RESUMO
BACKGROUND: Whole Slide Imagers or digital slide scanners have developed very rapidly in the last 8 years and went through three generations. Third generation instruments have just reached the market which have the stability and throughput to be used for routine clinical work. We describe in this article the technical background and reasoning behind engineering decisions we made during the development of 3DHISTECH's 3rd generation combined brightfield and fluorescent scanner. MATERIALS AND METHODS: The Panoramic 250 FLASH utilizes Plan-Apochromat 20x and 40x objectives, a 2 megapixel 3CCD camera for brightfield and a monochrome scientific CMOS camera for fluorescent scanning. A solid state light engine for fluorescent and a strobe light for bright field illumination are used. RESULTS: The system can scan 1cm2 including focusing at 45x resolution in 1 minute. It can scan a well stained DAPI, FITC, TRIC, 1cm2 fluorescent slide in 11 minutes. It can load and scan 250 slides in walk away mode. CONCLUSION: Using the latest camera technology and electronics, state of the art computer and standard microscope optical components high throughput high quality whole slide imaging is feasible and is sufficient for most of the routine diagnostic work. Extended depth of field and Z-stack scanning is possible with the use of area scan technology.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Patologia Clínica/métodos , Telepatologia/métodos , Registros Eletrônicos de Saúde , Humanos , IluminaçãoRESUMO
Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software package of the MIRAX viewer was used for image segmentation and quantitative analysis. The data was displayed by the histogram, scatter plot, and gallery functions of the same program. Fluorescent samples can be reliably detected, focused, and scanned. The measured integrated fluorescence showed linearity with exposure time and staining intensity. Automated fluorescent WSI with stable LED illumination and high-quality homogeneous fluorescent slides can be used conveniently for SBC.
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Automação/instrumentação , Citometria por Imagem/instrumentação , Fluorescência , Humanos , Microesferas , Fatores de TempoRESUMO
Automated and quantitative histological analysis can improve diagnostic efficacy in colon sections. Our objective was to develop a parameter set for automated classification of aspecific colitis, ulcerative colitis, and Crohn's disease using digital slides, tissue cytometric parameters, and virtual microscopy. Routinely processed hematoxylin-and-eosin-stained histological sections from specimens that showed normal mucosa (24 cases), aspecific colitis (11 cases), ulcerative colitis (25 cases), and Crohn's disease (9 cases) diagnosed by conventional optical microscopy were scanned and digitized in high resolution (0.24 mum/pixel). Thirty-eight cytometric parameters based on morphometry were determined on cells, glands, and superficial epithelium. Fourteen tissue cytometric parameters based on ratios of tissue compartments were counted as well. Leave-one-out discriminant analysis was used for classification of the samples groups. Cellular morphometric features showed no significant differences in these benign colon alterations. However, gland related morphological differences (Gland Shape) for normal mucosa, ulcerative colitis, and aspecific colitis were found (P < 0.01). Eight of the 14 tissue cytometric related parameters showed significant differences (P < 0.01). The most discriminatory parameters were the ratio of cell number in glands and in the whole slide, biopsy/gland surface ratio. These differences resulted in 88% overall accuracy in the classification. Crohn's disease could be discriminated only in 56%. Automated virtual microscopy can be used to classify colon mucosa as normal, ulcerative colitis, and aspecific colitis with reasonable accuracy. Further developments of dedicated parameters are necessary to identify Crohn's disease on digital slides.
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Colite Ulcerativa/patologia , Colite/patologia , Colo/patologia , Doença de Crohn/patologia , Inflamação/classificação , Inflamação/patologia , Colite/classificação , Colite Ulcerativa/classificação , Colite Ulcerativa/diagnóstico , Colo/ultraestrutura , Doença de Crohn/classificação , Citometria de Fluxo/métodos , Humanos , Aumento da Imagem/métodos , Inflamação/diagnóstico , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Estudos RetrospectivosRESUMO
BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.
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Imunofenotipagem/métodos , Citometria de Varredura a Laser/métodos , Pontos Quânticos , Benzimidazóis/química , Biotina/química , Complexo CD3/análise , Antígenos CD4/análise , Relação CD4-CD8 , Antígenos CD8/análise , DNA/análise , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Imunofenotipagem/instrumentação , Citometria de Varredura a Laser/instrumentação , Leucócitos/química , Leucócitos/citologia , Leucócitos/imunologia , Coloração e Rotulagem/métodos , Estreptavidina/químicaRESUMO
Conventional optical microscopy of specimens from colorectal biopsies commonly produces diagnostic errors due to incomplete sampling or poor orientation. Obtaining additional sections or re-embedding may help avoid these errors, but can prolong turnaround time. We describe new technology, which incorporates exhaustive sectioning, 3-dimensional reconstruction, and virtual microscopy, that may eliminate these problems by enabling pathologists to rapidly examine entire specimens and convert poorly oriented mucosa to well-oriented mucosa.
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Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Imageamento Tridimensional , Microscopia/métodos , Idoso , Biópsia , Humanos , MasculinoRESUMO
BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings. METHODS: For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining. RESULTS: In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01). CONCLUSIONS: SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Citometria de Varredura a Laser/métodos , Microscopia de Fluorescência/métodos , Antiporters , Humanos , Processamento de Imagem Assistida por Computador , Leucócitos Mononucleares/citologia , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/patologia , Proteínas de Saccharomyces cerevisiae , Estatística como AssuntoRESUMO
BACKGROUND: Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. METHODS: We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. RESULTS: With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. CONCLUSIONS: SFM is a valuable method for the evaluation of fluorescently labeled cells.