Dye free automated cell counting and analysis

Dehlinger, Dietrich; Suer, Lynn; Elsheikh, Maher; Peña, José; Naraghi-Arani, Pejman

doi:10.1002/bit.24757

Cited by 11 publications

(10 citation statements)

References 21 publications

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“…Confluence measurements were found to be consistent between different images obtained using the same microscope and magnification, which was the case for successive pictures in our time lapse sequences. This method is similar to the methods used by several other groups (Chen et al, ; Dehlinger et al, ). However, we have simplified the technique, foregoing an attempt to identify individual cells.…”

Section: Methodsmentioning

confidence: 85%

Myoblast alignment on 2D wavy patterns: Dependence on feature characteristics and cell‐cell interaction

Grigola

Dyck

Babacan

et al. 2014

Biotech & Bioengineering

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In this study, we investigate the effects of micron-scale surface patterns on the alignment of individual cells and groups of cells. Using a simple replication molding process we produce a number of micron-scale periodic wavy patterns with different pitch and depth. We observe C2C12 cells as they grow to confluence on these patterns and find that, for some geometries, cell-cell interaction leads to global alignment in a confluent culture when individual cells would not align on the same pattern. Three types of alignment behavior are thus defined: no alignment, immediate alignment, and alignment upon confluence. To further characterize this response, we introduce a non-dimensional parameter that describes the aligning power of a periodic pattern based on its geometry. The three types of alignment behavior can be distinguished by the value of the alignment parameter, and we identify values at which the transitions in alignment behavior occur. Applying this parameter to data from the current and several earlier studies reveals that the parameter successfully describes substrate aligning power over a wide range of length scales for both wavy and grooved features.

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Section: Methodsmentioning

confidence: 85%

Myoblast alignment on 2D wavy patterns: Dependence on feature characteristics and cell‐cell interaction

Grigola

Dyck

Babacan

et al. 2014

Biotech & Bioengineering

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“…Previously published strategies to enhance image contrast in bright-field microscopy have used contrast differentials from multiple z-stacked images [9, 10], or they have determined cell density through measuring the size of confluent areas and multiplying by a calculated density factor [17]; these strategies are best suited for automated image acquisition. The method presented here permits cell counting by use of a simple bright-field microscope with very minor hardware modification.…”

Section: Discussionmentioning

confidence: 99%

“…Counting cells is relatively easy in naturally round, individually growing cells such as yeast [7]. However, when flat, adherent cells must be quantitated, digital holography [8], z-projection of multiple z-stacked images [9] or intensity derivation [10] may all be required to improve contrast and allow cell counting. These methods require acquisition of multiple images followed by application of an image analysis algorithm, making them best suited to automated, high content screening microscopy.…”

Section: Introductionmentioning

confidence: 99%

Counting Unstained, Confluent Cells by Modified Bright-Field Microscopy

2013

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We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions; it does this via free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies by brief incubation in PBS. The procedure was benchmarked against manual counting and automated counting of fluorescently labeled cell nuclei.. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average bright-field images produced the same counts as fluorescent images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope, absent cell-line modification or cell staining.

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“…The binary image was further processed by applying size-constraints for potential objects of interest. Then, cells were detected from bright field stacks by determining the differential image of two focus frames [28] and successive application of thresholding and size constraints to remove small and large objects caused by noise, debris and visible background objects, such as device borders. Again, the threshold values were determined empirically such that the cells were preserved in the segmentation result.…”

Section: Methodsmentioning

confidence: 99%

Unidirectional P-Body Transport during the Yeast Cell Cycle

et al. 2014

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P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained “hovering” near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions.

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Dye free automated cell counting and analysis

Cited by 11 publications

References 21 publications

Myoblast alignment on 2D wavy patterns: Dependence on feature characteristics and cell‐cell interaction

Myoblast alignment on 2D wavy patterns: Dependence on feature characteristics and cell‐cell interaction

Counting Unstained, Confluent Cells by Modified Bright-Field Microscopy

Unidirectional P-Body Transport during the Yeast Cell Cycle

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