Elena Holden

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

DNA double‐strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis‐associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser‐139; the phosphorylated H2AX is denoted γH2AX. The intensity of immunofluorescence (IF) of γH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug‐induced (DI) IF of γH2AX, and in particular to distinguish it from AA γH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation.

Laser scanning cytometry: principles and applications.

The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF-kappaB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry.

Laser scanning cytometry: principles and applications-an update.

Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

Next-generation laser scanning cytometry.

Publisher Summary Flow cytometry (FC) has been at the forefront of quantitative cytometric analysis. Recent experimental needs in the life sciences demand a combination of quantitative cytometry and imaging cytometry. This demand has been fulfilled by the development of laser scanning cytometry (LSC). LSC is a combination of quantitative cytometry and imaging cytometry. LSC technology transforms the microscope from a qualitative to a quantitative tool for cell biology. Laser scanning cytometer and two newer, next-generation systems, the automated imaging cytometer (iCyte), and the research imaging cytometer (iCys) are a product line of laser scanning cytometers. The iCys and iCyte systems provide for either interactive (iCys) or walkaway (iCyte) analysis. The chapter gives an overview of obtaining the images; segmentation and feature extraction; and data analysis for these cytometers. The iNovator application development module adds significantly to the capabilities of the iCyte and iCys systems. With the iNovator, the user can (1) employ imaging tools to the segmentation and data analysis process, (2) control the process with visually oriented macros, and (3) perform multiscale scanning and analysis. The user has the ability to define and save numerous types of data files, both numerical and image. A number of applications have been developed for the new iCys and iCyte platforms.

Laser scanning cytometry for automation of the micronucleus assay

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.

Laser Scanning Cytometry and Its Applications: A Pioneering Technology in the Field of Quantitative Imaging Cytometry

Imaging cytometry plays an increasingly important role in all fields of biological and medical sciences. It has evolved into a complex and powerful discipline amalgamating image acquisition technologies and quantitative digital image analysis. This chapter presents an overview of the complex and ever-developing landscape of imaging cytometry, highlighting the imaging and quantitative performance of a wide range of available instruments based on their methods of sample illumination and the detection technologies they employ. Each of these technologies has inherent advantages and shortcomings stemming from its design. It is therefore paramount to assess the appropriateness of all of the imaging cytometry options available to determine the optimal choice for specific types of studies. Laser scanning cytometry (LSC), the original imaging cytometry technology, is an attractive choice for analysis of both cellular and tissue specimens. Quantitative performance, flexibility, and the benefits of preserving native sample architecture and avoiding the introduction of artificial signals, particularly in cell-signaling studies and multicolor tissue analysis, are speeding the adoption of LSC and opening up new possibilities for developing sophisticated applications.

Automation of the Buccal Micronucleus Cytome Assay Using Laser Scanning Cytometry

Laser scanning cytometry (LSC) can be used to quantify the fluorescence intensity or laser light loss (absorbance) of localized molecular targets within nuclear and cytoplasmic structures of cells while maintaining the morphological features of the examined tissue. It was aimed to develop an automated LSC protocol to study cellular and nuclear anomalies and DNA damage events in human buccal mucosal cells. Since the buccal micronucleus cytome assay has been used to measure biomarkers of DNA damage (micronuclei and/or nuclear buds), cytokinesis defects (binucleated cells), proliferative potential (basal cell frequency), and/or cell death (condensed chromatin, karyorrhexis, and pyknotic and karyolytic cells), the following automated LSC protocol describes scoring criteria for these same parameters using an automated imaging LSC. In this automated LSC assay, cells derived from the buccal mucosa were harvested from the inside of patients mouths using a small-headed toothbrush. The cells were washed to remove any debris and/or bacteria, and a single-cell suspension prepared and applied to a microscope slide using a cytocentrifuge. Cells were fixed and stained with Feulgen and Light Green stain allowing both chromatic and fluorescent analysis to be undertaken simultaneously with the use of an LSC.

Abstract 4553: Development of a 4-sample version of the Kolmogorov-Smirnov test for evaluating the temporal physiology of cells treated with test compounds in a label-free, high content, platform for quantitative analysis of adherent cell-culture models

Our objective is to develop multi-functional nanotechnology-based anti-tumor drug delivery systems for improving the efficacy of treatments and reducing undesirable side effects. The essential part of this process is the development and validation of un-biased, quantitative analytical techniques. We employed a newly developed holographic imaging cytometry system HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase Holographic Imaging, Sweden).In previous work we have demonstrated our ability to obtain quantitative, high content cellular feature data congruent to data obtained from traditional label-based systems. We applied a modified version of the Kolmogorov-Smirnov 2-sample test. The classic test takes control and test frequency distributions (histograms), and converts them to probability functions. The maximum vertical displacement between the two is reported as the D-value, to determine if the two distributions are significantly different. In modified versions of the test, it is a comparator; instead of a single value, we obtain histograms of the difference between the control and test normalized probability functions. These histograms are termed Brownian Bridges, where the end points are fixed at a value of zero, and the function is free to vary in between, in either up-going, down-going or mixed. Now, we report the development of a 4-sample Kolmogorov based method. Two modified K.S. tests are performed to compare distributions A to Ac and B to Bc. It is assumed that all the distributions have the same time basis, and each time point is processed sequentially. The resulting time point pairs are plotted as a vector in a two parameter scattergram, tracing the outlines of the probability distribution of the cell populations through time. Unlike, the Brownian Bridge histograms, here, the starting and the ending points are both at the same location (0,0), within the two dimensional scattergram, but are free to move in both positive and negative directions away from the origin. HeLa cells in a 12-well MatTek plate were adhered and treated with Paclitaxel (PCT) in concentrations from 0 to 40 nM in 5-fold increments, either pulsed for 4 hrs. followed by 48 hours of imaging or with continuous exposure for 48 hours of imaging. We obtained a variety of different plot types, including continuous vs. pulsed comparisons, inter dosage comparisons, as well as the ability to compare multiple features to each other. Of particular significance is the fact that in comparative dosage experiments over 10 nM, the pulsed and continuous probability zones are completely separated, reflecting live and dead cells, and allowing complex pharmacodynamic tracking. Citation Format: Ed Luther, Livia Mendes, Jiayi Pan, Elena Holden, Vladimir Torchilin. Development of a 4-sample version of the Kolmogorov-Smirnov test for evaluating the temporal physiology of cells treated with test compounds in a label-free, high content, platform for quantitative analysis of adherent cell-culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4553. doi:10.1158/1538-7445.AM2017-4553

Abstract B18: A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test

Our objective is to develop multi-functional nanotechnology-based anti-tumor drug delivery systems for improving efficacy of treatments and reducing undesirable side effects. The essential part of this process is the development of un-biased quantitative analytical techniques. We are reporting a successful validation of a very high content, medium throughput system in multi-well plates. We employed a newly developed holographic imaging cytometry system HoloMonitor® M4 for label-free time-lapse cellular analysis (Phase Holographic Imaging, Sweden), typically at 5 minute intervals for 48–72 hours. A low power red laser is split into sample and reference beams to obtain holograms of cells. The holograms are unwrapped by proprietary software into quantitative dark field images, with very precise calculation of the cell optical thickness. These images are segmented, and a vast variety of features are extracted for cellular events. In our evaluations we found that cell optical thickness, volume and area have high correlation with features used in traditional fluorescent DNA-stained analysis. We obtain full cell cycle profiles, including the separation of mitotic cells, cells undergoing mitotic dysfunction, and apoptotic cells—all in label-free environment. We recently developed a novel 4D image display of evaluated fields of view: X position, Y position, cell thickness coded as brightness over time in the Z direction. In these images, the history of the viewing area over time is displayed, and salient features such as cell proliferation, cell thickness, and cell motility, contact inhibition, and cell death are discernable. As an example, a combinatorial liposomal formulation containing paclitaxel and a P-gp inhibitor tariquidar was compared to free paclitaxel in SKOV3 TR (taxol resistant) cells seeded in Petri dishes. TR cells treated with free paclitaxel presented increased mitoses, while TR cells treated with the combinatorial preparation exhibited a complete abrogation of mitotic division. Cells were frozen in mitosis due to the polymerization of tubulin, the mechanism of paclitaxel toxicity. In this study Hela cells were seeded in a 6-well plate and allowed to acclimate overnight. Free doxorubicin (DOX) was added to 5 wells at concentrations ranging from 1.6 nM to 1 uM in 5-fold increments. Three fields (0.56 mm2) in each well were imaged for 48 hours at 5 min. intervals. We developed a modified version of the Kolmogorov-Smirnov 2-sample test to analyze the data. The classic test takes control and test frequency distributions (histograms), and converts them to probability functions. The maximum vertical displacement between the two is reported as the D-value, which quantifies the amount of difference between the two. In our version, we obtain histograms of the D-values. We set up our analysis so the X-axis is time, and the feature is any of the reported metrics from the HoloMonitorM4 software. Our results are briefly summarized in the following table indicating where the sample is greater than the control (+), less than the control (-), and a transition (/). Dox Con......(1.6 nM).(8 nM).....(40 nM)..(200 nM)..(1 uM) Cell Count.........(+).......(-)........(---).......(---).......(---) Area (-)......(+)....(+++++)..(++/-)...(++/-) Max. Thick.........(+)....(++)...(+++++)...(-/+)......(-/+) Volume (+)......(+)....(+++++)...(++)......(++) Perimeter........ (+/-)...(+/-).......(++)....(--/++)...(-/++) Roughness.........(-).......(-).......(++/-)....(++/-)......(--) The results are consistent with the known effects of Dox. At low concentration, unrepaired DNA strand breaks start to accumulate and cause slight increases in the volume and thickness of the cells. At mid-concentration the DNA repair process is overwhelmed, proliferation is halted, and vast changes in cellular morphology are evident. At the higher concentration, changes in morphology correspond with cell death followed by eventual deterioration. Citation Format: Ed Luther, Livia Mendes, Daniel Costa, Jiayi Pan, Elena Holden, Vladimir Torchilin. A label-free, high content, moderate throughput analytical platform for quantitative kinetic analysis of cell behavior upon drug activation in cell-culture models based on the Kolmogorov-Smirnov test. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B18.

Myron Melamed, 1927–2013

WITH deep sadness we learned that Myron Roy Melamed, known to his friends and colleagues as Mike, died on September 18 after a six and a half year battle against pancreatic cancer. His name is well-known among researchers in the fields of pathology, flow cytometry, and imaging cytometry and his contributions to these fields are many, varied, and impactful. Mike earned his MD from the University of Cincinnati in 1950 and followed it with residency and fellowship training that included internal medicine, hematology, pathology, and histochemistry at the University of Cincinnati, Duke University Hospital, Mount Sinai Hospital in New York, and Hammersmith Hospital in London. During the Korean War he served as a Captain in the Army Medical Corps. From 1979 to 1989 Mike was the Chairman of the Pathology Department at Memorial Sloan-Kettering Cancer Center and Professor of Pathology and Biology at Cornell University Medical College. From1991 to 2007 he was Chairman of the Department of Pathology at Westchester Medical Center in Valhalla, NY. His contribution to the field of cytometry is enormous. In 1965, Mike and his friend and colleague Louis (Lou) Kamentsky, who was then at the IBM Watson Laboratory at Columbia University published the seminal paper “Spectrophotometer: New Instrument for Ultra-rapid Cell Analysis” (18). The extremely fruitful collaboration between Mike and Lou, which continued into the current century, led to the development of the several generations of flow cytometers and subsequently to the laser scanning slide-based cytometer (LSC). Their work together is described in the continuing segment of this In Memoriam article by Lou himself. Mike’s interest in cytometry stems from his recognition of the importance of quantitative analysis of cellular attributes in pathology. He pioneered the development of numerous applications of flowand laser scanningcytometry for clinical diagnosis and prognosis, particularly for many types of cancer. A perusal of his publications reveals numerous original and review articles, extensively cited, utilizing cytometry for analyses of clinical material as well as in basic research in cell biology (1–28). From historical perspective it is likely that Mike will be recognized as the world most renowned pathologist contributing towards developments in cytometry. Mike’s contribution comes also from his work in organizing and coordinating developments in the field of cytometry worldwide. He was the originator, Founding Member and one of the first Presidents of the International Society for Analytical Cytology, currently known as the International Society for Advancement of Cytometry (ISAC). Under the auspices of the Engineering Foundation Mike organized one of the first In the Pantheon of Fathers of Cytometry: Mort Mendelsohn, Mike Melamed and Lou Kamentsky (ISAC Congress 1998)