CellTracks TDI: An image cytometer for cell characterization

Scholtens, T.M.; Schreuder, F.; Ligthart, Sjoerd; Swennenhuis, Joost F.; Tibbe, Arjan G.J.; Greve, Jan; Terstappen, Leon W.M.M.

doi:10.1002/cyto.a.21024

Cited by 29 publications

(22 citation statements)

References 28 publications

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“…This system uses EpCAM antibody labeled ferrofluids to immunomagnetically enrich cells of epithelial origin. The magnetically enriched cells are fluorescently labeled with the nucleic acid dye DAPI, Phycoerythrinlabeled monoclonal antibodies directed against Cytokeratin 8,18, and 19 (CK-PE), and Allophycocyanin-labeled monoclonal antibodies against CD45 (CD45-APC). After incubation, the sample is transferred into a CellTracks analysis cartridge, which is present inside the MagNest 1 cell presentation device.…”

Section: Sample Preparationmentioning

confidence: 99%

“…After the initial scan with the CellTracks Analyzer II, the analysis cartridge is kept inside the MagNest and the sample is rescanned the same day using the CellTracks TDI system (18,19). To increase the resolution and sampling density, the CellTracks TDI uses a 403/0.6 NA objective, instead of the 103/0.45 NA objective that is used in the CellTracks Analyzer II.…”

Section: Celltracks Tdi Analyzermentioning

confidence: 99%

“…introduce an image cytometer that uses lasers and a Time Delay Integration (TDI) camera to achieve automated classification of CTC (18,19).…”

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confidence: 99%

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Automated identification of circulating tumor cells by image cytometry

Scholtens

Schreuder

Ligthart³

et al. 2011

Cytometry Pt A

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Presence of circulating tumor cells (CTC), as detected by the CellSearch 1 System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 403/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated. '

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Section: Sample Preparationmentioning

confidence: 99%

Section: Celltracks Tdi Analyzermentioning

confidence: 99%

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Automated identification of circulating tumor cells by image cytometry

Scholtens

Schreuder

Ligthart³

et al. 2011

Cytometry Pt A

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“…A hardware solution that improves the CV without any of these disadvantages is desired. Previously, a double microlens array (MLA) was used to improve the uniformity of the illumination from a tungstenhalogen lamp (6) and from lasers (7). Here, we demonstrate that epifluorescence microscopes equipped with a mercury arc lamp can be retrofitted with a double MLA system to achieve the uniformity of Koehler alignment, while achieving the illumination intensity of semi-critical alignment.…”

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confidence: 92%

Flat‐top illumination profile in an epifluorescence microscope by dual microlens arrays

2012

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Low uniformity in illumination across the image plane impairs the ability of a traditional epifluorescence microscope to quantify fluorescence intensities. Two microlens arrays (MLAs) were introduced into the illumination path of two different epifluorescence microscope systems to improve the uniformity of the illumination. Measurements of the uniformity of illumination were performed with a CCD camera in the focal plane and with fluorescent beads in the image plane. In semi critical alignment, a uniformity of illumination of 15-23% was found compared with 1-2% in the modified system. Coefficient of variation (CV) of fluorescent beads measured on the unmodified system was 20.4% AE 5.3% in semi critical alignment and 10.8% AE 1.3% in Koehler alignment. On the MLA systems, CV was 7.9% AE 2.0% and on a flow cytometer, the CV was 6.7% AE 0.7%. Implementation of MLAs in an epifluorescence microscope improves the uniformity of illumination, thereby reducing the variation in detection of fluorescent signals of the measured objects and becomes equivalent to that of flow cytometry. ' 2012 International Society for Advancement of Cytometry Key terms microlens array; coefficient of variation; epifluorescence microscopy; homogeneous illumination profile; beam shaping; flat top IN a traditional epifluorescence microscope, there is a tradeoff between the illumination uniformity and power. Koehler illumination is used to achieve high uniformity at the expense of illumination intensity, whereas semi-critical alignment is used to achieve high illumination intensity at the expense of uniformity (1). In Koehler illumination, the light source is focused onto the back pupil of the objective, while in semi-critical illumination, the light source is focused slightly beyond the sample. Poor uniformity results in large spatial intensity variations at the focal plane, while low illumination intensity requires longer illumination times and results in additional bleaching of the sample. With Koehler alignment, an improvement in CV can be obtained (2). If a better CV is needed, the signal can be corrected by shading correction (3,4) or by using the middle 50% of the image (5) to reduce the CV down to 3%. Multiplication is effective in cases where the illumination profile is well known and the signals are bright. In cases where the signal is dim, multiplication is not effective because signal to noise ratio is not changed. Reduction of the field of view increases the time needed for acquisition. A hardware solution that improves the CV without any of these disadvantages is desired. Previously, a double microlens array (MLA) was used to improve the uniformity of the illumination from a tungstenhalogen lamp (6) and from lasers (7). Here, we demonstrate that epifluorescence microscopes equipped with a mercury arc lamp can be retrofitted with a double MLA system to achieve the uniformity of Koehler alignment, while achieving the illumination intensity of semi-critical alignment. The need for such improvements arises from our desire to i...

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“…Unlike conventional flow cytometry, only cells that have been immunomagnetically selected are used to count and to characterize rare cells, such as CTCs in the CellTracks system. The power of combining the flow cytometry-type of cell imaging and sorting with immunomagnetical selection has been demonstrated by detecting rare cells, including CTCs [21,22]. The CellTracks system is highly adaptable for the analysis of a wide variety of rare cells in biological fluids, provided that the immunomagnetical selection process can be modified to be specific to the cells of interest and fluorescence staining of the cells is compatible with the fluorescence optics.…”

Section: Fluorescence Imaging-based Techniquesmentioning

confidence: 99%