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Since the advent of microscopy imaging and flow cytometry, there has been an explosion in the number of probes, consisting of a component binding to an analyte and a detectable tag, to mark areas of interest in or on cells and tissue. Probe tags have been created to detect and/or visualize probes. Over time, these probe tags have increased in number. The expansion has resulted in arbitrarily created synonyms of probe tags used in publications and software. The synonyms are problematic for readability of publications, accuracy of text/data mining, and bridging data from multiple platforms, protocols, and databases for Big Data analysis. Development and implementation of a universal language for probe tags will ensure equivalent quality and level of data being reported or extracted for clinical/scientific evaluation as well as help connect data from many platforms. The International Society for Advancement of Cytometry Data Standards Task Force composed of academic scientists and industry hardware/software/reagent manufactures have developed recommendations for a standardized nomenclature for probe tags used in cytometry and microscopy imaging. These recommendations are shared in this technical note in the form of a Probe Tag Dictionary. © 2020 International Society for Advancement of Cytometry.

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.

Optical multiplexing plays an important role in applications such as optical data storage1, document security2, molecular probes3,4 and bead assays for personalized medicine5. Conventional fluorescent colour coding is limited by spectral overlap and background interference, restricting the number of distinguishable identities. Here, we show that tunable luminescent lifetimes τ in the microsecond region can be exploited to code individual upconversion nanocrystals. In a single colour band, one can generate more than ten nanocrystal populations with distinct lifetimes ranging from 25.6 µs to 662.4 µs and decode their well-separated lifetime identities, which are independent of both colour and intensity. Such ‘τ-dots’ potentially suit multichannel bioimaging, high-throughput cytometry quantification, high-density data storage, as well as security codes to combat counterfeiting. This demonstration extends the optical multiplexing capability by adding the temporal dimension of luminescent signals, opening new opportunities in the life sciences, medicine and data security. Control over the luminescence lifetimes of upconversion nanocrystals allows a new form of temporal multiplexing for imaging and data-storage applications.

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication’s adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

Many microorganisms have a very low threshold (10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a low-cost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels,100 μs, and the autofluorescence backgrounds,0.1 μs, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm(2) , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm × 0.075 mm) down to a few "elements of interest" containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences.

A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.

Background Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. Methods We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. Results The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). Conclusions The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML. Cytometry Part A 54A:56–65, 2003. © 2003 Wiley-Liss, Inc.

Significant multiplexing capacity of optical time-domain coding has been recently demonstrated by tuning luminescence lifetimes of the upconversion nanoparticles called ‘τ-Dots’. It provides a large dynamic range of lifetimes from microseconds to milliseconds, which allows creating large libraries of nanotags/microcarriers. However, a robust approach is required to rapidly and accurately measure the luminescence lifetimes from the relatively slow-decaying signals. Here we show a fast algorithm suitable for the microsecond region with precision closely approaching the theoretical limit and compatible with the rapid scanning cytometry technique. We exploit this approach to further extend optical time-domain multiplexing to the downconversion luminescence, using luminescence microspheres wherein lifetimes are tuned through luminescence resonance energy transfer. We demonstrate real-time discrimination of these microspheres in the rapid scanning cytometry, and apply them to the multiplexed probing of pathogen DNA strands. Our results indicate that tunable luminescence lifetimes have considerable potential in high-throughput analytical sciences., Accurately determining luminescence lifetimes for slow-decaying signals can be challenging. Here, the authors report a fitting algorithm and subsequently experimentally show a method for the rapid measurement of luminescence lifetimes in the microsecond region.

The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes. Three detailed, step-by-step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time-gated luminescence. Curr. Protoc. Cytom. 67:2.22.1-2.22.36. © 2014 by John Wiley & Sons, Inc. Keywords: lanthanide; time-gated luminescence; microscopy; autofluorescence

Background The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of “ultra-rare” cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. Methods Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate–conjugated anti-fetal hemoglobin and 4,6-diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 107 nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. Results Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. Conclusion This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection. Cytometry 39:285–294, 2000 © 2000 Wiley-Liss, Inc.

The focus of this Workshop, which occurred on October 20, 1999, was on extending the use of Ada into the commercial off-the-shelf (COTS) domain. The goal of this Workshop is to determine what should we do to both make Ada the dominant language for COTS and profit by doing so? The contents of Sections 2. Red Hat, Where the Money Went, a Case Summary and 3. How to Commercialize Ada and Profit have been updated.

Time-gated luminescence images were obtained by analog summation of a series of sequential images that were obtained with a cooled modified interline CCD camera, and a fluorescence microscope modified to use a UV LED for illumination. The interline CCD camera obtains an analog sum of a multi-frame image by not reading out the storage line after each frame is acquired; instead, the charges from the acquisition pixels are transferred to the storage pixels, which adds them to those previously stored; subsequently, the sum of the images is readout from the storage pixels and digitized. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise). Previously, the quality of the images obtained with the room temperature camera was degraded by the buildup of thermal noise. The interline transfer, electronically shuttered, cooled astronomy CCD camera, which was modified for analog summation rapidly produced low noise images; yet permitted long exposures. The past problems with lanthanide dyes of low extinction coefficients and equipment cost have now been solved.

CytometryML is an XML schema based translation, extension and amalgamation of the DICOM and ISAC standards. CytometryML consists of 4 major XML schemas: Series, Instance, Instrument, and Specimen; it also includes Image and List-Mode schemas. Series metadata, which is specific for an entire collection of images and/or list-mode files produced by a single instrument and derived from a single specimen, is stored together with associated metadata files in a container (ZIP) file. Each Instance container file includes binary image and/or list-mode files together with associated metadata files that are specific for a single or closely related group of instrument runs from a single specimen. The Archival Cytometry Standard (ACS) proposed Table of Contents schema including its Resource Description Framework (RDF) capabilities has been extended and modified for use in the Instance schema.

The goal of this work is to develop a safe software construction means for an XML based data standard for a class of medical devices, cytometry instruments. Unfortunately, the amount of empirical evidence to archive this goal is minimal. Therefore, technologies associated with high reliability were employed together with reuse of existing designs. The basis for a major part of the design was the Digital Imaging and Communications in Medicine (DICOM) standard and the Flow Cytometry Standard (FCS). Since the DICOM Standard is a Class II device, the safety of software should be maximized. The XML Schema Definition Language (XSDL) has been used to develop schemas that maximize readability, modularity, strong typing, and reuse. An instance and an instrument XML schema were created for data obtained with a microscope by importing multiple schemas that each consisted of a class that described one object. This design was checked by validating the schemas and creating XML pages from them.

Problem: Previous images of time-gated luminescence have been obtained with a cooled CCD camera by digitally summing a series of sequential images. The data acquisition rate of approximately 10 one millisecond exposure images per second was rate limiting and too slow for standard research and clinical use. An annoying undulating background was present, which could not be totally removed by subtraction of an unexposed, control image. Solution: An analog approach to this problem is to use an interline transfer, electronically shuttered camera. After each exposure, the storage line is not readout; instead, the electrons from the acquisition pixels are transferred to the storage pixels and thus are added to those previously stored. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise) electrons. This electronic concept was tested with a Point Grey Dragonfly2 640 by 480 pixel monochrome camera equipped with a Sony 1/3" progressive interline scan, electronically shuttered CCD, which since it did not have any cooling, was operated at room temperature. Pulsed excitation was from a Nichia UV LED. Results: Five and 0.5 micron uniform europium complex stained microspheres could at room temperature be imaged with time-gated excitation and acquisition times of 1 millisecond each and analog summation of 50 images. Conclusion: The analog integration solution apparently works; however, a cooled scientific grade camera with the same capacity for multiple transfers into storage pixels would be better suited for use with dimmer luminescent objects.

In advanced cytometry, a fundamental challenge for rapid specific detection of rare-event micro-organisms is the autofluorescence noise from the complex biological samples. Time-gated luminescence can effectively discriminate labeled cells from autofluorescence background. Recently, a real-time true-colour time-gated luminescence microscopy system has been developed based on the synchronization of a solid-state excitation source and a super-fast optical shutter. We also developed a variety of ultra-bright silica nano-biolabels with multiple luminescence colours and controllable lifetimes in microsecond range. These developments allowed the development of an advanced cell analysis system for real-time background-free imaging and rare-event counting of microsecond-lifetime multi-colour labelled water-borne pathogens.

Introduction: The International Society for the Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) is developing a new Advanced Cytometry Specification (ACS). DICOM has developed and is extending a pathology extension. The work of both groups is complementary with some overlap. Interoperation would benefit both groups and permit each to benefit from the other’s expertise. Methods: The design and implementation of the CytometryML version of the ACS schemas have been based on each schema describing one object (modularity), iterative (spiral) development, inheritance, and reuse of data-types and their definitions from DICOM, Flow Cytometry Standard, and other standards. Results: These schemas have been validated with two tools and XML pages were generated from highest level schemas. Binary image data and its associated metadata are stored together in a zip file based container. A schema for a table of contents, which is one of the metadata files of this container, has recently been developed and reported upon. The binary image data is placed in one file in the container; and the metadata associated with an image in another. The schema for the image metadata file includes elements that are based on the DICOM design. This image schema includes descriptions of the acquisition context, image (including information on compression), specimen, slide, transmission medium, major optical parts, optical elements in one or more optical channels, detectors, and pixel format. The image schema describes both conventional camera systems and scanning or confocal systems.

Introduction: The highest priority for the Advanced Cytometry Standard (ACS) is the interpretation of list-mode cytometry measurements. Other priorities of lesser importance are the capacity to reproduce a cytometry measurement and the implementation of a digital microscopy image standard. The sequential nature of these requirements is being accommodated by a flexible, modular design. A major feature of this modular design is the creation of a design for an Advanced Cytometry Standard Container (ACSC) that includes a Table of Contents (ToC) XML file, one or more binary data containing files and files that contain the meta-data that describes the binary data. Methods: The design and partial implementation of the CytometryML schemas have been based on the techniques of modularity (each schema describing one object), iterative (spiral) development, inheritance, and reuse. Data-types including their definitions have been reused from DICOM, FCS, and other standards. Results: A prototype ToC schema together with prototypes of many of the schemas that describe the contents of the ACSC have been created together with their supporting schemas. These schemas have been validated with two tools and XML pages were generated from the main element(s) of the highest level schemas. These elements describe the table of contents of the zipped container file and a flow-cytometry instrument. The zipped container file (ACSC) describes and contains the meta and binary data.

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type.The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

Purified hepatocytes (LH), Kupffer cells (LKu), and intrahepatic biliary duct cells (LD) were isolated from canine livers, as well as tubular cells from canine kidneys, by enzymatic digestion, gradient centrifugation, and tissue culture techniques. Incubation of LH, LKu, and LD for 48 hr in a two-compartment diffusion chamber opposite two-way mixed lymphocyte cultures, or with canine gamma interferon purified and standardized in our laboratory, resulted in a significant increase in class II expression. This was detected in the cell analyzer with directly fluoresceinated B1F6, a monoclonal antibody (mab) generated in our laboratory vs. a canine class II monomorphic epitope. An amplification of the allogeneic mixed lymphocyte liver cell cultures (MLLC) of at least 2-fold was observed by preinduction of canine class II expression with IFN-gamma on LKu and LD cells, but an autologous reaction could not be elicited. However, an autologous as well as allogeneic lymphoproliferation against kidney tubular cells (MLKC) could be easily observed without IFN-gamma and amplified with IFN-gamma to stimulation indices of at least 3 times that of noninduced cultures. Dependence of the allogeneic MLLC and allogeneic and autologous MLKC on class II gene expression was also evidenced by blocking of 3H-thymidine uptake seen by incubation with 5 micrograms of B1F6. Another mab, I1F6, generated against tubular cells and inhibiting the autologous and allogeneic MLKC, had no blocking effect on lymphoproliferation with any of the liver cell preparations. No such tissue-specific mab (analogous to I1F6) has thus far been found in response to mouse immunization with LH, LKu, or LD. In the absence of accepted defined molecular probes in the dog as yet, we conclude that, in contrast to kidney tubular cells, cells of the normal canine liver do not readily stimulate a primary lymphoproliferative autoimmune reaction in vitro despite class II amplification. Thus autoreactivity (as opposed to alloreactivity) is much less prominent in immune recognition of purified cellular components of nondiseased liver tissue than of kidney tissue in which tissue-associated epitopes are more operative.

This PDF file contains the front matter associated with SPIE Proceedings Volume 6859, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.© (2008) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.

Flow cytometric detection of specific rare-event targets within high-background samples such as water or food are frequently defeated by the extremely large population of non-target background particles. Time-gated detection of long lifetime fluorescence (>10μs) labeled microbial targets has been proven highly efficient in suppressing this non-target autofluorescent (10 million non-target particles). The recovery rate for counting these very-rare-event particles using the TGL flow cytometer was then found to be 100%±20% between bead concentrations evaluated.

The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, will be enhanced by the availability of narrow-band emitting lanthanide calibration beads. These beads can also be used to characterize spectrographic instruments, including microscopes. Methods: 0.5, 3, and 5 micron (µm) beads containing a luminescent europium-complex were manufactured and the luminescence distribution of the 5 µm beads was measured with a time-delayed luminescence flow cytometer and a timedelayed digital microscope. The distribution of the luminescence intensity from the europium-complex in individual beads was determined on optical sections by confocal microscopy. The emission spectra of the beads under UV excitation were determined with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation were measured under LED excitation with a fluorescence microscope. Results: The kinetics of UV bleaching were very similar for the 0.5, 3, and 5 µm beads. Emission peaks were found at 592, 616, and 685 nanometers (nm). The width of the principal peak at half-maximum (616 nm) was 9.9 nm. The luminescence lifetimes in water and in air were 340 and 460 microseconds (µs), respectively. The distribution of the europium- complex in the beads was homogeneous. Conclusions: The 5 µm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Background: Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, Leif et al., Cytometry 2006;69A:767–778, described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy. Methods: The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand or the sodium salt of TTFA in ethanol solution, the EuMac-labeled sample was allowed to dry. Both a conventional arc lamp and a time-gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image. Results: With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes®, as molecular diagnostics for cytological and histopathological microscopic imaging. © 2006 International Society for Analytical Cytology

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed. Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement.

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

Methods for increasing the luminescence intensity of lanthanide macrocycles, Quantum Dyes®, by the Fluorescence Res-onance Energy Transfer Enhanced Luminescence (FRETEL) effect in the solid state have been developed. A homoge-neous solution containing the europium or terbium Quantum Dye and an excess of selected energy transfer species isevaporated to dryness, resulting in a thin film that surrounds and embeds the Quantum Dye or its conjugates. Under theseconditions, in the presence of the gadolinium-thenoyltrifluoroacetonate complex as the energy transfer species, the lumi-nescence of the europium Quantum Dye increased approxima tely 6-fold upon drying. Howeve r, the presence of a non-emitting lanthanide such as gadolinium is not always required for this effect. In studies employing the 2,6-pyridinedicar-boxylate ion as the energy transfer species, where both the terbium and the europium Quantum Dyes could be simulta-neously excited at 280 nm, the presence of gadolinium actually decreased the luminescence compared to that obtainedwith the 2,6-pyridinedicarboxylate alone. The simplest explanation for the FRETEL effect is that fluorescence resonanceenergy transfer occurs between the ph oto-trapping energy transfer species, either unbound or complexed with the non-luminescent gadolinium ion. The energy being finally transferred to the luminescent lanthanide ion complexes with con-sequent increase in emission intensity. Th is new method for the enhancement of luminescence intensity in the solid statehas the significant advantage of eliminati ng the need for the previously required aqueous emulsion, which was difficult tomake and transport.

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe® Photoshop® RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

Improvements in the lanthanide enhanced luminescence (LEL) protocol have facilitated the use of the recently synthesized Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye, as a label. It was discovered that a homogeneous solution in ethanol or other solvent could be used to produce the lanthanide enhanced luminescence (LEL) effect, provided that the solution was permitted to evaporate. This protocol has been applied to the direct staining of cells in S phase, and was optimized for solid phase assays with Quantum Dye labeled streptavidin. Preliminary studies indicate that cells stained with the europium Quantum Dye can be observed both by conventional UV laser excitation and by infrared two-photon confocal microscopy. An enhancer has been found that enables the observation of simultaneous emissions from both the europium and terbium Quantum Dyes.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye, has been synthesized that has minimal contamination with the Eu(III)-macrocycle-di-isothiocyanate, which cross-links proteins. The mono-isothiocyanate has been conjugated to streptavidin (EuMac-Strept). An indirect assay with EuMac-Strept and biotinylated anti5BrdU has been used to observe apoptotic cells. This system and cells directly labeled with the Eu(III)-macrocycle-di-isothiocyanate have been employed in fading studies and reagent stability tests. The fading of cells mounted in a plastic medium was much slower than that observed when the cells were in the aqueous, micellar Lanthanide Enhanced Luminescence (LEL) solution. The fading was not the result of the photo-destruction of the Eu(III)-macrocycle, since the luminescence returned after a second addition of the LEL solution. A time-gated, peltier cooled, monochrome CCD camera has been combined with a flashlamp to eliminate imaging of the emission of fluorescein while maintaining the images of EuMAc staining. This was demonstrated with both separate preparations of fluorescein and EuMac stained cells and mixtures thereof. Time-gating was employed to produce an EuMac image of cells that were stained with both the EuMac and DAPI.

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.