A Microfluidic System for Dynamic Yeast Cell Imaging

Lee, Philip J.; Helman, N.C.; Lim, Wendell A.; Hung, Paul J.

doi:10.2144/000112673

Cited by 61 publications

(59 citation statements)

References 14 publications

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“…The development of microfluidic devices has raised expectations for their capability to cultivate yeast cells in controlled environments with continuous microscopic observation (14,15). Unfortunately, none of the currently existing microfluidic devices can be applied for long-term replicative aging studies.…”

Section: Saccharomyces Cerevisiaementioning

confidence: 99%

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Lee

Vizcarra

Huberts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

279

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Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

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Section: Saccharomyces Cerevisiaementioning

confidence: 99%

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Lee

Vizcarra

Huberts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

279

View full text Add to dashboard Cite

show abstract

“…Lee et al reported a microfluidic chip with a passive filtering-and-trapping mechanism that is capable of fixing cells in the same focal plane for image without any moving components on the chip. 9 Ohnuki et al developed an active cell trapping mechanism that uses flexible partical desportin mass spectrometry membranes to hold cells stationary in the same focal plane. 6 However, both designs lack the ability to isolate or manipulate single yeast cells.…”

Section: Introductionmentioning

confidence: 99%

Image processing and classification algorithm for yeast cell morphology in a microfluidic chip

et al. 2011

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Abstract. The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

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“…However, manual manipulation of cells is laborious, and accurately determining pedigree and protein expression by microscopy is challenging as cells grow out of the focal plane after only a few divisions. Various microfluidic devices maintain cells in a single focal plane as they grow (10,(16)(17)(18)(19)(20)(21), but many of these devices require sophisticated fabrication techniques such as multilayer fabrication with valves (16,18), channel height differences (17), or membranes (10,21). To optimize the statistical power of these techniques, the initial placement of cells should be controlled; several other microfluidic devices achieve single-cell trapping (22)(23)(24), but these trapping mechanisms are not conducive to the lineage analysis that we perform here.…”

mentioning

confidence: 99%

Tracking lineages of single cells in lines using a microfluidic device

Rowat

Bird²,

Agresti³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

117

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Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.epigenetics ͉ protein expression ͉ single cell assay ͉ yeast

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A Microfluidic System for Dynamic Yeast Cell Imaging

Cited by 61 publications

References 14 publications

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Image processing and classification algorithm for yeast cell morphology in a microfluidic chip

Tracking lineages of single cells in lines using a microfluidic device

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