm, and the uniformity is good. DSR test also shows that stabilizer can effectively improve the high temperature performance of SBS modified asphalt.

Application of Fluorescence In Situ Hybridization Assisted by Fluorescence Microscope in Detection of Her2 Gene in Breast Cancer Patients.

In order to study the important factors for evaluating the prognosis of breast cancer patients, a fluorescence microscopy-assisted fluorescence in situ hybridization technique was proposed. Compared with other detection techniques, fluorescence in situ hybridization (FISH) technology assisted by a fluorescence microscope has gradually gained favor in related fields due to its advantages of high detection specificity, high sensitivity, and strong experimental period. Combined with the basic overview of fluorescence microscopy and FISH technology, the advantages and application points of FISH technology assisted by fluorescence microscopy in the detection of the Her2 gene in breast cancer patients were studied and discussed. The results show that IHC can be used as the primary screening for HER2 gene status detection; IHC (2+) and IHC (3+) have false positives, which are related to chromosome 17 polysomy, so FISH should be done to confirm the diagnosis.

Bioelectricity Buzz

Bioelectricity Buzz

SBS morphology characteristics in asphalt binder and their relation with viscoelastic properties

Fluorescence microscopy technology was conducted to characterize the microscopic morphology of SBS modifier in asphalt binder, and the effect law of different SBS content, shear temperature and shear rate during the modification, on the microscopic morphology of SBS modifier is discussed. SBS area ratio, box dimension, area-weighted average axis ratio, coefficient of variation in axis ratio, average particle area and coefficient of variation in particle area were defined to characterize the SBS morphologies. Relation between SBS microscopic morphology and the viscoelastic properties of asphalt binder was also established. Results show that the area ratio of SBS particle has linear relationship with the SBS content, while higher SBS content has negative impact on the SBS distribution in asphalt binder. Increasing shear temperature can reduce the size of SBS particles from 7 to 10 μm at 130 °C to 4–5 μm at 175 °C. Higher shear rate brings stronger mechanical effect to SBS asphalt, which makes the physical dispersion of SBS more uniform, the specific surface area of fine particle size is larger, and the area ratio increases due to sufficient swelling effect. The rutting factor of SBS asphalt is greatly affected by the microstructure of SBS modifier. The modified Einstein model and modified Maron and Pierce and Quemada model can be successfully used to describe the relation between rutting factor of asphalt binder and morphologies of SBS modifier in the binder. This study provided a good technic to enhance the quality of SBS modified binder and its corresponding mixture by means of improving the SBS morphologies in asphalt binder.

Abstract 14072: Bioenergetic and Metabolic Impairments in Duchenne Muscular Dystrophy (DMD) Patients' Induced Pluripotent Stem Cell-derived Cardiomyocytes (iPSC-CMs)

Introduction: DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Since the DMD DCM treatment is limited, novel therapeutic modalities are urgently needed. Hypothesis: We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods: The study included iPSC-CMs from a healthy volunteer and 3 DMD patients (young male, YM; adult male, AM; adult female, AF) and mdx mice. We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of iPSC-CMs and heart tissues using the SeaHorse Flux Analyzer, patch clamp, confocal fluorescence microscopy and Liquid Chromatography Mass Spectrometry (LC-MS) technologies, respectively. Results: To test the hypothesis, we measured the iPSC-CM respiration rate using the SeaHorse. Compared to healthy, in both AM and AF DMD, but not in YM DMD cardiomyocytes, ATP production decreased by 75%, and basal respiration decreased by 80% and 45%, respectively. In agreement with impaired oxidative phosphorylation capacity, mitochondrial activity measured by 3D life confocal microscopy was attenuated by 35% in AM, but not in YM. Concurring with the healthy-like bioenergetic status, YM iPSC-CMs fired regularly with no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. Further, the LC-MS analysis demonstrated that the levels of 13%, 6% and 7% of the 120 metabolite measured, were lower in AM, AF and YM, while 10%, 18% and 43% were higher, respectively, compared to healthy iPSC-CMs ( p<0.05 ). For example, in AM and AF (but not in YM) the phosphocreatine (PCr) levels were diminished by >80%, compared to healthy iPSC-CMs, indicating a dysfunctional PCr energy system. Finally, the LC-MS analysis of mdx ventricular tissue demonstrated a statistically-significant different metabolic profile in 8 mdx mice vs 8 healthy mice. Conclusion: DMD iPSC-CMs and mdx mice exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients.

Bioenergetic and metabolic impairments in Duchenne Muscular Dystrophy (DMD) patients' iPSC-derived cardiomyocytes

Abstract Introduction DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. Treatments for DCM in DMD are limited to steroids and standard heart failure medications such as β-blockers and ACE-inhibitors, and therefore novel therapeutic modalities are urgently needed. Purpose We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function. Methods Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) were generated from healthy volunteer and 3 DMD patients: young male (YM), adult male (AM) and adult female (AF). We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of healthy and DMD iPSC-CMs using the Seahorse Flux analyzer, patch clamp, confocal fluorescence microscopy and Liquid chromatography mass spectrometry (LC-MS) technologies, respectively. Results To test the hypothesis, we measured respiration and glycolytic rates of healthy and DMD iPSC-CMs. Compared to healthy iPSC-CMs, in both AM and AF DMD, but not in YM DMD cardiomyocytes, there was a 75% decrease in ATP production, and 80% and 45% decrease in basal respiration, respectively. In agreement with the healthy-like bioenergetic status of YM, the iPSC-CMs showed no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. To determine whether the impairment in the phosphorylation pathway (OXPHOS) affects glycolysis, we measured the cardiomyocytes' response to glycolytic stress test. These experiments showed that the glycolytic rates were similar in healthy and DMD iPSC-CMs. In agreement with impaired OXPHOS, mitochondrial activity measured by 3D life confocal microscopy was attenuated in the DMD male by 35%, compared to healthy cardiomyocytes. Furthermore, the metabolomic LC-MS analyses demonstrated significant differences in metabolite levels in YM, AM and AF DMD iPSC-CMs relative to healthy iPSC-CMs. For example, compared to healthy iPSC-CMs, there was a dramatic fall to undetected levels in phosphocreatine in both AM and AF, but not in YM DMD, indicating a dysfunctional phosphocreatine energy system. Conclusions DMD iPSC-CMs exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): ISF - Israel Science Foundation

Metamaterial-Assisted Photobleaching Microscopy with Nanometer Scale Axial Resolution.

The past two decades have witnessed a dramatic progress in the development of novel super-resolution fluorescence microscopy technologies. Here, we report a new fluorescence imaging method, called metamaterial-assisted photobleaching microscopy (MAPM), which possesses a nanometer-scale axial resolution and is suitable for broadband operation across the entire visible spectrum. The photobleaching kinetics of fluorophores can be greatly modified via a separation-dependent energy transfer process to a nearby metamaterial. The corresponding photobleaching rate is thus linked to the distance between the fluorophores and the metamaterial layer, leading to a reconstructed image with exceptionally high axial resolution. We apply the MAPM technology to image the HeLa cell membranes tagged with fluorescent proteins and demonstrate an axial resolution of ∼2.4 nm with multiple colors. MAPM utilizes a metamaterial-coated substrate to achieve super-resolution without altering anything else in a conventional microscope, representing a simple solution for fluorescence imaging at nanometer axial resolution.

Optical tweezers and multimodality imaging: a platform for dynamic single‐molecule analysis

Utilization of single molecule technologies offer researchers the unique opportunity to perform direct real‐time observations and measurements for a plethora of biological mechanisms. Here, we present our efforts to facilitate biologically meaningful discoveries using fully integrated optical tweezers with single‐molecule fluorescence microscopy technology. Correlative fluorescence and optical tweezer microscopy (C‐trap™) is the only technology that truly integrates optical tweezers, confocal/STED microscopy and an advanced microfluidics. The C‐Trap ™ enables live, simultaneous and correlative visualization and manipulation of molecular interactions with sub‐piconewton (pN) force resolution and a kilo‐Hz (to mega‐Hz) temporal resolution, such that even the quickest cellular events can be captured. This technology can be utilized as an easy‐to‐use and stable instrument with the capacity to elucidate biologically meaningful mechanisms. Here we present applications detailing protein (un)folding and conformational changes; DNA‐protein interactions and genome modifications; effects of mechanical stress on DNA/RNA structure; motility of cytoskeletal molecular motors; and protein droplet and aggregation dynamics. These experiments demonstrate technological advances in hybrid single‐molecule methods and the potential for their use in future scientific breakthroughs.

Properties of ultraviolet phosphor for ion-beam-induced fluorescent microscopy technology

Abstract In this study, we have investigated the ultraviolet (UV) fluorescence properties of a phosphor (YPO4:Gd) sample from the irradiation of a scanning proton beam to evaluate its usefulness as a staining agent of ion-beam-induced fluorescence microscopy (IBIF). The phosphor was prepared using a co-precipitation method. Owing to the irradiation, the sample exhibited UV fluorescence with a peak wavelength of 312 nm. In the emission spectrum, the FWHM of the UV peak ≈ 6 nm. The light emission in the UV region and the narrow peak bandwidth are characteristics that are effective at minimizing the background fluorescence and allow high-sensitivity IBIF imaging. Moreover, it was confirmed that the phosphor had a considerable radiation endurance. In addition, we successfully obtained IBIF images and elemental maps simultaneously for a test sample containing YPO4:Gd powder. These results demonstrate that the prepared phosphor is suitable for IBIF.

AIEgens in cell-based multiplex fluorescence imaging

Fluorescence imaging is an important branch of bioimaging. It is non-invasive and provides superior spatial and temporal resolution during the real-time monitoring of biological samples of interest. Although the spatial resolution limit of optical microscopes is about 200 nm, due to the diffraction limit, with the application of super-resolution fluorescence microscopy technologies this limit has been pushed below 30 nm. This makes it feasible to visualize biological structures in subcellular levels and to monitor subcellular biological processes in real time. However, due to the complexity of the biological structure and components within cells, simultaneous staining and monitoring multiple intracellular components with different coloured fluorophores is often needed during multiplex imaging, to better understand biological processes. Aggregation-induced emission luminogens (AIEgen) and AIEgen based nanoparticles (NPs) have presented many advantages in fluorescence imaging, with strong potential for biological science and nano-medicine. Herein this review, we focus on the advantages of AIEgen and AIEgen NP in cell-based fluorescence imaging, and the latest advances of AIEgens in cell-based multiplex imaging are summarized and discussed. The future perspectives are proposed.

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells

Introduction: The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA?Materials and Methods: CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology.Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10−8). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M– arrest, micronuclei) increase. Expression of anti-apoptotic genes (BCL2, BCL2A1, BCL2L1, BIRC3, MDM2) is elevated, while expression of pro-apoptotic genes (BAX, BID, BAD, PMAIP1, BBC3) is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression.Conclusion: (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.

Neurosurgical Flexible Probe Microscopy with Enhanced Architectural and Cytological Detail

Microscopic delineation and clearance of tumor cells at neurosurgical excision margins potentially reduce tumor recurrence and increase patient survival. Probe-based invivo fluorescence microscopy technologies are promising for neurosurgical invivo microscopy. We sought to demonstrate a flexible fiberoptic epifluorescence microscope capable of enhanced architectural and cytological imaging for invivo microscopy during neurosurgical procedures. Eighteen specimens were procured from neurosurgical procedures. These specimens were stained with acridine orange and imaged with a 3-dimensional (3D)-printed epifluorescent microscope that incorporates a flexible fiberoptic probe. Still images and video sequence frames were processed using frame alignment, signal projection, and pseudo-coloring, resulting in resolution enhancement and an increased field of view. Images produced displayed good nuclear contrast and architectural detail. Grade 1 meningiomas demonstrated 3D chords and whorls. Low-grade meningothelial nuclei showed streaming and displayed regularity in size, shape, and distribution. Oligodendrogliomas showed regular round nuclei and a variably staining background. Glioblastomas showed high degrees of nuclear pleomorphism and disarray. Mitoses, vascular proliferation, and necrosis were evident. We demonstrate the utility of a 3D-printed, flexible probe microscope for high-resolution microscopic imaging with increased architectural detail. Enhanced invivo imaging using this device may improve our ability to detect and decrease microscopic tumor burden at excision margins during neurosurgical procedures.

Imaging Flies by Fluorescence Microscopy: Principles, Technologies, and Applications.

The development of fluorescent labels and powerful imaging technologies in the last two decades has revolutionized the field of fluorescence microscopy, which is now widely used in diverse scientific fields from biology to biomedical and materials science. Fluorescence microscopy has also become a standard technique in research laboratories working on Drosophila melanogaster as a model organism. Here, we review the principles of fluorescence microscopy technologies from wide-field to Super-resolution microscopy and its application in the Drosophila research field.

Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging.

The development of fluorogens with deep-red emission is one of the hottest topics of investigation in the field of bio/chemosensors and bioimaging. Herein, the tunable fluorescence of perylene diimide (PDI) derivatives was achieved by the incorporation of varied isolation groups linked on the PDI core. With the enlarged sizes of isolation groups, the conversion from aggregation caused quenching to aggregation-induced emission was obtained in their fluorescence variations from solutions to nanoparticles, as the result of the efficient inhibition of π-π stacking by the larger isolation groups. Accordingly, DCzPDI bearing 1,3-di(9H-carbazol-9-yl)benzene as the biggest isolation group exhibited the bright deep-red emission in the aggregated state with a quantum yield of 12.3%. Combined with the three-photon excited fluorescence microscopy (3PFM) technology, through-skull 3PFM imaging of mouse cerebral vasculature can be realized by DCzPDI nanoparticles with good biocompatibility, and the penetration depth can be as deep as 450 μm.

Erythrophagocytosis by plasmacytoid dendritic cells and monocytes is enhanced during inflammation.

Generation of antibodies against red blood cell (RBC) antigens can be a clinically significant problem. The underlying mechanisms that regulate the production of RBC antibodies are only partially understood; however, factors such as inflammation significantly increase the rates of RBC antibody generation. Humoral alloimmunization begins with consumption of transfused RBCs by antigen-presenting cells (APCs). Recently, it has become appreciated that there are multiple different types of APCs. The relative contribution of APC subsets to RBC antibodies has not been described in either the quiescent or the inflamed states. To evaluate the types of APCs that consume RBCs, and how inflammation affects this process, C56Bl/6 mice were treated with polyinosinic-polycytidylic acid (poly(I:C)) to induce an inflammatory response and/or were transfused with 3,3'-dihexadecyloxacarbocyanine perchlorate-labeled syngeneic RBCs. Erythrophagocytosis (both at baseline and during inflammation) was analyzed for different subsets of macrophages (MΦ), dendritic cells (DCs), B cells, and monocytes, by a combined approach using flow cytometry and fluorescent microscopy technology. In four independent experiments, erythrophagocytosis at baseline was predominately performed by red pulp MΦ; however, during inflammation both plasmacytoid DCs (pDCs) and monocytes increased RBC consumption. Furthermore, pDCs up regulated MHC-II and activation markers CD80 and CD86. In addition to changing patterns of erythrophagocytosis, inflammation also led to a significant decrease in CD11c+ conventional DC populations and an increase in granulocytes. The nature of APCs that consume transfused RBCs is changed by inflammation. Given that APCs initiate humoral immune responses, these findings provide potential mechanistic insight into how inflammation regulates RBC alloimmunization.

High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas.

The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists mainly based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High content imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Lymphocytes were obtained from lymph nodes of six lymphoma-free and five lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T-cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: (a) cell area, (b) nuclear area, (c) nuclear displacement, (d) cytoplasmic area, (e) nucleus to cell area ratio (N/Cell), (f) nucleus to cytoplasm ratio (N/Cyt), and (g) cell roundness. HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences and the means for each of the samples were not overlapping between the two groups. Mean/median/SD for control versus lymphoma samples were: (a) cell area (µm(2) ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, (b) nuclear area (µm(2) ): 33.4/34.8/3.5 versus 40.2/38.5/5, (c) cytoplasm area (µm(2) ): 22.1/21/6.2 versus 40/38.4/4.9, (d) N/cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, (e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30/0.056, (f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, and (g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (P < 0.05 for all). HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.

"High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas"

Background: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High Content Imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. Objective: We tested the hypothesis that HCI could semi-automatise the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Methods: Lymphocytes were obtained from lymph nodes of 6 lymphoma-free and 5 lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T- cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: a) cell area, b) nuclear area, c) nuclear displacement, d) cytoplasmic area, e) nucleus to cell area ratio (N/Cell), f) nucleus to cytoplasm ratio (N/Cyt), g) cell roundness. Results: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences without value overlap between groups. Mean/ median/SD for control versus lymphoma samples were: a) cell area (µm2 ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, b) nuclear area (µm2 ): 33.4/34.8/3.5 versus 40.2/38.5/5, c) cytoplasm area (µm2 ): 22.1/216.2 versus 40/38.4/4.9, d) N/Cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30//0.056, f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (p<0.05). Conclusion: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype. © 2014 Clinical Cytometry Society.