Computer Control of Microscopes Using µManager

Edelstein, Arthur; Amodaj, Nenad; Hoover, Karl; Vale, Ron; Stuurman, Nico

doi:10.1002/0471142727.mb1420s92

Cited by 1,641 publications

(1,457 citation statements)

References 1 publication

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“…Image data were recorded using either Solis image capture software (Andor) or µManager microscopy software 35 . Algorithms for image processing were implemented in Python 2.7.…”

Section: Deep-imaging With Two-photon Excitation (Tpe)mentioning

confidence: 99%

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

et al. 2014

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When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm) 2 image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, sPod). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, exPAn). this shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.In recent years the development of super-resolution techniques has had a profound impact on biology and other fields in which subdiffraction-limited resolution of fluorescently labeled samples is desired [1][2][3][4][5][6][7][8][9][10][11][12][13] . Prominent examples are stimulated emission depletion (STED) microscopy 1,4 , photoactivated localization microscopy (PALM) 3,5,6 and stochastic optical reconstruction microscopy (STORM) 2,7 . Generally, these techniques are based on reversible switching between two states. Whereas STED is based on a deterministic switching in a nanometric interference pattern, STORM and PALM are based on wide-field illumination and stochastic switching on the level of single isolated molecules, which are then localized. Other approaches, such as super-resolution optical fluctuation imaging (SOFI) 8 , reversible saturable optical fluorescence transitions (RESOLFT) microscopy 9,11 and saturated structured illumination microscopy (SSIM) 12,13 , are also based on stochastic or deterministic switching between two states and provide spatial resolution enhancement. Here we present an alternative approach that distinguishes adjacent molecules or nanoareas in the sample (arranged, for example, in a grid of 50 nm × 50 nm rectangular areas) by different average orientations of fluorescent dyes attached to rigid sample structures within these nanoareas. This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD). We also show that the range of polarization angles that results in effective excitation of differently oriented molecules can be substantially narrowed by rotating a second wide-field de-exciting stimulated emission beam of a polarization perpendicular to the excitation beam polarization (ExPAN), resulting in better spatial resolution ...

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“…Image data were recorded using either Solis image capture software (Andor) or µManager microscopy software 35 . Algorithms for image processing were implemented in Python 2.7.…”

Section: Deep-imaging With Two-photon Excitation (Tpe)mentioning

confidence: 99%

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

et al. 2014

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“…Microscopy of artificial liposomes and living E. coli cells was performed within 5 min after NaCl addition using a Nikon Eclipse Ti inverted epifluorescence microscope equipped with a CoolLED PrecisExcite light source and a Nikon 60x oil immersion objective providing a total magnification of 600-fold. Filters used for fluorescence imaging of FM1-43 were 470 ± 40 nm (excitation) and 525 ± 50 nm (emission), respectively, and exposure time was set to 2.5 s. Image acquisition and basic analysis (brightness and contrast optimization, image sharpening) were performed using µManager 52 and ImageJ 53 , respectively. For the liposome experiment, detection and classification of particles larger than 100 pixels was performed using Matlab.…”

Section: Metabolomicsmentioning

confidence: 99%

Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli

2014

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Bacteria are thought to cope with fluctuating environmental solute concentrations primarily by adjusting the osmolality of their cytoplasm. To obtain insights into underlying metabolic adaptions, we analyzed the global metabolic response of Escherichia coli to sustained hyperosmotic stress using non-targeted mass spectrometry. We observed that 52% of 1,071 detected metabolites, including known osmoprotectants, changed abundance with increasing salt challenge. Unexpectedly, unsupervised data analysis revealed a substantial increase of most intermediates in the ubiquinone-8 (Q8) biosynthesis pathway and a 110-fold accumulation of Q8 itself, as confirmed by quantitative lipidomics. We then demonstrate that Q8 is necessary for acute and sustained osmotic stress tolerance using Q8 mutants and tolerance rescue through feeding non-respiratory Q8 analogues. Finally, in vitro experiments with artificial liposomes reveal mechanical membrane stabilization as a principal mechanism of Q8-mediated osmoprotection. Thus, we find that besides regulating intracellular osmolality, E. coli enhances its cytoplasmic membrane stability to withstand osmotic stress. AbstractBacteria are thought to cope with fluctuating environmental solute concentrations primarily by adjusting the osmolality of their cytoplasm. To obtain insights into underlying metabolic adaptions, we analyzed the global metabolic response of Escherichia coli to sustained hyperosmotic stress using non-targeted mass spectrometry. We observed that 52% of 1,071 detected metabolites, including known osmoprotectants, changed abundance with increasing salt challenge. Unexpectedly, unsupervised data analysis revealed a substantial increase of most intermediates in the ubiquinone-8 (Q8) biosynthesis pathway and a 110-fold accumulation of Q8 itself, as confirmed by quantitative lipidomics. We then demonstrate that Q8 is necessary for acute and sustained osmotic stress tolerance using Q8 mutants and tolerance rescue through feeding non-respiratory Q8 analogues.Finally, in vitro experiments with artificial liposomes reveal mechanical membrane stabilization as a principal mechanism of Q8-mediated osmoprotection. Thus, we find that besides regulating intracellular osmolality, E. coli enhances its cytoplasmic membrane stability to withstand osmotic stress.

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“…All lenses used (apart from the objective) are made from UV fused silica to optimise UV transmission and the optical fibers are UV grade (OceanOptics). The camera is controlled using the opensource software Manager 1.4 22 .…”

Section: Optical Setupmentioning

confidence: 99%

A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes

et al. 2014

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With the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes is gaining widespread importance. We present a novel, label-free microfluidic assay that quantifies the permeability coefficient of a broad spectrum fluoroquinolone antibiotic, norfloxacin, across lipid membranes using the UV autofluorescence of the drug. We use giant lipid vesicles as highly controlled model systems to study the diffusion through lipid membranes. Our technique directly determines the permeability coefficient without requiring the measurement of the partition coefficient of the antibiotic.

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Computer Control of Microscopes Using µManager

Cited by 1,641 publications

References 1 publication

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli

A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes

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