Academic literature on the topic 'Microscopy-Based cytometry'
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Journal articles on the topic "Microscopy-Based cytometry"
1
Wessels, J. T., A. C. Busse, J. Mahrt, B. Hoffschulte, G. A. Mueller, A. Tárnok, and A. Mittag. "NorthernLights in slide-based cytometry and microscopy." Cytometry Part A 9999A (2010): NA. http://dx.doi.org/10.1002/cyto.a.20863.
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Ecker, Rupert C., and Georg E. Steiner. "Microscopy-based multicolor tissue cytometry at the single-cell level." Cytometry 59A, no. 2 (2004): 182–90. http://dx.doi.org/10.1002/cyto.a.20052.
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Kummrow, A., M. Frankowski, N. Bock, C. Werner, T. Dziekan, and J. Neukammer. "Quantitative assessment of cell viability based on flow cytometry and microscopy." Cytometry Part A 83A, no. 2 (October 18, 2012): 197–204. http://dx.doi.org/10.1002/cyto.a.22213.
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Mendoza, Maria Gracia Garcia, Alex Sutton, Raymond Kong, Matthew Rodrigues, and Haley Pugsley. "A rapid and fully automated in vitro micronucleus assay using imaging flow cytometry and convolutional neural network analysis." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 172.02. http://dx.doi.org/10.4049/jimmunol.208.supp.172.02.
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Abstract Micronuclei (MN) originate from whole chromosomes or chromosome fragments that lag behind during cell division and fail to be incorporated into one of the two main nuclei. As a result, scoring MN using the well-established in vitro micronucleus assay evaluates the ability of chemicals or other agents to induce DNA damage. This technique is typically performed by manual microscopy, which can be time-consuming and prone to variability. Additionally, automated methods lack cytoplasmic visualization when using slide-scanning microscopy, and conventional flow cytometry doesn’t provide visual confirmation of MN. The ImageStream®X Mk II (ISX) imaging flow cytometer combines the high-resolution imagery of microscopy with conventional flow cytometry’s speed and statistical robustness in a single system. Previously, we developed a rapid and automated MN assay based on high-throughput image capture and feature-based image analysis using IDEAS® Software. However, the feature-based analysis was not readily applicable to multiple cell lines and chemicals, so we developed a deep learning method based on convolutional neural networks to score imaging flow cytometry data in both the cytokinesis-blocked and unblocked versions of the MN assay using Amnis® AI Software. Our current study validates our previously established assay and analyses using three different chemicals (Mitomycin C, Cyclophosphamide, and Eugenol) and three different cell lines (TK6, L5178Y, CHO-K1). Here, we demonstrate how using Amnis AI to score imagery acquired on the ISX provides a rapid and fully automated in vitro MN assay with improved accuracy, reproducibility, and time-to-results in toxicity and biodosimetry applications across multiple cell lines.
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Sláma, Petr, Zbyšek Sládek, and Dušan Ryšánek. "Application of methods for detection of apoptosis and necrosis of bovine blood neutrophil granulocytes in vitro." Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 54, no. 5 (2006): 107–16. http://dx.doi.org/10.11118/actaun200654050107.
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The study was an in vitro analysis of bovine blood neutrophil apoptosis and necrosis based on the detection of their morphological and biochemical features. The experiment was carried out in six clinically healthy Holstein x Bohemian Red Pied crossbred virgin heifers aged 16 to 18 months. The fresh and in vitro cultivated blood were analysed by light and electron microscopy and flow cytometry. In fresh blood, non apoptotic or necrotic neutrophils were found by light microscopy. On the other hand, 3.34% apoptotic and 0.32% necrotic neutrophils were detected by flow cytometry. After four hours of incubation, 3.53% apoptotic and 1.37% necrotic neutrophils were found by light microscopy. In the same conditions, there were assessed 17.54% apoptotic and 0.58% necrotic neutrophils by flow cytometry. Correlation coefficient between light microscopy and flow cytometry in portion of apoptotic neutrophils was 0.625 (P<0.05) and correlation coefficient between light microscopy and flow cytometry in portion of necrotic neutrophils was 0.983 (P<0.05). There was found out that these detection methods (light microscopy and flow cytometry) are right combination for detection of apoptotic and necrotic bovine blood neutrophils.
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Asthana, Vishwaratn, Yuqi Tang, Adam Ferguson, Pallavi Bugga, Anantratn Asthana, Emily R. Evans, Allen L. Chen, Brett S. Stern, and Rebekah A. Drezek. "An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types." PeerJ 6 (June 5, 2018): e4937. http://dx.doi.org/10.7717/peerj.4937.
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Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.
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Adachi, Takahiro, and Takeshi Tsubata. "FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy." Biochemical and Biophysical Research Communications 367, no. 2 (March 2008): 377–82. http://dx.doi.org/10.1016/j.bbrc.2007.12.142.
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Wei, Shu-Gen, Ling-Yun Wan, Ying Wei, Li-Li He, Jin-E. Fu, and Li-Mei Pan. "Analysis of ploidy level of Artemisia annua L. based on flow cytometry and confocal laser scanning microscopy." Bangladesh Journal of Botany 50, no. 1 (March 27, 2021): 29–35. http://dx.doi.org/10.3329/bjb.v50i1.52668.
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Eighty nine Artemisia samples treated with different concentrations of colchicine were used as breeding samples, with diploid Artemisia as the control. The ploidy levels of samples were determined by flow cytometry and confocal laser scanning microscopy (CLSM). An analysis of the flow cytometry results identified three suspected tetraploid plants and seven suspected triploid plants. The results of this study may be useful for breeding new Artemisia lines.
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Heckmann, Mara, Gerald Klanert, Georg Sandner, Peter Lanzerstorfer, Manfred Auer, and Julian Weghuber. "Fluorescence microscopy-based quantitation of GLUT4 translocation." Methods and Applications in Fluorescence 10, no. 2 (January 21, 2022): 022001. http://dx.doi.org/10.1088/2050-6120/ac4998.
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Abstract Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.
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Lima, Ângela, Christina A. Muzny, and Nuno Cerca. "An Indirect Fluorescence Microscopy Method to Assess Vaginal Lactobacillus Concentrations." Microorganisms 12, no. 1 (January 5, 2024): 114. http://dx.doi.org/10.3390/microorganisms12010114.
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Lactobacillus species are the main colonizers of the vaginal microbiota in healthy women. Their absolute quantification by culture-based methods is limited due to their fastidious growth. Flow cytometry can quantify the bacterial concentration of these bacteria but requires the acquisition of expensive equipment. More affordable non-culturable methods, such as fluorescence microscopy, are hampered by the small size of the bacteria. Herein, we developed an indirect fluorescence microscopy method to determine vaginal lactobacilli concentration by determining the correlation between surface area bacterial measurement and initial concentration of an easily cultivable bacterium (Escherichia coli) and applying it to lactobacilli fluorescence microscopy counts. In addition, vaginal lactobacilli were quantified by colony-forming units and flow cytometry in order to compare these results with the indirect method results. The colony-forming-unit values were lower than the results obtained from the other two techniques, while flow cytometry and fluorescence microscopy results agreed. Thus, our developed method was able to accurately quantify vaginal lactobacilli.
Dissertations / Theses on the topic "Microscopy-Based cytometry"
1
Apichitsopa, Nicha. "Intrinsic cytometry based on computational microscopy." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/107030.
Full textAbstract:
Thesis: S.M., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 123-131).
With the goal of understanding cells, we propose to study the intrinsic properties of individual cells by combining visual observation from large field-of-view computational microscopy with separation of cells via an integrated label-free microfluidic platform. This intrinsic cytometry will benefit from the parallel and gentle separation of label-free cells via a microfluidic platform and parallel tracking of multiple cells via a large field-of view microscopy in contrast to the gold standard, flow cytometry, which is able to rapidly and singly identify multiple cell properties via scattering of external fluorescent cell markers. In this thesis, a prototype of this integrated platform was designed and fabricated. The prototype consisted of a large field-of-view digital in-line holographic microscopy system and a microfluidic deterministic lateral displacement array which separated particles based on size. Each system was first characterized separately and later integrated such that individual cells inside the deterministic lateral displacement array could be recorded and tracked with the large field-of-view digital in-line holographic microscopy system, showing the promise of our proposed intrinsic cytometry. In future studies, if a microfluidic platform can be designed to investigate multiple intrinsic properties of individual cells on the same platform, the intrinsic cytometer will enable a large pool of quantitative measurement data of cell intrinsic properties that can potentially be used for cell characterization and diagnostics.
by Nicha Apichitsopa.
S.M.
2
Schumacher, William Charles. "Development of Novel Fluorescence-Based Methods for Detection of Bacillus Anthracis Spores." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1222046829.
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Wu, Jianglai. "A light sheet based fluorescence imaging flow cytometer for phytoplankton analysis." HKBU Institutional Repository, 2014. https://repository.hkbu.edu.hk/etd_oa/36.
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Monitoring phytoplankton species composition and their abundance are routine tasks in marine ecological research and environmental monitoring. As phytoplankton populations are highly heterogeneous in terms of size, morphology, and most significantly, their abundance can change drastically in a very short time, it is extremely difficult to quantify and monitor them and there are demands on the instrumentation. Conventional optical microscopy and flow cytometry are the main tools to enumerate and identify phytoplankton, but they have a compromise between spatial information and acquisition speed. While imaging flow cytometry has the potential to integrate the benefit of high spatial resolution from optical microscopy and the advantage of high throughput from flow cytometry, two intrinsic blur sources, motion blur and out-of-focus blur, prevent imaging flow cytometers from obtaining high spatial resolution images with high throughput. To address these limitations, in this work, a novel light sheet based fluorescence imaging flow cytometer has been proposed, constructed, and tested for phytoplankton analysis. Both 2D and 3D imaging mode of the light sheet based fluorescence imaging flow cytometer have been investigated. In the 2D imaging mode, the instrument can screen untreated costal water samples at a volumetric throughput up to 1 ml/min. The instrument demonstrated shows a high immunity to motion blur, and all-in-focus fluorescence images are captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~ 1 µm to ~ 200 µm that includes pico-, nano-and microphytoplankton. This is made possible by suppressing the out-of-focus blur using thin light sheet illumination and image deconvolution, and by precluding the motion blur with a unique flow configuration. With these abilities, the instrument demonstrated has high potential as a practical field instrument for monitoring phytoplankton. In the 3D imaging mode, the instrument can scan a large number of phytoplankton cells in a short time with spatial resolution as achieved by light sheet microscopy. The lateral resolution is 0.81 ± 0.07 µm, and axial resolution in terms of FWHM of the axial scattering PSF is 1.42 ± 0.15 µm. The volumetric throughput of the instrument is 0.5 µl/min. This is benefitted from the improvement that 3D images can be acquired without the need of sample immobilization, in contrast to existing 3D imaging approaches, such as confocal fluorescence microscopy. Preliminary results from untreated coastal water samples and cultured samples show promising potentials of the instrument for phytoplankton monitoring and scientific research.
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Troisi, Lucie. "Development of a new class of synthetic gene circuits based on protein-protein interactions." Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS728.
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La biologie synthétique promet de révolutionner la façon dont les scientifiques manipulent et analysent les systèmes vivants. Dans ce projet, nous proposons de développer une nouvelle classe de réseaux de gènes synthétiques, basée sur la compétition entre la forme active et inactive d'un facteur de transcription synthétique. Afin de déterminer les paramètres moléculaires et les topologies requises pour une fonction voulue, nous utilisons une approche in silico évolutionnaire couplée à de la modélisation. Avec cette méthodologie, nous voulons construire des circuits à multiples entrées, ainsi que de nouveaux réseaux bistables et oscillatoires. Cette nouvelle classe de réseaux pourra par la suite être étendue à des réseaux multi-cellulaires montrant des motifs dissymétriques ou oscillatoires. Ce projet fondamental à l'interface entre la modélisation et la validation expérimentale permettra de promouvoir le développement de circuits avancés avec des applications prometteuses en diagnostique, en thérapie génique et en ingénierie tissulaire avancéeSynthetic biology, by its engineering approach, promise to revolutionize the way scientists manipulate and analyze living systems. In this project, we propose to develop a new class of synthetic gene circuits whose fine tuning rely on the affinity competition between active and inactive forms of a transcription factor. Modelling, together with an in silico evolutionary approach, will be used to determine molecular parameters and network topologies required for a given functionality. Circuits will be assembled accordingly and their expression in mammalian cells measured to confirm the expected response or correct our model. Using this methodology, we plan to build multi-inputs circuits with tunable response function, as well as new bistable and oscillatory circuits. The new investigated class of circuits will also be extended to multi-cellular networks exhibiting symmetry breaking or oscillating patterns. This fundamental project bridging modelling and experimental validation will promote the development of advanced targeting circuits with promising applications in diagnosis, gene therapy and complex tissue engineering
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Wu, Cheng-Ham, and 吳政翰. "Label-free Imaging Cytometry of Leukocytes Based on Third Harmonic Generation Microscopy." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/85016410448762663461.
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博士國立臺灣大學
醫學工程學研究所
105
Blood tests, which analyze the composition of species in blood, are usually the first stage of clinical examination in the hospital. This includes biochemical and complete blood count analysis. The indices of the latter provide information about the health status of a person. The blood cells include erythrocytes (red blood cells, RBCs), leukocytes (white blood cells, WBCs), and thrombocytes (platelets). For a healthy person, the quantities and percentage of these cells will remain in a steady state; thus, studying the variation of cells gives doctors an aspect of diagnosis. However, these processes require blood to be drawn for further analysis (e.g. flow cytometry, histology), which not only takes time and causes pain through an invasive method, but the in vitro samples are affected by the environment. Therefore, an instrument with real-time and non-invasive analysis is highly desired for blood-cell counting. In current studies, nonlinear optical microscopy, such as harmonic generation microscopy (HGM) and two-photon microscopy (2PFM), has been widely used in biological and material studies due to its sub-micron three-dimensional (3D) spatial resolution. Obeying the energy conservation of harmonic generation, there is no energy deposition in tissue, making it more suitable for long-term observation. Additionally, the greatest advantage of HGM is that it needs no extra labeling for samples, making it more suitable for studying living biology, especially in clinics. In this thesis, we developed a real-time and label-free in vivo third-harmonic generation (THG) flow cytometer, and we further distinguished and differentiated different types of WBCs (neutrophils, monocytes, and lymphocytes). The laser source in this study was a homemade femtosecond Cr: forsterite (Cr: F) with a Kerr-lens mode-locking technique. As the wavelength falls within the second optical penetration window, this laser penetrates deeper without out-of- and on-focus photodamage. With the help of high-speed imaging acquisition software and a set of 16-kHz resonant galvanometer mirrors, the rolling cells within vessels could be revealed individually with 30 frames per second (fps) and 512 x 512 pixels in the image output. The results showed that neutrophil shows a stronger THG signal than monocyte and lymphocyte, with a dark multi-lobed nucleus within the cell. In contrast, monocyte and lymphocyte have identical THG intensity, but less than neutrophil, with a single nucleus roughly equal to the cell’s size. Taking the size of each cell into account, WBCs could be differentiated into three groups by a k-means clustering algorithm in a THG intensity-size plot. The cell count and percentage of each type of leukocyte agreed with the regular complete blood count readout, showing that the in vivo THG imaging flow cytometer will be applicable to clinical diagnosis in the future.
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Regmi, Raju. "Light Sheet Based Microfluidic Flow Cytometry Techniques for High throughput Interrogation and High-resolution Imaging." Thesis, 2014. https://etd.iisc.ac.in/handle/2005/3108.
Full textAbstract:
Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go.
The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations.
The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability.
Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel.
The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique.
Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective.
This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.
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Regmi, Raju. "Light Sheet Based Microfluidic Flow Cytometry Techniques for High throughput Interrogation and High-resolution Imaging." Thesis, 2014. http://hdl.handle.net/2005/3108.
Full textAbstract:
Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go.
The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations.
The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability.
Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel.
The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique.
Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective.
This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.
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Hyll, Kari. "Image-based quantitative infrared analysis and microparticle characterisation for pulp and paper applications." Doctoral thesis, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-180722.
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Measurements of process variations and particle morphology are widely employed in the pulp and paper industry. Two techniques with high potential, infrared thermography and microparticle characterisation, are mainly used qualitatively. Quantitative thermography requires knowledge of the emittance, a material property which has not been measured under many process-relevant conditions. Quantitative characterisation of microparticles, e.g. pulp fines and mineral fillers, requires the analysis of a large number of particles, which can be accomplished using flow microscopes. Flow microscopes for pulp analysis have had insufficient spatial resolution to resolve fines and fillers. Additionally, there has been a lack of methods which can differentiate between fines and fillers in a mixed suspension. State-of-the-art instruments for particle image analysis were evaluated and compared to laser diffractometry, a measurement method based on scattering by diffraction. Laser diffractometry was found to be highly sensitive to the complex refractive index of the particles, and especially to its change due to moisture absorption. A high-resolution imaging flow cytometer and a high-resolution fibre analyser were found to be complementary for characterisation of pure fines and fines/filler mixtures, and superior to laser diffractometry. A method for differentiating between fines and fillers in a suspension based on their autofluorescence and side-scattering was proposed and qualitatively evaluated. Furthermore, a method for measuring the directional and integrated emittance of paper was developed and its accuracy was determined. Measurements on a wide range of samples showed that the emittance of fibre-based materials vary significantly with wavelength, pulp type, observation angle, and moisture content. By applying measured quantitative values of the emittance, the thermal energy emitted by sack paper samples during mechanical deformation could be quantitatively calculated. The increase in thermal energy at the time of rupture was found to correlate well with the elastic share of the mechanical energy that was stored in the sample during its elongation. In summary, the results of this work have facilitated the use of quantitative microparticle analysis and infrared thermography for pulp and paper applications.Mätningar av processvariationer och partiklars form och storlek utförs i stor skala inom massa- och pappersindustrin. Två mättekniker med stor potential, infraröd termografi och mikropartikel-karaktärisering, används mest kvalitativt idag. Kvantitativ termografi kräver att provets emittans är känd. Emittansen är en materialegenskap som inte har mätts för många förhållanden som är relevanta inom papperstillverkning. Kvantitativ karaktärisering av partiklar kräver att ett tillräckligt stort antal partiklar analyseras, något som kan göras med flödesmikroskop. Flödesmikroskop för mäldanalys har haft otillräcklig upplösning för att karaktärisera mikrometerstora partiklar, t.ex. fines och fyllmedel. Det har heller inte funnits någon metod som kan särskilja mellan fines och fyllmedel i en blandning. Högupplösta mätinstrument för bildbaserad mikropartikelkaraktärisering utvärderades och jämfördes med en laserdiffraktometer, en mätmetod baserad på ljusspridning genom diffraktion. Laserdiffraktometerns mätresultat påverkades starkt av det brytningsindex som antogs för provet, och hur brytningsindexet ändrades med fukthalt. En högupplöst bildbaserad flödescytometer och en högupplöst fibermätare konstaterades komplettera varandra vid mätningar av mäldens finmaterial. De var även pålitligare än laserdiffraktometern vid mätningar av organiskt finmaterial. En metod för att skilja mellan organiskt och oorganiskt finmaterial i en mäld baserat på deras autofluorescens och ljusspridning presenterades och utvärderades kvalitativt. En metod för att mäta den vinkelberoende och våglängdsintegrerade emittansen hos fiberbaserade material utvecklades och dess mätnoggrannhet utvärderades. Mätningar på ett stort antal prover visade att emittansen varierade betydligt med våglängd, mäldtyp, observationsvinkel, och fukthalt. Genom att använda den uppmätta emittansen kunde den termiska energin som frigjordes av ett säckpappersprov vid brottögonblicket beräknas. Denna energi korrelerade väl med den elastiska energi som lagrades i provet medan det töjdes, fram till tidpunkten för brottet. Sammanfattningsvis har resultaten av detta arbete möjliggjort kvantitativ användning av mikropartikel-karaktärisering och infraröd termografi i massa- och papperstillämpningar.
QC 20160122
Books on the topic "Microscopy-Based cytometry"
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Zrazhevskiy, P., and X. Gao. Bioconjugated quantum dots for tumor molecular imaging and profiling. Edited by A. V. Narlikar and Y. Y. Fu. Oxford University Press, 2017. http://dx.doi.org/10.1093/oxfordhb/9780199533060.013.17.
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This article discusses the use of bioconjugated quantum dots (QDs) for tumor molecular imaging and profiling. The need for personalized diagnostics and therapy is becoming apparent in all areas of medicine, and especially urgent and sought after in treating cancer. Mechanisms of cancerogenesis and cancer response to therapy remain poorly understood, thus precluding accurate cancer diagnosis, prognosis, and effective treatment. Accurate molecular profiling of individual tumors is one key to effective treatment. This article first considers the photophysical properties of QDs before reviewing the most common methods for engineering QD-based probes for biomedical applications, including water solubilization and bioconjugation approaches. It also describes a number of techniques for molecular imagingand profiling of tumors, ranging from QD-based multicolor flow cytometry and applications of QDs in high-resolution correlated fluorescence/electron microscopy, QD bioprobes for molecular profiling of tumor-tissue sections and microarrays, and QD-oligonucleotide bioconjugates for in-situ hybridization.
Book chapters on the topic "Microscopy-Based cytometry"
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van Schaik, Tom, Stefano G. Manzo, and Bas van Steensel. "Genome-Wide Mapping and Visualization of Protein–DNA Interactions by pA-DamID." In Methods in Molecular Biology, 215–29. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2140-0_12.
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AbstractSeveral methods have been developed to map protein–DNA interactions genome-wide in the last decades. Protein A-DamID (pA-DamID) is a recent addition to this list with distinct advantages. pA-DamID relies on antibody-based targeting of the bacterial Dam enzyme, resulting in adenine methylation of DNA in contact with the protein of interest. This m6A can then be visualized by microscopy, or mapped genome-wide. The main advantages of pA-DamID are an easy and direct visualization of DNA that is in contact with the protein of interest, unbiased mapping of protein–DNA interactions, and the possibility to select specific subpopulations of cells by flow cytometry before further sample processing. pA-DamID is particularly suited to study proteins that form large chromatin domains or that are part of distinct nuclear structures such as the nuclear lamina. This chapter describes the pA-DamID procedure from cell harvesting to the preparation of microscopy slides and high-throughput sequencing libraries.
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Zhu, Hongying, and Aydogan Ozcan. "Opto-Fluidics Based Microscopy and Flow Cytometry on a Cell Phone for Blood Analysis." In Methods in Molecular Biology, 171–90. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-2172-0_12.
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De Vos, W. H., B. Dieriks, G. Joss, and P. Van Oostveldt. "High content image-based cytometry as a tool for nuclear fingerprinting." In EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany, 783–84. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-85156-1_392.
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Nazeer, Nazim, Pooja Ratre, Kaniz Zehra Zaidi, Vikas Gurjar, Rakhi Dewangan, Arpit Bhargava, and Pradyumna Kumar Mishra. "Exploring Semiconductor Quantum Dots for Nanophotonic Applications." In Reshaping Healthcare with Cutting-Edge Biomedical Advancements, 235–88. IGI Global, 2024. http://dx.doi.org/10.4018/979-8-3693-4439-2.ch011.
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In the realm of nanophotonics, there has been a noticeable increase in demand for semiconductor nanomaterials over the past few years. Quantum dots (QDs) have captured global scientific attention due to their unique optical properties. QDs comprise a semiconductor material core surrounded by a shell with a wider bandgap, which determines their optoelectrical properties. This chapter looks closely at various types of QDs, including carbon-based, perovskite, colloidal, magnetic QDs, and metal-based QDs. The authors examine different methods of surface functionalization and doping to optimize the performance of QDs. QDs have numerous applications in biomedical imaging and nanosensing. QDs are highly suitable as they demonstrate excellent photostability and bright emission, making them ideal for use as both fluorescent detectors and reporter molecules in laser scanning confocal microscopy, super-resolution microscopy, molecular spectroscopy, and flow cytometry.
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X. Chen, Cindy, Hillel B. Price, and Adam Wax. "Holography Cytometry: Imaging of Cells in Flow." In Holography - Recent Advances and Applications [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.106581.
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Holographic cytometry (HC) has been developed as an ultra-high throughput implementation of quantitative phase microscopy (QPM). While QPM has been well developed for studying cells based on endogenous contrast, few implementations have imaged cells in flow or provided high throughput measurements. Although QPI offers high resolution imaging, experiments are limited to examining a single cell at a time. The HC approach enables high throughput by imaging cells as they are flowed through microfluidic devices. Stroboscopic illumination is used in an off-axis interferometry configuration to produce holographic images of flowing cell samples without streaking artifact. The ability to profile large number of cells using individual images has been demonstrated in red blood cell and cancer cell samples. The large volume of data provides suitable training data for developing machine learning algorithms, producing excellent accuracy in classifying cell type. Analysis of the adherent cells to flow also produces diagnostically useful information in the form of biomechanical cell properties. Introduction of a new parameter, disorder strength, a measure of the variance of phase fluctuations across a cell, provides an additional window into the cell mechanical properties.
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Muhammad, Ismail, Micah Pukuma Sale, and Tanko Mahmoud Mohammed. "Advanced Techniques and Unusual Samples for Malaria Diagnosis." In Infectious Diseases. IntechOpen, 2023. http://dx.doi.org/10.5772/intechopen.113756.
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Successful malaria control, treatment, and prevention depends on successful diagnosis using appropriate equipment with high sensitivity and specificity. In most tropical countries where the disease is endemic, malaria diagnosis is still based on the conventional techniques (Microscopy and RDT) which have so many shortcomings, hence the need to switch to the most advanced diagnostic technique for better results. In this review, several serological and molecular malaria diagnostic techniques like Polymerase Chain Reaction (PCR), Flow cytometry, Loop-mediated Isothermal Amplification (LAMP), Indirect Immunofluorescence, Enzyme-Linked Immunosorbent Assay (ELISA), Radioimmunoassay (RIA), Quantitative Buffy Coat (QBC) and Laser Desorption Mass Spectrometry (LDMS) were systematically discussed in simple and direct language for easier understanding of the principle involved in each case scenario. In addition, some unusual samples for malaria diagnosis like Urine and saliva were also discussed.
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Schwietzer, Ysabel Alessa, Katharina Helene Susek, Ziqing Chen, Evren Alici, and Arnika Kathleen Wagner. "A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids." In Methods in Cell Biology. Elsevier, 2022. http://dx.doi.org/10.1016/bs.mcb.2022.07.011.
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Gakamsky, Dmitry M., and Israel Pecht. "Evaluating receptor stoichiometry by fluorescence resonance energy transfer." In Receptors: Structure and function, 113–36. Oxford University PressOxford, 2001. http://dx.doi.org/10.1093/oso/9780199638819.003.0005.
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Abstract Elucidating mechanisms of molecular interactions among receptors and their ligands is a central problem in cell and molecular biology. Many different methods, including gel filtration, radiolabelling, immunoprecipitation, two-hybrid system analysis, circular dichroism, fluorescence and absorption spectroscopy, and surface plasmon resonance, have been developed to reveal the proximity of proteins in cells, their organization into molecular complexes, and to determine their stoichiometry. The present review focuses on the use of fluorescence resonance energy transfer (FRET), which is a universal, versatile, and powerful approach for evaluating/assaying distances between and within molecules. In comparison with other methods, FRET has numerous advantages. The exquisite sensitivity provided by a fluorescence detection method (single molecule detection is possible), coupled with the wide range of fluorescence-based instrumentation (e.g. microscopy, flow cytometry, steady-state fluorimetry, fluorescence polarization, fluorescence lifetime, rapid mixing devices) and a large variety of commercially available fluorescent probes, allows numerous possible approaches for studying biological structure and function. Moreover, the ability to measure the concentration of bound complexes, without the need to separate them from the unbound fraction, together with the potential for high spatial resolution, make FRET an excellent tool for the study ofreal time receptor-ligand interactions in individual cells (1-3). This is illustrated by the recent application of FRET to the design of high throughput screening instruments in drug design (4, 5). In this chapter we describe FRET applications in the determination of complex molecular organization and stoichiometry in solutions and in living cells.
Conference papers on the topic "Microscopy-Based cytometry"
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Shaked, Natan T. "Quantitative imaging flow cytometry based on digital holographic microscopy." In Optical Methods for Inspection, Characterization, and Imaging of Biomaterials VI, edited by Pietro Ferraro, Simonetta Grilli, and Demetri Psaltis. SPIE, 2023. http://dx.doi.org/10.1117/12.2674432.
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Pan, Feng, Wen Xiao, Lu Xin, Hao Wang, Ran Peng, and Xi Xiao. "High-throughput and high-precision flow cytometry based on digital holographic microscopy." In Optical Methods for Inspection, Characterization, and Imaging of Biomaterials VI, edited by Pietro Ferraro, Simonetta Grilli, and Demetri Psaltis. SPIE, 2023. http://dx.doi.org/10.1117/12.2674497.
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Kong, Lingjie, Jianyong Tang, and Meng Cui. "In vivoimaging flow cytometry based on laser scanning two-photon microscopy at kHz cross-sectional frame rate." In SPIE BiOS, edited by Ammasi Periasamy, Peter T. C. So, and Karsten König. SPIE, 2016. http://dx.doi.org/10.1117/12.2211418.
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Picazo-Bueno, Jose Angel, Álvaro Barroso, Steffi Ketelhut, Vicente Micó, Jürgen Schenekenburger, and Björn Kemper. "Multimodal imaging flow cytometry based on single capture bright field and digital holographic microscopy for the analysis of living cells." In Optical Methods for Inspection, Characterization, and Imaging of Biomaterials VI, edited by Pietro Ferraro, Simonetta Grilli, and Demetri Psaltis. SPIE, 2023. http://dx.doi.org/10.1117/12.2673841.
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Dmitrieva, Valentina Viktorovna, and Evgeny Valeryevich Polyakov. "THE MEDICAL INFORMATION SYSTEM CONCEPT FOR ACUTE LYMPHOBLASTIC LEUKEMIA DIAGNOSING." In NEW TECHNOLOGIES IN MEDICINE, BIOLOGY, PHARMACOLOGY AND ECOLOGY. Institute of information technology, 2021. http://dx.doi.org/10.47501/978-5-6044060-1-4.06.
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Continuous improvement of software and hardware platforms makes it possible to significant-ly expand the development capabilities of modern medical information systems (MIS) to reach a qualitatively new level associated with the transition to the creation of universal cross-platform web-applications. The article discusses the concept of the implementation of MIS for the diagnosis of acute leu-kemia (AL) and minimal residual disease (MRD) through the complex integration of systems based on laser flow cytometry (an integrated method for diagnosing hemoblastosis), intelli-gent computer microscopy based on expert neural network analyzer for recognition of blast cell images based on formed reference knowledge base. When making a diagnosis, this MIS will allow doctors to work with information about patients directly through the system inter-face by comparing data about new patients with images of an expert database and then sub-sequent generate the diagnostic conclusion based on the results of the studies performed. Data security in the system is ensured by the web server administration regulations and the division of access rights for various categories of users. MIS provides an opportunity for a comprehensive diagnostic study of patients making decisions about the tactics of treatment.
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Xue, Liang, Nan Sun, Keding Yan, Fei Liu, and Shouyu Wang. "High-speed quantitative interferometric microscopy based phase imaging cytometer." In SPIE/COS Photonics Asia, edited by Qingming Luo, Xingde Li, Ying Gu, and Yuguo Tang. SPIE, 2014. http://dx.doi.org/10.1117/12.2070398.
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Darabi, Jeff, and Joseph Schober. "A Microfluidic Platform for On-Chip Analysis of Circulating Tumor Cells." In ASME 2021 Fluids Engineering Division Summer Meeting. American Society of Mechanical Engineers, 2021. http://dx.doi.org/10.1115/fedsm2021-65766.
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Abstract Studies have shown that primary tumor sites begin shedding cancerous cells into peripheral blood at early stages of cancer, and the presence and frequency of circulating tumor cells (CTCs) in blood is directly proportional to disease progression. The challenge is that the concentration of the CTCs in peripheral blood may be extremely low. In the past few years, several microfluidic-based concepts have been investigated to isolate CTCs from whole blood. However, these devices are generally hampered by complex fabrication processes and very low volumetric throughputs, which may not be practical for rapid clinical applications. This paper presents a high-performance yet simple magnetophoretic microfluidic chip for the enrichment and on-chip analysis of rare CTCs from blood. Microscopic and flow cytometric assays developed for selection of cancer cell lines, selection of monoclonal antibodies, and optimization of bead coupling are discussed. Additionally, on-chip characterization of rare cancer cells using high resolution immunofluorescence microscopy and modeling results for prediction of CTC capture length are presented. The device has the ability to interface directly with on-chip pre and post processing modules such as mixing, incubation, and automated image analysis systems. These features will enable us to isolate rare cancer cells from whole blood and detect them on the chip with subcellular resolution.