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PubMed2006

Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.

Cell Line, Tumor↗
PubMed2002

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death.

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.

Cell Death↗
PubMed1994

A disposable-chamber temperature-regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital-imaging microscopy.

Utilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the [Ca2+]i transient involved in T-cell activation. We wished to extend these studies to single-cell transients using florescence digital-imaging microscopy (DIM). However, the most currently available temperature-regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual-temperature regulation system that will maintain Nunc, eight-well, coverglass-bottom, disposable chambers, and three disposable addition pipets at 37 degrees C for physiological studies on an inverted digital-imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX-inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow-cytometric measurement indicated. The correlation between flow cytometry and DIM permits the amalgamation of these results in the interpretation of studies on the regulation of T-cell activation.

Calcium↗
PubMed2003

Evaluation of SDF-1/CXCR4-induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry.

BACKGROUND: The chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell-derived factor 1 [SDF-1], regulated on activation normal T-cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1alpha and 1beta, MIP-1alpha and MIP-1beta) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti-HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine-induced transient intracellular Ca(2+) flux. We evaluated two cell-based-fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF-1-induced intracellular Ca(2+) mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system-dependent limitations. METHODS: CXCR4(+) lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca(2+) indicator Fluo-3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca(2+) concentration after stimulation with SDF-1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca(2+)-bound Fluo-3. Surface CXCR4 expression was also determined flow cytometrically. RESULTS: In all five CXCR4(+) cell lines, SDF-1 elicited a transient increase in intracellular Ca(2+) concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca(2+) response. AMD3100 effected a dose-dependent inhibition of the SDF-1-induced Ca(2+) flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca(2+) responses. In freshly isolated PBMCs, the Ca(2+) response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR. CONCLUSIONS: The FLIPR system is a most adequate to monitor intracellular Ca(2+) mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor-mediated signal transduction.

Aniline Compounds↗
PubMed1996

DNA cytometry in renal cell carcinoma.

The course of patients suffering from renal cell carcinoma depends on various factors. The measurement of the nuclear DNA content has been considered as a prognostic indicator in addition to morphologic and clinical parameters. The meaning of this parameter is under discussion today. The aim of the present study is to investigate the influence of nuclear DNA parameters of renal cell carcinoma on the prognosis. It should be searched for such parameters which give us the possibility to divide the patients into a low-risk-group and a high-risk-group. 110 clear cell renal carcinomas were investigated by means of image analysis and flow cytometry. Stemline ploidy, 5c-exceeding-rate, DNA grade of malignancy, S-phase-fraction, proliferating fraction, and nuclear area have been determined. A significant correlation between the size of the tumor, lymph node metastases and distant metastases on the one hand and the computed DNA parameters on the other hand was found. A similar correlation between histologic grading and DNA parameters could be found. There were pieces of information about the clinical follow-up in 87 patients (mean follow-up time 15,5 months). Local recurrence occurred in 12 cases. The computed differences between DNA parameters and local recurrence were not significant. Further clinical follow-up and clinical re-examination of all patients will be performed. Then we can decide, if it is possible to make a division into low-risk and high-risk cases based on DNA parameters.

Adenocarcinoma, Clear Cell↗
PubMed1985

Compartmentalization of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulax as studied with image-intensified video microscopy and flow cytometry.

Compartmentalization of specialized functions to discrete locales is a fundamental theme of eucaryotic organization in cells. We report here that bioluminescence of the dinoflagellate alga Gonyaulax originates in vivo from discrete subcellular loci that are intrinsically fluorescent. We demonstrate this localization by comparing the loci of fluorescence and bioluminescence as visualized by image-intensified video microscopy. These fluorescent particles appeared to be the same as the previously described in vitro "scintillons." We attribute the endogenous fluorescence to that of the bioluminescence substrate, luciferin, because (a) the fluorescence excitation and emission characteristics are comparable, (b) the autofluorescence is lost after exhaustive stimulation of bioluminescence, and (c) the fluorescence of discharged particles in vitro can be restored by adding luciferin. The fluorescence in vivo exhibits a standard property of circadian (daily) rhythmicity: under constant environmental conditions, the intensity of the particle fluorescence fluctuates cyclically (it is maximal during the night phase and is low during the day). Thus, luciferin is localized within the cell at discrete loci from which the bioluminescence emanates; the cellular quantity of luciferin is rhythmically modulated by the circadian clock.

Dinoflagellida↗
PubMed1996

Quantitative DNA ploidy analysis of breast carcinoma: a study of the effects of joint photographer experts group (JPEG) compression on DNA ploidy images.

Telepathology usage in the past has typically been a qualitative procedure rather than a quantitative measurement. DNA ploidy using image analysis has been favorably compared to DNA ploidy analysis by flow cytometry in numerous publications. A step from DNA ploidy analysis using conventional image analysis to DNA ploidy analysis using stored images allows DNA ploidy analysis by image cytometry to become a powerful tool in telepathology. Remote DNA ploidy analysis using stored images has an impact on the field of pathology, as not every hospital or laboratory can afford to perform this type of specialized testing. However, images have large data files and require lengthy transmission times over communication systems to other computers. Joint Photographer Experts Group (JPEG) compression is a computer algorithm that allows the file size of an image to be reduced in order to decrease transmission times to another computer. A study was initiated to investigate the effects of JPEG compression on images of Feulgen stained breast tumor touch preps and the resulting DNA ploidy histograms.

Animals↗
PubMed1991

Nuclear distribution of centromeres during the cell cycle of human diploid fibroblasts.

The distribution of centromeres in the interphase nuclei of human diploid fibroblasts was analyzed using anti-centromere immunofluorescence and laser scanning confocal microscopoy. The positions of the centromeres were placed within the nuclear chromatin distribution and presented some aspects of the dynamics of nuclear structure during the cell cycle. During the G1 phase of the cell cycle many of the centromeres were located in association with nucleoli or fused in chromocenters. A few centromeres were dispersed singly in the euchromatin. During S phase, the fused centromeres dispersed, often forming distinct patterns of rings or lines. At prophase, the centromere immunofluorescence condensed into distinct double dots upon the formation of the prophase chromosomes. Quantitative analysis by both image and flow cytometry showed that the intensity of immunofluorescence started to duplicate in mid S phase, well before the appearance of the double dots. The coalesence of the centromeres during G1 indicated that regions of the chromosome domains remain compacted and possibly sequestered from transcriptional activity. During S phase the chromatin and the coalesced centromeres dispersed for DNA replication. The dynamics of the centromeres and chromatin during the cell cycle seen here are evidence for a higher-order organization of nuclear structure that accompanies DNA transcription and replication.

Cell Nucleus↗
PubMed2003

CytometryML, an XML format based on DICOM and FCS for analytical cytology data.

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.

Computer Communication Networks↗
PubMed1991

[Correlations between nuclear DNA content and nuclear morphometry versus histological grading of prostatic carcinoma].

The nuclear area, N/C ratio and anisonucleosis by morphometry and the DNA content by image photo-cytometry of paraffin-embedded tissues of 9 cases of prostatic hypertrophy and 48 cases of carcinoma obtained by open surgery, needle biopsy or by autopsy were correlated with histological grading. In histological grading, in addition to 9 benign hypertrophy cases, 11 were classified as well differentiated adenocarcinoma, 19 as poorly differentiated adenocarcinoma and 1 as small cell carcinoma. All 14 tumors taken from distant metastasis were classified as poorly differentiated adenocarcinoma. Correlation of the data by tumor cell morphometry with those of nuclear image photo-cytometry was carried out on surgical materials alone. In autopsy materials, DNA ploidy patterns were compared between primary lesions and metastases. The DNA histogram patterns of most cases were classified into 4 groups. Hypertrophy was differentiated from carcinoma by the histogram patterns. There were significant correlations between mean DNA value and morphometric factors. One of the 4 histogram patterns had a higher mortality than others. The DNA histograms did not distinguish between the primary and metastatic lesions of the same patients. However, the DNA histograms constructed from metastasis frequently showed a single stem-line of the primary lesion. This study disclosed several correlations between histological grading and morphometrical factors.

Adenocarcinoma↗
PubMed2001

Evolution of DNA ploidy during squamous cell carcinogenesis in the esophagus.

Image and flow cytometry was used to study the nuclear DNA content (ploidy) during the squamous cell carcinogenesis in the esophagus. The present retrospective study comprised 26 surgical specimens of squamous cell carcinomas (SCC) in patients who underwent surgery alone at the Department of Surgery in CHUV Hospital in Lausanne, between January 1992 and December 1999. We analyzed 53 healthy tissues, 43 tumors, and six lymph node metastases. Diploid DNA histogram patterns were observed in all non-pathologic tissues analyzed, either distant or proximal to the lesion. Aneuploidy was observed in 30 (70%) of 43 lesions; 20 (62.5%) of 32 early squamous-cell carcinomas; and 10 (91%) of 11 advanced carcinomas. In patients with various tumor stages or with multicentric synchronous or metachronous tumors, DNA content was not different among different tumor stages. Four of six lymph node metastases had the same DNA content as the primary tumor. In four patients, discordance between image and flow cytometry analysis was observed for malignant lesions only. Ploidy status was not statistically associated with the differentiation of the tumor, but it was associated with the stage of tumor (P < 0.001). These findings suggest that early malignant changes in the esophagus are already associated with alteration in DNA content, and aneuploidy tends to correlate with progression to invasive SCC. This cell kinetic information could help clinicians in selecting the optimal treatment for the individual patient.

Aged↗
PubMedDate not supplied

Interactive static image analysis: a major enhancement to histopathology and cytology.

Recent developments in computerized data processing and digital video technology have made it possible to perform cytology and histopathology in quantitative as well as qualitative terms. The most common method for studying the DNA content of tumors in clinical laboratories is flow cytometry. The development of image analysis (IA) to study DNA content by Feulgen staining and microspectrophotometry actually preceded the development of flow cytometry, but the complexity of the method was so great that it never received widespread usage. In recent years computerized interactive static IA on microscope-based instruments has been developed. These instruments involve microscopy, a television camera and monitor, and a computer to analyze the data. They can be put together from separate components or are available as complete instrument packages from commercial companies. In addition to the study of DNA content, software is also available to perform quantitative immunoperoxidase determination and morphometry. These commercial instruments emphasize user-friendly software. Companies such as CAS (Cell Analysis Systems, Elmhurst, Illinois) provide operator training and annual user meetings. There is a growing interest among laboratories in bringing this technology into their institutions. This review will deal with the following aspects of IA: what it can do, how it works, what skills and training are required for an operator to use it, financial considerations, and how image cytometry compares to flow cytometry.

Cell Biology↗
PubMed1987

DNA cytometry and cytology by quantitative fluorescence image analysis in symptomatic bladder cancer patients.

A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orange-stained cells was quantitated using (1) QFIA and (2) simple filter microspectrofluorophotometry (SFM). Both methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFIA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting low-grade transitional-cell carcinoma were 86% for SFM, 76% for QFIA, and 33% for Pap cytology. QFIA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 greater than p greater than 0.001). Comparison of sensitivity obtained with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFI also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.

Carcinoma, Transitional Cell↗
PubMed1997

Ala-Pro-cresyl violet, a synthetic fluorogenic substrate for the analysis of kinetic parameters of dipeptidyl peptidase IV (CD26) in individual living rat hepatocytes.

A new type of fluorogenic substrates for proteases based on the leaving group cresyl violet has been synthesized. Cresyl violet is not fluorescent when amino acids or peptide groups are attached but becomes highly fluorescent after proteolytic liberation. Its fluorescence shows linearity with concentration and barely any fading. The properties of Ala-Pro-cresyl violet as substrate for dipeptidyl peptidase IV (DPPIV) (CD26) for localization and quantification of its activity in individual freshly isolated living rat hepatocytes were investigated using confocal microscopy, image analysis, and flow cytometry. DPPIV activity was localized exclusively in patches at plasma membranes likely being bile canalicular domains. Activity was analyzed quantitatively in individual cells by capturing series of images in time. Production of fluorescence was analyzed on the basis of the series of digital images and it appeared to be nonlinear with time. By calculation of the initial velocity at time zero, activity of DPPIV per individual hepatocyte was calculated. Cresyl violet-dependent fluorescence appeared in a similar way when cells were analyzed by flow cytometry. A dipeptide phosphonate inhibitor inhibited production of fluorescence competitively with a Ki of 7 microM. K(m) values in individual hepatocytes varied in the range of 6-22 microM depending on the individual rat from which the hepatocytes were obtained, whereas the Vmax varied in the range of 4-16 nU. K(m) and Vmax values per individual rat were inversely correlated indicating posttranslational regulation of the kinetic parameters of DPPIV. This relationship was lost when membrane fractions of the same hepatocyte suspensions were analyzed. It is concluded that cresyl violet-based protease substrates are the compounds of choice to localize and quantify protease activity in living cells and tissues.

Animals↗
PubMed1992

[Prognostic significance of DNA content in transitional cell carcinoma of the urinary bladder].

The cellular DNA content in paraffin-embedded specimens of recurrent transitional cell carcinoma of the urinary bladder were analyzed by quantitative image analysis in 48 and flow cytometry techniques in 44 cases. The results showed a positive correlation of cellular ploidy with malignant proliferation of the tumors. The aneuploidy was increased in cases of higher clinical stages and histological grades. The prognosis of patients with diploid cells was better than those with aneuploidy, while patients with aneuploidy and higher grading showed very poor prognosis. The recurrence rate of diploid tumors was considerably lower than that of aneuploid tumors. The results indicate that such a correlation might be of clinical significance in prognosis and predicting and controlling recurrence of tumors.

Adult↗
PubMedDate not supplied

DNA cytometry and morphometry by TV based image analysis system (TAS) in the diagnosis of gastric carcinoma.

Cytological smears from gastric biopsies were studied by two types of TAS. Böcking's algorithm and a great number of form factors were used to separate the intact cells from the tumourous ones. The numerically expressed density features and morphometric parameters are able to indicate the possibility of malignant transformation even in dysplasia. These values become much higher in tumours and lead to unequivocal diagnosis. Both types of TAS render valuable help in assessing cases not reliably diagnosed by routine staining procedures.

Carcinoma↗