scholarly journals Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

2018 ◽  
Author(s):  
Mohammed Mahamdeh ◽  
Steve Simmert ◽  
Anna Luchniak ◽  
Erik Schäeffer ◽  
Jonathon Howard
Keyword(s):  
High Speed ◽  
Signal To Noise Ratio ◽  
Dark Field ◽  
Fluorescent Labeling ◽  
Wide Field ◽  
Differential Interference Contrast ◽  
Label Free ◽  
High Speed Imaging ◽  
Highly Sensitive

SummaryWhen studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

Redox-Type Label-Free ATP Image Sensor for Highly Sensitive in Vitro Imaging of Extracellular ATP

2021 ◽  
Author(s):  
Hideo Doi ◽  
Tomoko Horio ◽  
Young-Joon Choi ◽  
Kazuhiro Takahashi ◽  
Toshihiko Noda ◽  
...  
Keyword(s):  
Image Sensor ◽  
Extracellular Atp ◽  
Label Free ◽  
Highly Sensitive

scholarly journals Label Free High Speed Wide Field Imaging of Single Microtubules using Interference Reflection Microscopy

2018 ◽  
Vol 114 (3) ◽  
pp. 504a-505a
Author(s):  
Mohammed Mahamdeh ◽  
Steve Simmert ◽  
Anna Łuchniak ◽  
Erik Schäffer ◽  
Jonathon Howard
Keyword(s):  
High Speed ◽  
Wide Field ◽  
Label Free ◽  
Field Imaging ◽  
Wide Field Imaging

scholarly journals Coherent Raman Scattering Microscopy in Oncology Pharmacokinetic Research

2021 ◽  
Vol 12 ◽  
Author(s):  
Junjie Zeng ◽  
Wenying Zhao ◽  
Shuhua Yue
Keyword(s):  
Raman Scattering ◽  
High Speed ◽  
Cancer Drug ◽  
Cancer Drugs ◽  
High Speed Imaging ◽  
Coherent Raman Scattering ◽  
Anti Cancer ◽  
Coherent Raman

The high attrition rates of anti-cancer drugs during clinical development remains a bottleneck problem in pharmaceutical industry. This is partially due to the lack of quantitative, selective, and rapid readouts of anti-cancer drug activity in situ with high resolution. Although fluorescence microscopy has been commonly used in oncology pharmacological research, fluorescent labels are often too large in size for small drug molecules, and thus may disturb the function or metabolism of these molecules. Such challenge can be overcome by coherent Raman scattering microscopy, which is capable of chemically selective, highly sensitive, high spatial resolution, and high-speed imaging, without the need of any labeling. Coherent Raman scattering microscopy has tremendously improved the understanding of pharmaceutical materials in the solid state, pharmacokinetics of anti-cancer drugs and nanocarriers in vitro and in vivo. This review focuses on the latest applications of coherent Raman scattering microscopy as a new emerging platform to facilitate oncology pharmacokinetic research.

Ca2+ spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K+ channels, and coupling ratio between them

2004 ◽  
Vol 287 (6) ◽  
pp. C1577-C1588 ◽  
Author(s):  
Ronghua ZhuGe ◽  
Kevin E. Fogarty ◽  
Stephen P. Baker ◽  
John G. McCarron ◽  
Richard A. Tuft ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
High Speed ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Bk Channels ◽  
Wide Field ◽  
Coupling Ratio ◽  
Patch Clamp Recording ◽  
High Speed Imaging

Ca2+ sparks are highly localized Ca2+ transients caused by Ca2+ release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca2+ sparks activate nearby large-conductance, Ca2+-sensitive K+ (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca2+ sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca2+ sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca2+ released at a site and to estimate the Ca2+ current flowing through RyR [ ICa(spark)]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca2+ signal mass, ICa(spark), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca2+ spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.

scholarly journals Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering

2019 ◽  
Vol 116 (32) ◽  
pp. 15842-15848 ◽  
Author(s):  
Yuta Suzuki ◽  
Koya Kobayashi ◽  
Yoshifumi Wakisaka ◽  
Dinghuan Deng ◽  
Shunji Tanaka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Raman Scattering ◽  
Cancer Biology ◽  
High Speed ◽  
Stimulated Raman Scattering ◽  
Chemical Imaging ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Imaging Flow Cytometry ◽  
Stimulated Raman

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Preliminary Results on the Influence of Engineered Artificial Mucus Layer on Phonation

2014 ◽  
Vol 57 (2) ◽  
Author(s):  
Michael Döllinger ◽  
Franziska Gröhn ◽  
David A. Berry ◽  
Ulrich Eysholdt ◽  
Georg Luegmair
Keyword(s):  
High Speed ◽  
Polymer Concentration ◽  
Physiological Saline ◽  
Vocal Folds ◽  
Oscillatory Behavior ◽  
Linear Polymers ◽  
High Speed Imaging ◽  
Pass Filter ◽  
Low Pass Filter

Purpose Previous studies have confirmed the influence of dehydration and an altered mucus (e.g., due to pathologies) on phonation. However, the underlying reasons for these influences are not fully understood. This study was a preliminary inquiry into the influences of mucus architecture and concentration on vocal fold oscillation. Method Two excised human larynges were investigated in an in vitro setup. The oscillations of the vocal folds at various airflow volume rates were recorded through the use of high-speed imaging. Engineered mucus containing polymers (interconnected polymers and linear polymers) was applied to the vocal folds. From the high-speed footage, glottal parameters were extracted through the use of objective methods and were compared to a gold standard (physiological saline solution). Results Variations were found for all applications of mucus. Fundamental frequency dropped and the oscillatory behavior (speed quotient [SQ], closing quotient [CQ]) changed for both larynges. The 2 applied mucus architectures displayed different effects on the larynges. The interconnected polymer displayed clear low-pass filter characteristics not found for the linear polymer. Increase of polymer concentration affected parameters to a certain point. Conclusion The data confirm results found in previous studies. Furthermore, the different effects—comparing architecture and concentration—suggest that, in the future, synthetic mucus can be designed to improve phonation.

602 Label-free, high-speed imaging with real-time CARS microscope

2008 ◽  
Vol 2008.83 (0) ◽  
pp. _6-2_
Author(s):  
Takeo MINAMIKAWA ◽  
Mamoru HASHIMOTO ◽  
Tsutomu ARAKI
Keyword(s):  
Real Time ◽  
High Speed ◽  
Label Free ◽  
High Speed Imaging

scholarly journals MiniFAST: A sensitive and fast miniaturized microscope for in vivo neural recording

2020 ◽  
Author(s):  
Jill Juneau ◽  
Guillaume Duret ◽  
Joshua P. Chu ◽  
Alexander V. Rodriguez ◽  
Savva Morozov ◽  
...  
Keyword(s):  
Open Source ◽  
High Speed ◽  
High Sensitivity ◽  
High Gain ◽  
Image Sensor ◽  
High Speed Imaging ◽  
Light Power ◽  
Excitation Light

AbstractObserving the activity of large populations of neurons in vivo is critical for understanding brain function and dysfunction. The use of fluorescent genetically-encoded calcium indicators (GECIs) in conjunction with miniaturized microscopes is an exciting emerging toolset for recording neural activity in unrestrained animals. Despite their potential, current miniaturized microscope designs are limited by using image sensors with low frame rates, sensitivity, and resolution. Beyond GECIs, there are many neuroscience applications which would benefit from the use of other emerging neural indicators, such as fluorescent genetically-encoded voltage indicators (GEVIs) that have faster temporal resolution to match neuron spiking, yet, require imaging at high speeds to properly sample the activity-dependent signals. We integrated an advanced CMOS image sensor into a popular open-source miniaturized microscope platform. MiniFAST is a fast and sensitive miniaturized microscope capable of 1080p video, 1.5 µm resolution, frame rates up to 500 Hz and high gain ability (up to 70 dB) to image in extremely low light conditions. We report results of high speed 500 Hz in vitro imaging of a GEVI and ∼300 Hz in vivo imaging of transgenic Thy1-GCaMP6f mice. Finally, we show the potential for a reduction in photobleaching by using high gain imaging with ultra-low excitation light power (0.05 mW) at 60 Hz frame rates while still resolving Ca2+ spiking activity. Our results extend miniaturized microscope capabilities in high-speed imaging, high sensitivity and increased resolution opening the door for the open-source community to use fast and dim neural indicators.

scholarly journals Characterization and Performance of Minimum Quantity Lubricants in Through-Tool Drilling

2020 ◽  
Vol 5 (4) ◽  
pp. 98-115
Author(s):  
Amla Patil ◽  
Jay Raval ◽  
Tim Bangma ◽  
Immanuel Edinbarough ◽  
Bruce Tai ◽  
...  
Keyword(s):  
High Speed ◽  
Minimum Quantity Lubrication ◽  
Air Pressure ◽  
Cluster Method ◽  
High Concentration ◽  
High Speed Imaging ◽  
Minimum Quantity ◽  
Drilling Performance ◽  
Highly Sensitive ◽  
And Performance

This study characterized airborne microdroplet diameters and size distribution from two commercially available lubricants A and B for internal minimum quantity lubrication (MQL). The effects of air pressure, oil channel size, physical properties of lubricants on the resultant microdroplets and through-tool MQL drilling performance were studied. Airborne microdroplet diameters were highly sensitive to the coolant channel sizes and air pressure. Cluster method was used to divide microdroplets into smaller clusters for comparison. Experimental data show that the average airborne microdroplet of lubricant B was larger than that of lubricant A at different air pressures and channel sizes. The contact angle of lubricant A was at least 10° less than that of lubricant B when depositing on glass or aluminium. High-speed imaging showed the tendency of more viscous lubricant B sticking to the drill tip, and higher pressure and longer time was required to atomize this viscous oil. Built-up-edges were less significant when drilling A380 aluminium with lubricant A. Due to high machinability of A380 aluminium, variation of hole diameter and hole cylindricity were minimal when drilling with different lubricants. Insignificant improvement in hole quality was observed when drilling with excessive amount of MQL lubricants or high concentration of lubricant C in flood coolant.

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