Cellular analysis by open‐source software for affordable cytometry

Mittag, Anja; Pinto, Fernanda E.; Endringer, Denise Coutinho; Tárnok, Attila; Lenz, Dominik

doi:10.1002/sca.20220

Cited by 13 publications

(5 citation statements)

References 37 publications

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“…In addition to Fiji/ImageJ, Cellprofiler is another open-source system for high-throughput image analysis [ 45 ]. It has been proven very useful for cell image analysis [ 46 – 48 ], but also for morphological phenotypes measured in C . elegans [ 49 ].…”

Section: Discussionmentioning

confidence: 99%

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

et al. 2016

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The morphology of synapses is of central interest in neuroscience because of the intimate relation with synaptic efficacy. Two decades of gene manipulation studies in different animal models have revealed a repertoire of molecules that contribute to synapse development. However, since such studies often assessed only one, or at best a few, morphological features at a given synapse, it remained unaddressed how different structural aspects relate to one another. Furthermore, such focused and sometimes only qualitative approaches likely left many of the more subtle players unnoticed. Here, we present the image analysis algorithm ‘Drosophila_NMJ_Morphometrics’, available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt. We demonstrate that gender, genetic background and identity of abdominal body segment consistently and significantly contribute to variability in our data, suggesting that controlling for these parameters is important to minimize variability in quantitative analyses. Correlation and principal component analyses (PCA) were performed to investigate which morphometric parameters are inter-dependent and which ones are regulated rather independently. Based on nine acquired parameters, we identified five morphometric groups: NMJ size, geometry, muscle size, number of NMJ islands and number of active zones. Based on our finding that the parameters of the first two principal components hardly correlated with each other, we suggest that different molecular processes underlie these two morphometric groups. Our study sets the stage for systems morphometry approaches at the well-studied Drosophila NMJ.

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Section: Discussionmentioning

confidence: 99%

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

et al. 2016

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“…Compared with the original approach developed by Clatch (57–59), this micro fluidic device is an important advancement that allows one to semi-automatically analyze samples having minimal numbers of cells. The open-source software adaptation for the analysis of LSC generated data (138) is another recent upgrade of LSC capabilities. It also offers the possibility of comparing LSC data with other cytometric measurements.…”

Section: Utility Of Lsc In Other Applicationsmentioning

confidence: 99%

Laser Scanning Cytometry: Principles and Applications—An Update

Pożarowski

Holden²,

Darżynkiewicz

2012

Methods in Molecular Biology

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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.

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“…In the past, separate instrumentation has been used to obtain quantitative and visual information about cells. While quantitative information about cells within a heterogeneous population has been garnered through the use of flow cytometry, visual or qualitative information has been gathered through the use of fluorescence microscopy [1][2][3][4] . Here, we present a technology that bridges the gap between population and individual cell studies.…”

Section: Discussionmentioning

confidence: 99%

“…Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations 2 . Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer 3 .…”

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confidence: 99%

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Remple

Stone

2011

JoVE

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Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer 3 . The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis [4][5][6] and cell viability analysis with propidium iodide (PI) [7][8][9][10][11] .Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit -Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

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Cellular analysis by open‐source software for affordable cytometry

Cited by 13 publications

References 37 publications

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

Laser Scanning Cytometry: Principles and Applications—An Update

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

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