Focus Drift Research Articles

Design and beam dynamics simulation of an 8 MeV compact accelerator-driven neutron source

A compact accelerator-driven neutron source is proposed at Sino-French Institute of Nuclear Engineering and Technology, Sun Yat-Sen University, called Sun Yat-Sen University Proton Accelerator Facility (SYSU-PAFA). The proton accelerator is composed of a proton electron cyclotron resonance source, a four-vane radio frequency quadrupole (RFQ), and an alternative phase focusing drift tube linac (APF-DTL). It can accelerate 10 mA proton beam to 8 MeV. Due to the high current, beam matching is particularly important. In order to achieve beam matching between various components, beam transport sections are needed. The beam transport line is divided into three segments. The Low Energy Beam Transport (LEBT) ensures that the beam parameters are matched before entering the RFQ. The Medium Energy Beam Transport (MEBT) segment efficiently transfers the beam between the RFQ and DTL. The High Energy Beam Transport (HEBT) focuses on transporting the beam to the targets. The design goal of beam transport line is as short as possible while ensuring high efficiency of beam transportation. SYSU-PAFA has an overall transmission efficiency of 99%, with optimal transverse matching conditions between beam transport and RFQ or DTL accelerators. The efficient use of solenoids and magnets allows for a compact transmission section, resulting in a total length of 13.6 meters, shorter than most accelerators at the same beam energy. This paper will provide the detailed beam dynamics of the compact accelerator.

Quantitative Imaging of Genetically Encoded Fluorescence Lifetime Biosensors.

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)-FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy.

High-accuracy differential autofocus system with an electrically tunable lens.

We propose a quasi-confocal microscopy autofocus system incorporating an electrically tunable lens (ETL) to achieve differential detection. The ETL changes its focal length to collect differential curves at speeds <300 Hz, allowing selective locking onto desired focal layers and high-speed differential operations close to the locked focal plane. By segmenting the system's pupil, the interference between the outgoing and incoming near-infrared beams is avoided, thereby greatly improving the signal-to-noise ratio. This ultra-sensitive system, with a focus drift accuracy better than 1/22 focal depth (∼20 nm @100× objective), provides a new, to the best of our knowledge, implementation pathway to meet the requirements of various microscopy techniques.

SEM3De: image restoration for FIB-SEM.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

Focus variation due to near infrared laser in a confocal microscope.

Focus precision and stability is crucial in confocal microscopy not only for image sharpness but also to avoid radiometric fluctuations that can wrongly be interpreted as variations of the fluorescence intensity in the sample. Here we report a focus variation provoked by a continuous wave laser of 810-nm wavelength introduced along the optical path of an inverted confocal microscope with an oil immersion ×60 objective. When the laser is turned on or off, the focus position drifts toward lower or high values of the vertical coordinate z, respectively. The maximum drift observed was 2.25𝜇m for a laser power of 40 mW at the sample and over a 600-s exposure time. The temporal evolution of the focus position is well fitted by exponential curves that mimic temperature variations due to a heat source. Our analysis strongly suggests that the focus drift is due to heating of the immersion oil.

Simulation of space-charge effects using a quantum Schrödinger approach

Space-charge effects play an important role in high intensity and high brightness particle accelerators. These effects were generally studied self-consistently by solving the Vlasov-Poisson equations using a particle-in-cell method in the accelerator community. In this paper, we propose an alternative method to simulate the space-charge effects in the accelerator. Instead of solving the Vlasov-Poisson equations, the proposed approach solves the Schr\"odinger-Poisson equations to study the space-charge effects in accelerators. Using a quantum Schr\"odinger approach reduces the original problem from six- or four-dimensional phase space down to three or two spatial dimensions. It also provides a possibility to simulate accelerator beam physics on quantum computers by evolving the wave function through quantum gates. Benchmarks of a coasting proton beam through a focusing drift defocusing drift lattice show excellent agreement between the quantum Schr\"odinger method and the particle-in-cell method.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

Coupling of Sub-Terawatt Laser Into Hollow Core Waveguide for High-Harmonic Generation Above 200 eV

Hollow core waveguides are widely used in extreme field research to guide laser pulses. The laser peak intensity is usually several orders higher than the damage threshold of waveguide cladding material in these applications. The nonlinear drift of the spatial focus of such high power laser with the power or intensity fluctuation can easily destroy the waveguide in an avalanche manner. In this letter, we experimentally investigate the nonlinear drift of focal position and size of a sub-terawatt laser beam with respect to pulse energy and pulse duration. A theoretical model is derived and qualitatively explains the experimental observations. A reliable procedure to couple 3 mJ laser pulses at 27 fs into a hollow core waveguide with 150 $\mu \text{m}$ bore diameter is developed. When the waveguide is filled with Helium, the high harmonics up to 212 eV are generated which confirms that the intense laser pulses with peak intensity of $1.8\times 10 ^{\mathbf {15}}$ W/cm $^{\mathbf {2}}$ are successfully coupled into the waveguide.

Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen.

Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na+]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na+]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na+], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na+] between 0 and 150 mM, exhibiting an apparent K d of ∼80 mM. Based on these calibrations, a [Na+] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na+] of 13.0 mM, whereas [Na+] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na+/K+-ATPase by removal of extracellular K+ caused significant [Na+] increases in all cellular subcompartments. Using an alternative approach for data analysis ("Ratio FLIM") increased the temporal resolution and revealed a sequential response to K+ removal, with cytosolic [Na+] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na+]. This approach thus represents a valuable tool for quantitative determination of [Na+] and changes thereof in different subcellular compartments.

Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry.

A new instrument configuration for native ion mobility-mass spectrometry (IM-MS) is described. Macromolecule ions are generated by using a static ESI source coupled to an RF ion funnel, and these ions are then mobility and mass analyzed using a periodic focusing drift tube IM analyzer and an Orbitrap mass spectrometer. The instrument design retains the capabilities for first-principles determination of rotationally averaged ion-neutral collision cross sections and high-resolution measurements in both mobility and mass analysis modes for intact protein complexes. Operation in the IM mode utilizes FT-IMS modes (originally described by Knorr ( Knorr , F. J. Anal. Chem . 1985 , 57 ( 2 ), 402 - 406 )), which provides a means to overcome the inherent duty cycle mismatch for drift tube (DT)-IM and Orbitrap mass analysis. The performance of the native ESI-FT-DT-IM-Orbitrap MS instrument was evaluated using the protein complexes Gln K (MW 44 kDa) and streptavidin (MW 53 kDa) bound to small molecules (ADP and biotin, respectively) and transthyretin (MW 56 kDa) bound to thyroxine and zinc.

Novel aspects of live intestinal epithelial cell function revealed using a custom time-lapse video microscopy apparatus.

Many aspects of cell physiology, including migration, membrane function, and cell division, are best understood by observing live cell dynamics over time using video microscopy. To probe these phenomena in colon epithelial cells using simple components with a limited budget, we have constructed an inexpensive (<$410) self-contained apparatus, consisting of a closed-loop, feedback-controlled system regulated by a PID (proportional-integrative-derivative) controller contained within a 0.077 m3 insulated acrylic box. Temperature, humidity, pH, and proliferative capacity of colon epithelial cells in this system mimic those in a standard tissue culture incubator for over four days. Our system offers significant advantages over existing cost-prohibitive commercially available and custom-made devices because of its very low cost, use of PID temperature control, lack of reliance on constant infusion of external humidified, heated air or carbon dioxide, ability to directly measure cell culture medium temperature, and combination of exquisite cellular detail with minimal focus drift under physiological conditions for extended periods of time. Using this apparatus, coupled with an inverted microscope equipped with phase contrast optics and a programmable digital camera, we have observed many events in colon epithelial cells not visible by static imaging, including kinetics of normal and abnormal mitoses, dynamic membrane structures, intracellular vesicle movements, and cell migration. © 2018 International Society for Advancement of Cytometry.

Evaluation of initiating characteristics of osteoblastic calcium signaling responses to stretch by video rate time-course observation

Osteoblasts change their intracellular calcium ion concentration in response to mechanical stimuli. Although it has been reported that osteoblasts sense and respond to stretching of a substrate on which osteoblastic cells have adhered, the details of the dynamic characteristics of their calcium signaling response remain unclear. Motion artifacts such as loss of focus during stretch application make it difficult to conduct precise time-course observations of calcium signaling responses. Therefore, in this study, we observed intracellular calcium signaling responses to stretch in a single osteoblastic cell by video rate temporal resolution. Our originally developed cell-stretching microdevice enables in situ observation of a stretched cell without excessive motion artifacts such as focus drift. Residual minor effects of motion artifacts were corrected by the fluorescence ratiometric method with fluorescent calcium indicator Fluo 8H and fluorescent cytoplasm dye calcein red-orange. We succeeded to detect the intracellular calcium signaling response to stretch by video rate temporal resolution. The results revealed a time lag from stretch application to initiation of the intracellular calcium signaling response. We compared two time lags measured at two different cell areas: central and peripheral regions of the cell. The time lag in the central region of the cell was shorter than that in the peripheral region. This result suggests that the osteoblastic calcium signaling response to stretching stimuli initiates around the central region of the cell.

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal - used as a measure of priming 2) in vivo neutrophil labeling - achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Compact multi-band fluorescent microscope with an electrically tunable lens for autofocusing.

Autofocusing is a routine technique in redressing focus drift that occurs in time-lapse microscopic image acquisition. To date, most automatic microscopes are designed on the distance detection scheme to fulfill the autofocusing operation, which may suffer from the low contrast of the reflected signal due to the refractive index mismatch at the water/glass interface. To achieve high autofocusing speed with minimal motion artifacts, we developed a compact multi-band fluorescent microscope with an electrically tunable lens (ETL) device for autofocusing. A modified searching algorithm based on equidistant scanning and curve fitting is proposed, which no longer requires a single-peak focus curve and then efficiently restrains the impact of external disturbance. This technique enables us to achieve an autofocusing time of down to 170 ms and the reproductivity of over 97%. The imaging head of the microscope has dimensions of 12 cm × 12 cm × 6 cm. This portable instrument can easily fit inside standard incubators for real-time imaging of living specimens.

Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction

Total internal reflection fluorescence microscopy (TIRFM) is becoming an increasingly common methodology to narrow the illumination excitation thickness to study cellular process such as exocytosis, endocytosis, and membrane dynamics. It is also frequently used as a method to improve signal/noise in other techniques such as in vitro single-molecule imaging, stochastic optical reconstruction microscopy/photoactivated localization microscopy imaging, and fluorescence resonance energy transfer imaging. The unique illumination geometry of TIRFM also enables a distinct method to create an excitation field for selectively exciting fluorophores that are aligned either parallel or perpendicular to the optical axis. This selectivity has been used to study orientation of cell membranes and cellular proteins. Unfortunately, the coherent nature of laser light, the typical excitation source in TIRFM, often creates spatial interference fringes across the illuminated area. These fringes are particularly problematic when imaging large cellular areas or when accurate quantification is necessary. Methods have been developed to minimize these fringes by modulating the TIRFM field during a frame capture period; however, these approaches eliminate the possibility to simultaneously excite with a specific polarization. A new, to our knowledge, technique is presented, which compensates for spatial fringes while simultaneously permitting rapid image acquisition of both parallel and perpendicular excitation directions in ∼25 ms. In addition, a back reflection detection scheme was developed that enables quick and accurate alignment of the excitation laser. The detector also facilitates focus drift compensation, a common problem in TIRFM due to the narrow excitation depth, particularly when imaging over long time courses or when using a perfusion flow chamber. The capabilities of this instrument were demonstrated by imaging membrane orientation using DiO on live cells and on lipid bilayers that were supported on a glass slide (supported lipid bilayer). The use of the approach to biological problems was illustrated by examining the temporal and spatial dynamics of exocytic vesicles.

High resolution autofocus for spatial temporal biomedical research

Maintaining focus has been a critical but challenging issue in optical microscopy, particularly for microscopic imaging systems currently used in biomedical research. During live cell imaging, environmental temperature fluctuations and other factors contribute to the unavoidable focus drift. For single molecular imaging and super resolution, focus drift can be significant even over short durations. The current commercial and experimental solutions are either optically complicated, expensive, or with limited axial resolution. Here, we present a simple autofocus solution based on low cost solid state laser and imaging sensor. By improving the optical train design and using real-time data analysis, improvement in axial resolution by approximately two orders of magnitudes over the focal depth of microscope objectives can be achieved. This solution has been tested for prolonged live cell imaging for fast ramping up in environmental chamber temperature and large daily swing in room temperature. In addition, this system can be used to spatial-temporally measure the surface for three-dimensional cell culture and tissue engineering, with flexibility that exceeds commercially available systems.

Building a live cell microscope: what you need and how to do it.

In this post-genomic era, the predominant focus of biomedical research has moved towards elucidating the functionality of molecules at the cellular and subcellular level in integrated living systems. As such, biologic imaging has moved beyond the collection of static "snapshots" of the cellular state to the real-time visualization of cellular and molecular behavior in three-dimensional (3D) space. Live-cell imaging techniques can be used to assess protein abundance, as well as determine the functional role(s) and interactions of multiple unique molecules concurrently within the cellular environment, and determine the effects of these molecules on cell development, organization, and fate over extended periods of time. Such studies require advanced systems that allow multiparametric analysis of cells while maintaining their functional viability. This unit will focus on the design and implementation of modern live-cell imaging systems. We will examine the principle problems facing live-cell microscopists including: focus drift, cell viability, photo-toxicity, and data acquisition, and suggest appropriate solutions to these issues. Our goal in this unit is to provide individual scientists with the information required to implement live-cell imaging solutions within their own laboratories.

Nanometric locking of the tight focus for optical microscopy and tip-enhanced microscopy

We have successfully stabilized the tight focus onto the sample surface of an optical microscope within ±1.0 nm for a virtually unlimited time duration. The time-dependent thermal drift of the tight focus and the mechanical tilt of the sample surface were simultaneously sensed by a non-optical means based on a capacitive sensor and were compensated for in real-time. This non-optical scheme is promising for the suppression of background light sources for optical microscopy. The focus stabilization is crucial for microscopic measurement at an interface, particularly when scanning a large surface area, because there is always a certain amount of mechanical tilt of the sample substrate, which degrades the contrast of the image. When imaging nanoscopic materials such as carbon nanotubes or silicon nanowires, more stringent nanometric stabilization of the focus position relative to such samples is required, otherwise it is often difficult to interpret the results from the observations. Moreover, the smaller the sample volume is, the smaller the signal becomes, resulting in a long exposure time at each position. In this sense, long-term stability of the tight focus is essential for both microscopic large area scanning and nanosized sample scanning (high-resolution/large-area imaging). In addition, the recently developed tip-enhanced microscopy requires long-term stability of the relative position of the tip, sample and focus position. We were able to successfully demonstrate a stability improvement for tip-enhanced microscopy in the same manner. The stabilization of the tight focus enables us to perform long-term and robust measurements without any degradation of optical signal, resulting in the capability of true nanometric optical imaging with good reproducibility and high precision. The technique presented is a simple add-on for any kind of optical microscope.

Differential internal multi-reflection method for nano-displacement measurement

By utilizing the internal-reflection effect at air–glass boundary, we have proposed a novel method of nano-displacement measurement. Excellent linearity of the reflectance versus the angle of incidence in internal reflection is achieved by making use of a differential detection scheme. Thereby, the target's displacement which causes the change of incident angle can be determined accurately by measuring reflectance. Using a differential detector such as quadrant detector, a compact system based on the above method has been developed, with advantages of simple configuration and low costs compared with interferometers and traditional confocal methods (CMs). The resolution of this system is about 2nm in a wide measurement range. Factors such as the initial angle of incidence, the number of reflections, and the polarization state of the light determine the resolution and measurement range. It is very suitable for many potential applications, especially in the automatic focus of the state-of-art microscopy, in which the traditional problem of focus drift may be solved. In addition, theoretical analysis and experimental results of a prototype sensor are presented.

Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system

Single-point laser scanning confocal imaging produces signals with high spatial resolution in living organisms. However, photo-induced toxicity, bleaching, and focus drift remain challenges, especially when recording over several days for monitoring circadian rhythms. Bioluminescence imaging is a tool widely used for this purpose, and does not cause photo-induced difficulties. However, bioluminescence signals are dimmer than fluorescence signals, and are potentially affected by levels of cofactors, including ATP, O2, and the substrate, luciferin. Here we describe a novel time-lapse confocal imaging technique to monitor circadian rhythms in living tissues. The imaging system comprises a multipoint scanning Nipkow spinning disk confocal unit and a high-sensitivity EM-CCD camera mounted on an inverted microscope with auto-focusing function. Brain slices of the suprachiasmatic nucleus (SCN), the central circadian clock, were prepared from transgenic mice expressing a clock gene, Period 1 (Per1), and fluorescence reporter protein (Per1::d2EGFP). The SCN slices were cut out together with membrane, flipped over, and transferred to the collagen-coated glass dishes to obtain signals with a high signal-to-noise ratio and to minimize focus drift. The imaging technique and improved culture method enabled us to monitor the circadian rhythm of Per1::d2EGFP from optically confirmed single SCN neurons without noticeable photo-induced effects or focus drift. Using recombinant adeno-associated virus carrying a genetically encoded calcium indicator, we also monitored calcium circadian rhythms at a single-cell level in a large population of SCN neurons. Thus, the Nipkow spinning disk confocal imaging system developed here facilitates long-term visualization of circadian rhythms in living cells.