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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.

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.

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.

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.

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.

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.

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye®, has been coupled to a monoclonal antibody against 5BrdU. Since Quantum Dyes do not undergo concentration quenching, the coupling conditions were optimized to achieve the maximum number of Eu(III) macrocycles bound to the antiBrdU, without decrease in solubility or loss of antigen-binding ability. In order to optimize the coupling conditions, a colorimetric method for the quantitation of the Eu(III)- macrocycle-mono-isothio-cyanate has been developed. A simple mixture composed of an ethanolic solution and a Gd(III)- containing aqueous solution is now used to provide lanthanide enhanced luminescence, LEL. Under LEL conditions, the specific binding of Eu(III) macrocycles to apoptotic cells has been observed in both aqueous and mounted slide preparations. A comparison between measurements of the same LEL model system, obtained in both time-gated luminescence and standard fluorescence modes, has demonstrated that time- gating significantly improves the signal to noise ratio.

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).© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Fluorescence in situ hybridization (FISH) uses labeled DNA probes to determine the copy number of the specific DNA sequence targeted. After denaturation of the probe and target DNA, the probe is allowed to hybridize to an intact nucleus. The hybridization can then be visualized by direct conjugation with fluorescent dyes, or indirect conjugation, such as biotinavidin systems.

At present, the microscopic visualization of luminescent labels containing lanthanide(III) ions, primarily europium(III), as light-emitting centers is best performed with time-gated instrumentation, which by virtually eliminating the background fluorescence results in an improved signal to noise ratio. However, the use of the europium(III) macrocycle, Quantum Dye{trademark}, in conjunction with the strong luminescence enhancing effect (cofluorescence) of yttrium(III) or gadolinium(III), can eliminate the need for such specialized instrumentation. In the presence of Gd(III), the luminescence of the Eu(III)-macrocycles can be conveniently observed with conventional fluorescence instrumentation at previously unattainable low levels. The Eu(III) {sup 5}D{sub 0} {r_arrow} {sup 7}F{sub 2} emission of the Eu(III)-macrocycles was observed as an extremely sharp band with a maximum at 619 nm and a clearly resolved characteristic pattern. At very low Eu(III)-macrocycle concentrations, another sharp emission was detected at 614 nm, arising from traces of Eu(III) present in even the purest commercially available gadolinium products. Discrimination of the resolved emissions of the Eu(III)-macrocycle and Eu(III) contaminant should provide a means to further lower the limit of detection of the Eu(III)-macrocycle.

The development of peripherally substituted europium(III)-macrocycles suitable as luminescent bio-markers was continued in three related areas. (1) Protocols were established for the coupling of NCS-substituted Eu-macrocycles to proteins and for the mounting on microscope slides of particles labeled with luminescent Eu-macrocycles. The emission/excitation spectra of the dried, slide-mounted particles were investigated. (2) A procedure was developed for the synthesis of lanthanide-macrocycles having a single peripheral functionality. The structure and properties of the mono-functionalized macrocyclic complexes were established. (3) A study was undertaken to explore whether the emission intensity of the Eu-macrocycles can be increased by energy transfer from yttrium(III) complexes. Preliminary results have shown that a considerable luminescence enhancement can be achieved by this method. The results obtained in these three areas are evaluated in the light of the research reported by other investigators.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The need to detect increasingly low levels of antigens or polynucleotides in cells requires improvements in both the preparation and the staining of samples. The combination of centrifugal cytology with the use of glyoxal as cross-linking fixative produces monolayers of cells having minimum background fluorescence. Detection can be further improved by the use of a recently developed type of luminescent tag containing a lanthanide(III) ion as the light- emitting center. These novel tags are macrocyclic complexes functionalized with an isothiocyanate group to allow covalent coupling to a biosubstrate. The Eu(III) complex possesses a set of properties -- water solubility, inertness to metal release over a wide pH range, ligand-sensitized narrow-band luminescence, large Stoke's shift, and long excited-state lifetime -- that provides ease of staining as well as maximum signal with minimum interference from background autofluorescence. Luminescence efficiency studies indicate significant solvent effects.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (1 micros) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-microm calibration beads in environmental water dirt samples. Europium-labeled 5-microm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.

The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.

The electrokinetic properties of doxorubicin (DOX)-resistant (P388/R) and -sensitive (P388/S) murine leukemic cells were studied in a free-flow electrophoresis (FFE) system. The electrophoretic mobilities (EM) of P388/S and P388/R cells were 1.07 and 1.35 x 10(-4) cm2 V-1 s-1, respectively, suggesting a higher net negative charge on the P388/R cells. Neuraminidase treatment decreased the EM of both the P388/S and P388/R cells by 15-20% but had no effect on cellular doxorubicin retention. Total and cell surface sialic acid contents were similar in both the cell lines. Our studies show that no direct correlations may exist among surface charge, cell surface sialic acid content, and doxorubicin retention in DOX-resistant and -sensitive P388 cells; however, differences in cell surface charge between these cell types were used to separate them by preparative FFE.

The hydrodynamic artifacts of turning over and droplet formation associated with isopycnic swinging-bucket rotor centrifugation have been virtually eliminated by increasing the viscosity of the layering solution. A 0.25% DNA solution was utilized for this purpose. Since the cells are diluted as they enter the gradient column, they enter and act as single particles. Two sizes (8 ml and 25 ml) of special centrifuge tubes are described. Polyethylene washers which contain the sample and float on top of the gradient column are utilized to restrict the radial trajectory of the cells so that they miss the walls of the centrifuge tube. The utilization of a special swinging-bucket rotor together with programmed acceleration eliminates swirling of the gradients. Thus it is now possible to achieve virtually artifact-free centrifugation with a swinging-bucket rotor. This is demonstrated by the results of a rebanding experiment.

A special peristaltic pump is described that has functioned as part of a system for density gradient formation and fractionation. Twenty-five pumping tubes are actuated on both the top and bottom of the pump. A Mylar diaphragm interposed between the rollers and the pumping tubes filters out the horizontal, stretching component of the forces imparted to the tubes. This greatly prolongs tube life, increases the allowable pressures that can be achieved with such a pump, and thus permits accurate delivery of viscous solutions. An explanation of the cause of the pulsations produced by peristaltic pumps is presented and the virtual elimination of these pulsations is demonstrated. Both velocity and direction of flow of the pump are controlled. By means of two independent bidirectional digital counters, preset volumes of fluid can be delivered and the total volume of liquid determined. Studies demonstrating the relative independence of fluid volume delivered at a preset count versus flow velocity and composition are presented. Other possibilities for use of the pump in automating density gradient analysis are discussed. Possibilities for employment of the pump for autoanalysis and in artificial organs are indicated.

The Committee on Nomenclature of the Society for Analytical Cytology presents guidelines for the analysis of DNA content by cytometry. These guidelines cover staining of DNA, cytogenetic and cytometric terminology, DNA index, resolution of measurements, and cytometric standards.

Bovine adenohypophysial tissue was dissociated by sequential enzymatic incubation in a continuous flow system. Dispersed cells separated into discrete fractions after centrifugation in isopycnic bovine serum albumin gradients. The dispersed and separated cells were prepared for microscopic identification and differential counts by centrifugal cytology. Radioimmunoassays for LH, FSH, TSH, and Prl were used to corroborate the differential counts and determine the homogeneity of the fractions. The thyrotrophs banded at an average density (\(\bar \rho \)) of 1.0417, the FSH-secretory cells at\(\bar \rho = 1.0597\), the LH-secretory cells at\(\bar \rho = 1.0458\), and the Prl-secretory cells at\(\bar \rho = 1.0126\). A 7–16 fold enrichment of different cell populations was possible. In bovine hypophyses each hormone appears to be formed by specific cells: the average TSH concentrations of the thyrotrophs were 5.1 pg/cell and the average LH and FSH concentrations were 4.7 and 4.9 pg/cell for LH-and FSH-secreting cells, respectively. The average Prl concentration was 4.9 pg/cell for Prl-secreting cells.

An alternate method to the common technique of evaporating a metallic coating on cells to render them conductive for scanning electron microscopy is described. This wet chemical technique is less expensive and easier to use, but is not as widely applicable as the evaporative technique. It should prove invaluable in the identification of artifacts by comparison of micrographs of material prepared in both manners. The first step in our application of this wet chemical method is to use the Centrifugal Cytology bucket to deposit the cells onto conductive polyethylene. After centrifugation and fixation with 4% glutaraldehyde, the sample is placed in a 50% glutaraldehyde solution. After a rinse in distilled water it is treated with ammoniacal silver nitrate. The reduced silver renders the sample conductive and, thus, for erythrocytes, eliminates artifacts due to charging and reduces these artifacts to virtually acceptable levels for larger cells. The surfaces of erythrocytes appear smooth with this technique while those coated by conventional vapor deposition of Au-Pd alloys often appear slightly wrinkled. Ehrlich ascites cells apparently can be divided into two classes by surface morphology. The surface structure of Ehrlich ascites cells rendered conductive by the wet method appears to be finer than conventionally prepared ones.

The last review of the work of my laboratory (Leif, 1970a) described the technique for buoyant density separation of cells in isotonic gradients of bovine serum albumin (BSA) and the application of that technique to the separation of human and rabbit erythrocytes. The various gradient media, BSA, Ficoll, iodothalamate, and colloidal silica, were reviewed. The method of preparation of the BSA, including deionization, filtration, and addition of salts and tissue culture media, were described in detail. The biological studies presented in that review were limited to the first class of cells studied, erythrocytes (human and rabbit). Briefly, reproducible buoyant density distributions of human erythrocytes were obtained, and individual fractions were shown to be capable of being rebanded. The average buoyant density of the erythrocytes from four individuals was 1.0808 ± 0.0004 g cm-3. The average density from a fifth individual was 0.0028 g cm-3 greater. The entire buoyant density distribution shifts with salt content; the erythrocytes were shown to behave as “perfect” osmometers. Salt gradients were shown to spread or narrow the distribution predictably, and it was suggested that these gradients might be used to increase the resolving power of the density separations.

This paper describes the modification of Centrifugal Cytology for the preparation of permanent, fixed, stained dispensions for both light and scanning electron microscopy of cells which have been isolated on bovine serum albumin (BSA) boyant density gradients. The principal problem with BSA gradient fractions is that the albumin which is present even after dilution is precipitated by the glutaraldehyde fixative. This problem has been solved by the layering of an intermediate D2O solution under the BSA and subsequent removal of the BSA solution and the underlaying with D2O containing glutaraldehyde. A special layering machine facilitates and expedites these operations. This technique has also been applied to BSA-seperated guinea pig and chicken bone marrow cells, as well as Ehrlich ascites tumor cells, hen and human blood cells. The number of celll present in each area of the slide is maintained at a constant value by utlizing a table of dilution factors. This table was generated by a computer program which calculates the concentration of cells present in the rractions and divides it by the number of celll desired.

An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 X 10(5) cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of "labeled" cells can be followed through the cell cycle.

Several reports have appeared in the literature of the variation of "density" among different types of dispersed cells and of physical separations based upon this property. (1,2) Two general separation procedures have been used. Packed cell methods: cells are centrifuged from a suspension to form a viscous mass of packed cells, which is separated layerwise. Neutral density separations: cells are centrifuged in a dense liquid medium and are segregated into two fractions, one denser and one lighter than the suspending medium. With such methods the order of density of certain types of blood cells has been established. (3-5) It has also been shown that erythrocytes are heterogeneous in density and that young erythrocytes are less dense than old. (6-9)

Well-characterized phantom objects are necessary for investigating the performances of optical imaging systems based on time-resolved transmittance and fluorescence. For this purposes we have prepared inhomogeneous phantoms made of gelatinous objects placed in aqueous solutions of 10% Intralipid with different concentrations. The gelatinous objects have been prepared using a mixture of 10% Intralipid with agar at which absorbing ink have been added for transmittance based optical imaging system. For fluorescence measurements proper fluorescent dyes (rhodamine6G and IR125) have been added. Conventional optical characterization by spectrophotometric and spectrofluorimetric measurements have been performed. In addition, time-resolved transmittance and fluorescence measurements have been carried out. In particular, time-correlated single photon counting system has been used for time-resolved transmittance measurements. For time-resolved fluorescence measurements an optical imaging system based on a Ti:Sa laser and streak camera has been employed.