Proceedings Volume 4260

Optical Diagnostics of Living Cells IV

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Proceedings Volume 4260

Optical Diagnostics of Living Cells IV

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Volume Details

Date Published: 10 May 2001
Contents: 9 Sessions, 28 Papers, 0 Presentations
Conference: BiOS 2001 The International Symposium on Biomedical Optics 2001
Volume Number: 4260

Table of Contents

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Table of Contents

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  • Functional Imaging
  • New Methodologies for Optical Imaging
  • Poster Session
  • New Methodologies for Optical Imaging
  • Biological and Optical Manipulation
  • Functional Imaging
  • Biological and Optical Manipulation
  • Poster Session
  • Flow
  • Microscopy I
  • New Technologies
  • Microscopy II: Poster Session
  • Analytical Cytology Informatics
  • Biological and Optical Manipulation
Functional Imaging
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Digital imaging of diabetic endothelial cells
Jolanta Paltauf-Doburzynska, Sepp D. Kohlwein, Wolfgang F. Graier
Endothelial cells release factors that regulate dilatation and contraction of the vessels. They play an important role in modulating both the inflammatory response and vasomotor abnormalities that occur in coronary artery diseases. This endothelial function is associated with changes of intracellular Ca2+ concentration. For this study we used spatially and temporally resolved measurements of local Ca2+ concentration in human endothelial cells cultured in high glucose containing medium. Deconvolution techniques procedure allowed determination of intracellular Ca2+ concentration and its distribution into cellular compartments. We also used a confocal microscope for visualization of intracellular compartments (endoplasmatic reticulum, mitochondria) under normal and pathological conditions. We showed that the interrupted connection between superficial compartments and membrane channels is already the beginning of the cell damage in diabetes.
Dynamic studies on living cells with an atomic force fluorescence microscope
Jens Struckmeier, Erk Klopp, Matthias Born, et al.
Analysis of the reaction of bone structure to mechanical stimulation is a key issue in understanding the origins of osteoporosis and mechanical adaptation of living bone to external forces. This is thought to be regulated on a cellular level. We have investigated quantitative mechanical stimulation of single bone cells and their immediate intracellular calcium responses using a combination of an atomic force microscope (AFM) and a fluorescence microscope, developed in our laboratory. The force stimulation system can apply quantified forces in the pico- and nano-newton regime on exactly defined positions of a cell. We present here the first measurements using this system on the mechanically induced free calcium response of primary osteoblasts. The threshold forces for stimulation and the temporal behavior of the free calcium response are analyzed and the signal transmission to adjacent cells is investigated. An immediate increase of the intracellular calcium concentration is observed after dynamic stimulation with forces between 110nN and 550nN. Forces exceeding 700nN destroy the cell and the free calcium concentration of the neighboring cells react as a consequence.
Spatially-resolved cerebral blood flow imaging using laser speckle
Andrew K. Dunn, Hayrunnisa Bolay, Michael J. Moskowitz, et al.
Cerebral blood flow measurements are crucial for studying neuronal activity and cerebrovascular responses. Most optical techniques for monitoring real time blood flow rely on laser Doppler measurements which are limited to a localized region in space (point measurements). We have used the time-varying speckle pattern produced by coherent light scattered from moving particles to provide real-time, two-dimensional maps of capillary blood flow dynamics. The speckle images of relative blood flow during cortical spreading depression showed a 2-3 mm area of increased blood flow propagation with a velocity of 2.5 mm/min. The speckle technique therefore, provides a relatively simple method of obtaining spatially resolved cerebral blood flow changes in the exposed cortex with high temporal resolution (milliseconds).
New Methodologies for Optical Imaging
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Confocal microscopy using a InGaN laser diode at 406 nm
John M. Girkin, Allister I. Ferguson, David L. Wokosin, et al.
We report on the application of a novel blue laser diode source to confocal microscopy. The source has the potential to be a replacement for argon lasers in a range of fluorescence based imaging systems. It has been demonstrated that with the use of a minimal number of optical components, high quality confocal images can be obtained from laser diodes operating around 406nm. Improvements in image quality through the use of anamorphic prisms to modify the beam profile have been investigated. Living mammalian cells stained with a range of biologically significant compounds have been imaged with high resolution. The stains excited range from fluorescein based compounds to green fluorescent protein. Through the use of the absorption wings a wide range of shorter wavelength fluorophores have been excites, including those more normally excited using UV laser systems. It is expected that this will lead to reduced photo-toxicity within the sample and conventional rather than UV transmitting objective lenses can be used.
Ultrahigh-resolution imaging of biological systems based on standing-wave total internal reflection microscopy
Light microscopy is widely used in biomedical research. Its power derives from the ability to study living biological systems on the microscopic scale. Since its inception over three centuries ago, there has been little improvement in the lateral resolution of optical microscopy. Today, electron microscopy is one of the few methods capable of imaging on length scales below 100nm. Although electron microscopy is a powerful high resolution imaging technique, it is restricted to study fixed specimens. Clearly, there is a need for developing an in vivo optical technique with significantly improved lateral resolution. We have recently developed a new technique, Standing Wave Total Internal Reflection Microscopy (SW-TIRM), that can reach a resolution of about 50 nm. However, the theoretical basis for resolution enhancement in standing-wave total internal reflection microscopy has not been completely examined. SW- TIRM technique relies on the formation of an excitation field containing super-diffraction limited spatial frequency components. While the fluorescence generated at the object planes contains high frequency information of the object distribution, this information is lost at the image plane where detection optics act as a low pass filter. From the perspectives of point spread function engineering, one can show that if this excitation field is translatable experimentally, the high frequency information can be extracted from a set of images where the excitation fields have different displacement vectors. We have developed algorithms for this task that combine this image set to generate a composite image with an effective point spread function that is equal to the product of the excitation field and the Fraunhofer point spread function.
Poster Session
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Continuous fluorescence depletion anisotropy (CFDA) measurement of protein rotation
B. George Barisas, Hongyan Zhang
Fluorescence depletion anisotropy (FDA) measurements of protein rotation combine the long lifetime of chromophore triplet states with the sensitivity of the fluorescence excitation and detection. Frequency domain (FDA) addresses certain practical limitations of time-domain procedures, such as the need for detector gating, but presents its own difficulties. We have combined time- and frequency-domain FDA methods into an efficient continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repetitive waveform and fluorescence signals are averaged over recurring waveform periods by a low rearm time signal averager. Methods for extracting triplet decay and absorption anisotropy decay kinetics from data traces generated by arbitrary waveforms have been developed. For a sample of eosin-BSA in 86% glycerol at 9 degree(s)C, rotational correlation times of 77 micrometers and 137 microsecond(s) , initial anisotropies of 0.109 and 0.125 and limiting anisotropies of 0.017 and 0.022, were obtained by CFDA and cuvet FDA, respectively. Differences in results apparently arise from the weighing of decay components in CFDA by triplet lifetimes of individual components.
New Methodologies for Optical Imaging
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Precise separation of us and ua in tissue by use of 3D to 5D imaging
Manfred D. Kessler, Markus Boehnert, Andre Nicklas, et al.
Imaging in the microcosm of capillaries and cells in intact tissues opened new fields for research and patient monitoring. The experimental separation of scattering ((mu) s) and absorption ((mu) a) in organs can be improved drastically by visualization of subcellular structures. Improved evaluation techniques which apply matrices for storage of determined optical signals are very favorable.
Three-dimensional modeling of light scattering in biological tissue by the spectral method
Andrei V. Maximov, Clarence E. Capjack, Wojciech Rozmus, et al.
For the first time the three dimensional modeling of laser light scattering in biological tissue has been performed using the spectral technique. The accuracy of the spectral numerical method has been verified by comparison with linear perturbation theory and Mie theory. Comparison with Mie theory has validated that the three-dimensional scalar wave equation is a good approximation to the full Maxwell's set of equations for light scattering at moderate angles. The computational requirements for the spectral method in modeling laser interaction with biological samples are much lower than the requirements for other existing numerical methods: finite-difference time-domain and Monte Carlo. Yet the new logarithm is capable of resolving the variations in the scattered signal with a contrast in intensity of up to six orders of magnitude. The spectral technique can be successfully applied to address to address scattering from individual cells and from biological samples containing many cells. The new method is well suited to recognize the size and composition of biological cells, making it a valuable tool in cell cytometry, for example, in the detection of rare event cells and cancerous cells.
Biological and Optical Manipulation
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Integration of optoelectronic array devices for cell transport and sorting
Mark M. Wang, Mihrimah Ozkan, Erhan P. Ata, et al.
Current biochip technologies typically rely on electrostatic or mechanical forces for the transport and sorting of biological samples such as single cells. In this paper we have investigated how optical pressure forces can be effectively used for the manipulation of cells and switching in a microfluidic system. By projecting the optical beams externally non-contact between the control devices and the sample chip is possible thus allowing the sample chips to be disposable which reduces the chance of cross-contamination. In one implementation we have shown that vertical cavity surface emitting laser (VCSEL) array devices used as parallel optical tweezer arrays can increase the parallelism of sample manipulation on a chip. We have demonstrated the use of a high-order Laguerre-Gaussian mode VCSEL for optical tweezing of polystyrene microspheres and live cells. We have also shown that optical pressure forces from higher- power sources can be used for the switching of particles within microfluidic channels. Both the attractive gradient force and the scattering force of a focused optical beam have been used to direct small particles flowing through junctions molded in PDMS. We believe that by integrating optical array devices for simultaneous detection and manipulation, highly parallel and low-cost analysis and sorting devices may be achieved.
Functional Imaging
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Behavior of platelets stained by 5,6-CF-encapsulated PEGylated liposomes after laser irradiation of vessel wall: an in-vivo model for studying site-selective delivery of diagnostic or therapeutic a
Serge R. Mordon, Sylvie Begu, Bruno Buys, et al.
Vascular endothelium serves as an extensive interface between circulating blood and various tissues and organs of the body. As such, it offers an accessible target for blood-borne pharmacological and genetic manipulations that can mediate both local and systemic effects. Thus, targeting of liposomes to activated vascular endothelial cells may provide a strategy for site-selective delivery in the vascular system with broad therapeutic applicability. This study aimed to evaluate an intravital fluorescence imaging technique to visualize in-situ and in real-time the activation of platelets after staining by 5,6-CF- encapsulated PEGylated liposomes injected intravenously. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. The skin micro circulation was observed with an intravital microscope (using x25 and x40 magnification) fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for play-back analysis with a digital video cassette recorder. An inflammatory response was induced by an Argon laser emitting at 514.5nm. The 80micrometers laser beam was focused on a vessel and its position was controlled with the microscope imaging system, it was possible to see individual platelets flowing in blood vessels. As liposomes were labeled with a fluorescent probe which was hydrophilic (located in the aqueous phase), the fluorescence of platelets was due only to the uptake of liposomes. After laser irradiation, platelets activation at sites of vascular injury was obtained. Tethering, translocation of some platelets inside the irradiated zone were clearly seen. At last, detachment and extravasation of platelets were observed. A perivascular fluorescence confirmed that platelets migrated across the basal lamina into the dermal connective tissue. In conclusion, staining of platelets using 5,6-CF-encapsulated PEGylated liposomes injected intravenously presents the following advantages: i) in-situ labeling, ii) use of hydrophilic marker located in an aqueous compartment within the platelet, iii) as the release of the fluorescence marker is slow due to the formulation of liposomes, labeling of platelets could be observed during the whole experiment. Laser irradiation of blood vessels in vivo can induce the different phases of platelet activation: i) recruitment, ii) adhesion, iii) detachment, iv) transmigration. The combination of these techniques (platelet staining with PEGylated liposomes, intravital fluorescence microscopy, laser irradiation) provides a powerful tool to study local inflammation, platelet activation and behavior of liposomes in situ and in real time at an inflammation site. These observations could be considered as a preliminary approach to study the targeting of drugs to an endothelium under inflammation environment.
Biological and Optical Manipulation
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Numerical simulations of optical breakdown for cellular surgery at nanosecond to femtosecond time scales
Alfred Vogel, Joachim Noack
We have shown by experimental investigations that cellular surgery (microdissection, optoporation, and optoinjection) with Nd:YAG laser pulses of 1064 nm and 532 nm wavelength relies on nonlinear absorption leading to optical breakdown and plasma formation at the laser focus. The present study explores possibilities of refining the breakdown effects by employing shorter pulse durations and irradiances that generate plasmas below the threshold for shock wave and bubble formation. Optical breakdown in water at NA=0.9 and NA=1.3 was simulated numerically for wavelengths of 1064 nm and 532 nm and 355 nm, and pulse durations of 6 ns, 30 ps and 100 fs. We used a rate equation model that allows the calculation of the temporal evolution of the free electron density (rho) during breakdown. (rho) (t) could be followed separately for the free electrons generated by multi photon ionization and avalanche ionization. We obtained excellent agreement between the calculated and measured threshold values for breakdown with 6-ns pulses. The simulations predict that the energy threshold for cellular surgery can be reduced by a factor of 350-2600 (depending on wavelength) when the pulse duration is reduced from 6 ns to 100 fs. The calculated breakdown energies for 100 fs pulses focused by an objective with NA=1.3 are 0.6 nJ at 355 nm, 1.6 nJ at 532 nm, and 3.9 nJ at 1064 nm. With ns-pulses at 1064 nm, the breakdown threshold is very sharp, i.e. there is either no effect at all, or a dense plasma is formed causing a micro- explosion. With shorter wavelengths and pulse durations, the threshold is smoother, and electron densities may be produced that stay below the threshold for explosive evaporation and bubble formation. This creates the possibility of achieving highly localized plasma-mediated chemical or thermal changes in the cell. We conclude that both the reduced energy threshold and the smoother breakdown process with fs pulses bear a large potential for the refinement of intracellular surgery.
Studying cellular mechanics using magnetic tweezers and two-photon microscopy
Hayden Huang, Chen-Yuan Dong, Hyuk-Sang Kwon, et al.
The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron resolution 3-D two-photon imaging. Three dimensional maps of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses is not always a local deformation.
Dynamic tether force measurements of the outer hair cells plasma membrane with optical tweezers
Bahman Anvari, Zhiwei Li, Masayoshi Takashima, et al.
The mechanical characteristics of the outer hair cells (OHCs) plasma membrane was studied by forming tethers with optical tweezers. The average force to and from a plasma membrane tether at the lateral wall of the OHC was about 3.5 times greater than that at the basal end of the cell, consistent with the presence of a more extensive cytoskeleton support beneath the PM at the site of the lateral wall. The apparent viscosity of the PM was measured by pulling tethers at different rates while continuously recording the tether force, and was estimated in the range of 13-33 pN*s/micrometers .
Poster Session
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Application of computerized interference microscope for monitoring oscillations of dry cell weight and morphology of living cells
Gennady G. Levin, Theodor V. Bulygin, Eugene Kalinin, et al.
Interference microscopy technique allows implementing the quantitative analysis of a living cell, for example, to measure dry cell weight and cell density spatial distribution. The authors have created the microscope for quantitative analysis of living cells and observation of the dynamics of cell vital activity. Computer-aided processing of interferograms using phase shift technique, allows carrying out continuous (over 12 hours and more) monitoring of quantitative parameters of living cells with time interval between measurements less than one minute. RMS error for dry weight of non-living fixed test object using this microscope is about 1%. Numerous experiments on living lymphocytes and erythrocytes resulted in detecting the oscillations of dry weight. Spectral analysis of obtained time series has allowed allocating harmonics with the period from 2 hours to 15 minutes. Morphometric measurements of the same cells and cross correlation analysis of these data with oscillations of a dry mass were carried out also.
Flow
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Flow cytometer using a fiber optic detection system
Masahiko Kanda, Motomi Nakata, Misako Osoegawa, et al.
A flow cytometer with a new hybrid flow cell has been developed. The hybrid flow cell was constructed closed-type quarts curvets which produces a parabolic profile of flow velocities. It was involved a fiber-optic detecting unit for a high sensitivity collection and had a jet nozzle and a PZT device for droplet formation aimed to cell sorting. A optical fiber that consists of a glass core and have a core diameter of 200 micrometers with NA of 0.5 have been successfully used for the collection of side scatter and fluorescent signals. Two detecting fibers mounted in a hybrid flow cell with pith of 250 micrometers are designed to individually detect fluorescent signals excited by 2 lasers, which are a compact air-cooled AR laser (488 nm: 20 mW) and a semiconductor laser (640 nm: 15 mW). As a result, a sensitivity of our flow cytometer is 300 MESF determined using fluorescein-labeled beads and 2 scatter and 6 fluorescent signals could successfully be detected. Typical patterns of cell cycle were observed with Daudi human Burkitt Lymphoma cell lines stained with PL. Furthermore, the alignment requirements are more simplified because of a hybrid flow cell stably mounted.
Fluorescence lifetime discrimination of cellular DNA and RNA using various intercalating dyes and flow cytometric analysis
H. Helen Cui, Joseph G. Valdez, John A. Steinkamp, et al.
Previously we showed that the fluorescence lifetimes of ethidium and propidium iodide were different when intercalated into cellular double-stranded DNA or RNA. Current studies of four ethidium derivatives showed that the lifetime difference of DNA and RNA bound dyes existed in all four compounds, and that the magnitude of the difference was dependent on the dye structure, the staining concentration, and the conditions in which flow cytometry (FCM) analysis of stained cells was performed. The fluorescence lifetime of both DNA and RNA bound fluorochromes was reduced with increasing dye concentration; however the level of decrease varied in different dyes. Further analysis of stained cellular DNA in dye-free solution, which favors only high affinity binding, led to elevated fluorescence lifetime values compared to lifetime values obtained from analysis in the dye solution under equilibrium binding conditions. The changes of fluorescence lifetime value reflect the difference in dye structure and distinct interaction between the dyes and nucleic acids. The staining and analysis variables used in these studies may potentially provide additional information on differences in nucleic acid conformation and function in cell populations.
Flow cytometric time-resolved measurements by frequency heterodyning of fluorescence emission signals
John A. Steinkamp, Jimmie D. Parson
A first generation phase-sensitive flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making time-resolved fluorescence measurements in the frequency-domain or particles/cells labeled with fluorescent probes. Cells are analyzed as they flow through a chamber and intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence emission signals are currently processed by analog homodyne methods to quantify lifetimes and resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase shifts, while maintaining the capability to make conventional FCM measurements. in this study we report the current status of our phase flow cytometer using homodyne signal processing, including recent applications, along with the description of a flow cytometric method for quantifying fluorescence lifetimes by frequency heterodyning techniques. Lifetimes are determined from homodyned and heterodyned (1 MHz difference frequency) signals using analog signal processing electronics. These signals are then digitized and displayed as frequency distribution histograms in real time.
Microscopy I
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Increasing the luminescence of lanthanide(III) macrocyclic complexes by the use of polymers and lanthanide enhanced luminescence
Robert C. Leif, Margie C. Becker, Alfred J. Bromm Jr., et al.
A Eu (III)-macrocycle-isothiocyanate, Quantum DyeTM, has been reacted with lysine homo- and hetero-peptides to give polymers with multiple luminescent side chains. Contrary to the concentration quenching that occurs with conventional organic fluorophores, the attachment of multiple Quantum Dyes to a polymer results in a concomitant increase in luminescence. The emission intensity of the peptide-bound Quantum Dye units is approximately linearly related to their number. The attachment of peptides containing multiple lanthanide (III) macrocycles to analyte-binding species is facilitated by employing solid-phase technology. Bead-bound peptides are first labeled with multiple Quantum Dye units, then conjugated to an antibody, and finally released from the bead by specific cleavage with Proteinase K unedr physiological conditions. Since the luminescence of lanthanide(III) macrocycles is enhanced by the presence of GD(III) or Y(III) ions in a micellar system, a significant increase in signal can be achieved by attaching a polymer labeled with multiple Quantum Dye units to an analyte- binding species, such as a monoclonal antibody, or by taking advantage of the luminescence enhancing effects of Gd(III) or Y(III), or by both approaches concomitantly. A comparison between the integrated intensity and lifetime measurements of the Eu(III)-macrocycle under a variety of conditions show that the signal increase caused by Gd(III) can not be explained solely by the increase in lifetime, and must result in significant part from an energy transfer process invloving donors not directly bound to the Eu(III).
New Technologies
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Mass spectrometry technique for the analysis of metabolic changes in soft x-ray irradiated cells
Marziale Milani, Monica Ballerini, Guido Baroni, et al.
Mass spectrometry identifies atomic and molecular species and relative concentrations in a given atmosphere. The analysis of the composition and of the atmosphere variations in a batch system, that contains a suspension of yeast cells or lymphocytes, allows to identify and to track cell metabolic processes. Such a technique has proven to be efficient in radiobiology experiments to investigate soft X- ray non-nuclear damages, as complimentary to other physical and chemical assessments.
Monitoring the fluorescence intensity and anisotropy decay of individual cells within a population
Biological systems are heterogeneous in general and when stained with fluorescent molecules they emit signals that may consist of a number of lifetimes and rotational correlation times that result from the system heterogeneity and define the measured fluorescence intensity and anisotropy decays, respectively. All currently used instruments which measure these parameters lack the ability to re-measure specific cells, and give only an average value over the entire population yielding poor statistics. In this work a special setup was designed and built comprising a stroboscopic laser-based time domain Model C-720T fluorescence lifetime apparatus from PTI, Inc., and a modified Cellscan system. The Cellscan apparatus, has the unique ability to perform repeated measurements on individual cells within a population. Using this new system, different solutions (fluorescein, labeled beads and acridine orange with different concentrations of DNA) were measured and analyzed. Combining kinetic measurements along with the application of appropriate analysis portions of a given macromolecule. To the best of our knowledge, this is the first time fluorescence intensity and anisotropy decays of individual cells within a population are being monitored.
Microscopy II: Poster Session
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New method for investigation of cells and other biological objects in analytical cytology
Olexander I. Bilyi, Vasyl B. Getman, Sergey A. Kostyukevych
New method and instrument intended for checking the content of biology particles in dimension band 0.1-10.0 mkm in water flow is described in this report. The instrument measures the intensity of light scattered by the particles suspended in liquid flow when they are crossing the laser beam. The results of studying light scattered by platelet and bacterium's such a Pseudomonas aeruginosa, Escherichia coli, Micrococcus lutteus in such a liquid mediums as physiological solution and glucose solution are described.
Analytical Cytology Informatics
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Real-time decision making for high-throughput screening applications
James F. Leary, James A. Hokanson, Judah I. Rosenblatt, et al.
An overlooked need of High-Throughput-Screening (HTS) technologies is the ability to rapidly classify incoming events in real-time. Proper real-time classification of events frequently requires more than simple threshold, or even multiple-threshold, gating logic. Usually for HTS the desired event is ultra-rare and requires more complex decision making than simple gating on raw parameters. Otherwise, the number of false positives can lead to unacceptable results. Similarly, the number of false negatives can lead to unacceptable loss of ultra-rare events. Storage of all data for off-line analysis can lead to requirements for huge off-line storage capabilities with the need to still replay all the data. A better approach is a real-time, multi-queuing, multistage decision-making process that keeps up with the incoming data stream and stores only data of probable interest. A multistage classification system permits faster simpler decisions in early stages leaving more time for more sophisticated decision-making at later stages when the remaining event data stream is much slower. Complex linear and nonlinear mathematical and multi variate statistical classifier systems can be implemented in real-time using linked lookup tables operating at memory speeds. Overall event classifications rate in excess of 100,000 events per second can be achieved for HTS of billions of total events.
Flow cytometric determination of quantitative immunophenotypes
Douglas Redelman, Wayne Ensign, Don Roberts
Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.
DICOM-compatible format for analytical cytology data that can be expressed in XML
Robert C. Leif, Suzanne B. Leif
Flow cytometry data can be directly mapped to the Digital Imaging and Communications in Medicine, DICOM standard. A preliminary mapping of list-mode data to the DICOM Waveform information Object will be presented. This mapping encompasses both flow and image list-mode data. Since list- mode data is also produced by digital slide microscopy, which has already been standardized under DICOM, both branches of Analytical Cytology can be united under the DICOM standard. This will result in the functionality of the present International Society for Analytical Cytology Flow Cytometry Standard, FCS, being significantly extended and the elimination of the previously reported FCS design deficiencies. Thus, the present Flow Cytometry Standard can and should be replaced by a Digital Imaging and Communications in Medicine, DICOM, standard. Expression of Analytical Cytology data in any other format, such as XML, can be made interoperable with DICOM by employing the DICOM data types. A fragment of an XML Schema has been created, which demonstrates the feasibility of expressing DICOM data types in XML syntax. The extension of DICOM to include Flow Cytometry will have the benefits of 1) retiring the present FCS, 2) providing a standard that is ubiquitous, internationally accepted, and backed by the medical profession, and 3) inter-operating with the existing medical informatics infrastructure.
Biological and Optical Manipulation
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Laser ablation cell sorting in scanning cytometry
Feimo Shen, Jeffrey H. Price M.D.
Flow cytometry has been an important tool for automated cells sorting. However, the lack of good sensitivity prevents it from being used for rare events sorting; furthermore, fragile cells, anchorage-dependent cells, and clump forming cells cannot be sorted this way. A fully automated, high-speed scanning cytometer with autofocus and image segmentation is capable of accurately locating contaminant cells in a monolayer cell population. A laser ablation system was incorporated into the cytometer to negatively sort out the unwanted cells by applying a focused, ultra-short laser pulse (sub-micron diameter, pulse duration = 4 nsec, wavelength - 500 nm) to each targeted cell. Due to the high power density (approximately 1010 W/cm2) that was present at the focal point, disruptive mechanical forces were generated and were responsible for the kill. Fluorescently stained NIH-3T3 fibroblast cells were used as a model contaminant target ells in an unstained NIH-3T3 population to determine the identification-kill effectiveness. The contaminant cells were stained with the fluorochrome CellTracker Blue CMAC, whereas the background cells were left intact. Ablation pulses were applied in frame-by-frame increment batches to the cell culture on the microscope. The negative sorting effectiveness was analyzed by automatically re-scanning the post-ablation cell culture in phase contrast and propidium iodide stained epi fluorescent fields to verify cell death.
Experimental investigation of optical breakdown using nanosecond 532-nm and 1064-nm laser pulses delivered at high numerical aperture
Arnold Guerra III, Vasan Venugopalan, Kester Nahen, et al.
We have conducted time-resolved studies of optical breakdown produced by the irradiation of water using 6 ns Nd:YAG laser pulses of 1064 nm and 532 nm wavelength focused at a numerical aperture of NA=0.9. We determined pulse energy threshold values for plasma formation to be 1.89 (mu) J and 18.3 (mu) J for 532 and 1064 nm irradiation, respectively. These energy thresholds correspond to irradiance thresholds of 0.77 x 109 W/mm2 for 532 nm irradiation and 1.87 x 109 W/mm2 for 1064 nm irradiation. For pulse energies 1x, 2x, 5x, and 10x above threshold, we determined the length of the laser induced plasma, the propagation speed and peak pressures of the emitted shock wave, and the mechanical energy dissipated by subsequent cavitation bubble formation, growth and collapse. This analysis demonstrates that both the breakdown threshold as well as the conversion efficiency of the incident laser energy into mechanical energy is smaller for irradiation at 532 nm than for 1064 nm. These results are consistent with laser parameters employed for a variety of nanosecond pulsed micro irradiation procedures using 1064 nm and 532 nm radiation focused by microscope objectives with large numerical apertures (NA >0.8). These results suggest that laser- induced breakdown is the primary mechanism that drives a variety of cellular micro manipulation techniques which employ nanosecond visible and near-infrared laser pulses.
Virtual spatially modulated illumination microscopy predicts nanometer precision of axial distance measurment
A. V. Failla, Christoph G. Cremer
Far field optical light microscopy with its unique capability for contactless, non destructive imaging inside thick transparent specimen such as cell nuclei has contributed widely to the present knowledge of the three- dimensional (3D-) architecture of the interphase nucleus. A serious drawback, however, is the limited optical resolution. A recently introduced light microscopical approach, Spectral Precision Distance Microscopy (SPDM) allows the measurement of distances between point-like fluorescent objects of different spectral signature far below the optical resolution criterion as defined by the Full Width at Half Maximum (FWHM) of the point spread function (PSF). Here, an aspect of the theoretical limits of this method was studied by virtual microscopy. The precision of the axial distance measurements was studied, taking into account photon statistics and image analysis. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, a precision of distance measurements in the nanometer range can be determined.
Online visualization of axial intensity distribution in spatially modulated illumination microscopy
Benno Albrecht, Rainer Heintzmann, Christoph G. Cremer
Recently, several light microscopical approaches, such as point spread function (PSF) engineering, Spectral Precision Distance Microscopy (SPDM) and related methods have demonstrated the feasibility of measurements considerably beyond the conventional Abbe-Limit of optical resolution in far field light microscopy. The Heidelberg Spatially Modulated Illumination (SMI) microscope is based on the generation of a standing wave light field in the direction of the optical axis of the system. The theoretical PSF is obtained by the manipulation of the sin2-shaped illumination PSF and the epifluorescent detection PSF. Here, we report a software method to obtain online visualization of the axial light distribution of any object detected, developed using Microsoft Visual C++ and based on Windows NT. This strongly facilitates routine application of SMI Microscopy.