To improve the performance of particle image velocimetry in measuring instantaneous velocity fields, direct cross-correlation of image fields can be used in place of auto-correlation methods of interrogation of double- or multiple-exposure recordings. With improved speed of photographic recording and increased resolution of video array detectors, cross-correlation methods of interrogation of successive single-exposure frames can be used to measure the separation of pairs of particle images between successive frames. By knowing the extent of image shifting used in a multiple-exposure and by a priori knowledge of the mean flow-field, the cross-correlation of different sized interrogation spots with known separation can be optimized in terms of spatial resolution, detection rate, accuracy and reliability.

Theory of cross-correlation analysis of PIV images : Image analysis as measuring technique in flows

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

/pdf/super-resolution-microscopy-demystified-3pneht3a4l.pdf

Super-resolution microscopy demystified

Deep learning is becoming an increasingly important tool for image reconstruction in fluorescence microscopy. We review state-of-the-art applications such as image restoration and super-resolution imaging, and discuss how the latest deep learning research could be applied to other image reconstruction tasks. Despite its successes, deep learning also poses substantial challenges and has limits. We discuss key questions, including how to obtain training data, whether discovery of unknown structures is possible, and the danger of inferring unsubstantiated image details.

Applications, promises, and pitfalls of deep learning for fluorescence image reconstruction

Fluorescent nanothermometers can probe changes in local temperature in living cells and in vivo and reveal fundamental insights into biological properties. This field has attracted global efforts in developing both temperature-responsive materials and detection procedures to achieve sub-degree temperature resolution in biosystems. Recent generations of nanothermometers show superior performance to earlier ones and also offer multifunctionality, enabling state-of-the-art functional imaging with improved spatial, temporal and temperature resolutions for monitoring the metabolism of intracellular organelles and internal organs. Although progress in this field has been rapid, it has not been without controversy, as recent studies have shown possible biased sensing during fluorescence-based detection. Here, we introduce the design principles and advances in fluorescence nanothermometry, highlight application achievements, discuss scenarios that may lead to biased sensing, analyze the challenges ahead in terms of both fundamental issues and practical implementations, and point to new directions for improving this interdisciplinary field.

Advances and challenges for fluorescence nanothermometry

Structured illumination microscopy (SIM) allows rapid, super-resolution (SR) imaging in live specimens. We review recent technical advances in SR-SIM, with emphasis on imaging speed, resolution, and depth. Since its introduction decades ago, the technique has grown to offer myriad implementations, each with its own strengths and weaknesses. We discuss these, aiming to provide a practical guide for biologists and to highlight which approach is best suited to a given application.

Faster, sharper, and deeper: structured illumination microscopy for biological imaging.

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy.

Insulin is a key metabolic regulator in health and diabetes In pancreatic beta cells, insulin release is regulated by the major second messengers Ca2+ and cAMP: exocytosis is triggered by Ca2+ and mediated by the cAMP/protein kinase A (PKA) signalling pathway However, the causal link between these two processes in primary beta cells remains undefined Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells Membrane depolarisation-induced Ca2+ influx caused an increase in cytosolic PKA activity via activating a Ca2+-sensitive adenylyl cyclase 8 (ADCY8) subpool Glucose stimulation triggered coupled Ca2+ oscillations and PKA activation ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release Our findings suggest that depolarisation-induced Ca2+ influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release Therefore, Ca2+ influx may be important for glucose-stimulated insulin secretion

/pdf/calcium-influx-activates-adenylyl-cyclase-8-for-sustained-xzyyacysts.pdf

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells

How pancreatic β-cells acquire function in vivo is a long-standing mystery due to the lack of technology to visualize β-cell function in living animals. Here, we applied a high-resolution two-photon light-sheet microscope for the first in vivo imaging of Ca2+activity of every β-cell in Tg (ins:Rcamp1.07) zebrafish. We reveal that the heterogeneity of β-cell functional development in vivo occurred as two waves propagating from the islet mantle to the core, coordinated by islet vascularization. Increasing amounts of glucose induced functional acquisition and enhancement of β-cells via activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling. Conserved in mammalians, calcineurin/NFAT prompted high-glucose-stimulated insulin secretion of neonatal mouse islets cultured in vitro. However, the reduction in low-glucose-stimulated insulin secretion was dependent on optimal glucose but independent of calcineurin/NFAT. Thus, combination of optimal glucose and calcineurin activation represents a previously unexplored strategy for promoting functional maturation of stem cell-derived β-like cells in vitro.

In vivo imaging of β-cell function reveals glucose-mediated heterogeneity of β-cell functional development.

We demonstrate a new MEMS-Based Shadowless-illuminated variable-angle TIRF (SIVA-TIRF), which can realize two-plane, two-color TIRF imaging at the speed of 25Hz.

MEMS-based shadowless-illuminated variable-angle TIRF (SIVA-TIRF)

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Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy.

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells

In vivo imaging of β-cell function reveals glucose-mediated heterogeneity of β-cell functional development.

MEMS-based shadowless-illuminated variable-angle TIRF (SIVA-TIRF)