Autonomic regulation and comorbid symptoms in children with attention deficit hyperactivity disorder.

OBJECTIVE: Vagally-mediated heart rate variability (vmHRV) is regarded as transdiagnostic marker of emotion regulation and cognitive control capacity. We analysed vmHRV of children with attention deficit/hyperactivity disorder (ADHD). Based on previous research, we expected to find comorbid symptom dimensions (i.e. internalizing symptoms, conduct problems (CP), and callous unemotional (CU) traits) to relate to vmHRV measures. METHODS: The sample comprised 100 (70 boys) medication naïve children with ADHD. Children were 6 to 11 years old. High frequency HRV (HF-HRV) was measured at rest and during a delay of gratification task. Additionally, sympathetic reactivity was assessed via skin conductance responses (SCR). Comorbid symptoms were assessed by parent-report questionnaires and clinical interviews. RESULTS: The multiple correlation between CU traits and the HF-HRV scores proved statistically significant. Higher CU traits were associated with higher HRV resting-state and response scores. CP were positively associated with the SCR score. CONCLUSION: In children with ADHD, increased CU traits might point to a comparatively less impaired self-regulation capacity in the reward-related context. The result corresponds to findings from previous studies. In the future, CU traits should be considered in analyses of autonomic regulation in ADHD.

Roadmap on computational methods in optical imaging and holography [invited].

Computational methods have been established as cornerstones in optical imaging and holography in recent years. Every year, the dependence of optical imaging and holography on computational methods is increasing significantly to the extent that optical methods and components are being completely and efficiently replaced with computational methods at low cost. This roadmap reviews the current scenario in four major areas namely incoherent digital holography, quantitative phase imaging, imaging through scattering layers, and super-resolution imaging. In addition to registering the perspectives of the modern-day architects of the above research areas, the roadmap also reports some of the latest studies on the topic. Computational codes and pseudocodes are presented for computational methods in a plug-and-play fashion for readers to not only read and understand but also practice the latest algorithms with their data. We believe that this roadmap will be a valuable tool for analyzing the current trends in computational methods to predict and prepare the future of computational methods in optical imaging and holography. Supplementary Information: The online version contains supplementary material available at 10.1007/s00340-024-08280-3.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Long-term cortisol secretion in attention deficit hyperactivity disorder: roles of sex, comorbidity, and symptom presentation.

Low activity of the hypothalamic-pituitary-adrenal axis (HPAA) has been found in children with attention deficit hyperactivity disorder (ADHD). The condition may be related to the reduced attention regulation capacity and/or to comorbid oppositional defiant or conduct disorder (ODD/CD). Sex differences are probable but not sufficiently studied. We analyzed the HPAA activity and sympathetic nervous system reactivity (SR) in children with ADHD while accounting for ADHD symptom presentation, comorbidity, and sex differences. The sample comprised 205 children, 98 (61 boys, 37 girls) with ADHD and 107 (48 boys, 59 girls) healthy controls. DSM-5 phenotypic symptom presentation and comorbid ODD/CD were assessed using clinical interviews. Hair cortisol concentration (HCC) was used to assess the long-term, cumulative activity of the HPAA. SR was assessed via skin conductance response (SCR). For control purposes, comorbid internalizing symptoms and indicators of adverse childhood experiences (ACE) were assessed. Children were medication naive. Boys presenting with predominantly inattentive symptoms (ADHD-I) showed lower HCC than healthy boys. Girls presenting with combined symptoms (ADHD-C) showed higher HCC than did healthy girls (p's < 0.05, sex-by-group interaction, F (2,194) = 4.09, p = 0.018). Boys with ADHD plus ODD/CD showed a blunted SR (p < 0.001, sex-by-group interaction, F (2,172) = 3.08, p = 0.048). Adjustment for ACE indicators led to non-significant differences in HCC but did not affect differences in SR. HCC constitutes an easily assessable, reliable, and valid marker of phenotypic ADHD-related features (i.e. symptom presentation and comorbidity). It indicates more homogenous subgroups of ADHD and might point to specifically involved pathophysiological processes.

Clay nanoparticles efficiently deliver small interfering RNA to intact plant leaf cells.

RNA interference is triggered in plants by the exogenous application of double-stranded RNA or small interfering RNA (siRNA) to silence the expression of target genes. This approach can potentially provide insights into metabolic pathways and gene function and afford plant protection against viruses and other plant pathogens. However, the effective delivery of biomolecules such as siRNA into plant cells is difficult because of the unique barrier imposed by the plant cell wall. Here, we demonstrate that 40-nm layered double hydroxide (LDH) nanoparticles are rapidly taken up by intact Nicotiana benthamiana leaf cells and by chloroplasts, following their application via infiltration. We also describe the distribution of infiltrated LDH nanoparticles in leaves and demonstrate their translocation through the apoplast and vasculature system. Furthermore, we show that 40-nm LDH nanoparticles can greatly enhance the internalization of nucleic acids by N. benthamiana leaf cells to facilitate siRNA-mediated downregulation of targeted transgene mRNA by >70% within 1 day of exogenous application. Together, our results show that 40-nm LDH nanoparticle is an effective platform for delivery of siRNA into intact plant leaf cells.

DH-GAN: a physics-driven untrained generative adversarial network for holographic imaging.

Digital holography is a 3D imaging technique by emitting a laser beam with a plane wavefront to an object and measuring the intensity of the diffracted waveform, called holograms. The object's 3D shape can be obtained by numerical analysis of the captured holograms and recovering the incurred phase. Recently, deep learning (DL) methods have been used for more accurate holographic processing. However, most supervised methods require large datasets to train the model, which is rarely available in most DH applications due to the scarcity of samples or privacy concerns. A few one-shot DL-based recovery methods exist with no reliance on large datasets of paired images. Still, most of these methods often neglect the underlying physics law that governs wave propagation. These methods offer a black-box operation, which is not explainable, generalizable, and transferrable to other samples and applications. In this work, we propose a new DL architecture based on generative adversarial networks that uses a discriminative network for realizing a semantic measure for reconstruction quality while using a generative network as a function approximator to model the inverse of hologram formation. We impose smoothness on the background part of the recovered image using a progressive masking module powered by simulated annealing to enhance the reconstruction quality. The proposed method exhibits high transferability to similar samples, which facilitates its fast deployment in time-sensitive applications without the need for retraining the network from scratch. The results show a considerable improvement to competitor methods in reconstruction quality (about 5 dB PSNR gain) and robustness to noise (about 50% reduction in PSNR vs noise increase rate).

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Initial Clinical Needs Among Transgender and Non-binary Individuals in a Large, Urban Gender Health Program.

BACKGROUND: Transgender and gender-diverse individuals are particularly vulnerable to healthcare discrimination and related health sequelae. OBJECTIVE: To demonstrate diversity in demographics and explore variance in needs at the time of intake among patients seeking care at a large, urban gender health program. DESIGN: We present summary statistics of patient demographics, medical histories, and gender-affirming care needs stratified by gender identity and sexual orientation. PARTICIPANTS: We reviewed all intake interviews with individuals seeking care in our gender health program from 2017 to 2020. MAIN MEASURES: Clients reported all the types of care in which they were interested at the time of intake as their "reason for call" (i.e., establish primary care, hormone management, surgical services, fertility services, behavioral health, or other health concerns). KEY RESULTS: Of 836 patients analyzed, 350 identified as trans women, 263 as trans men, and 223 as non-binary. The most prevalent sexual identity was straight among trans women (34%) and trans men (38%), whereas most (69%) non-binary individuals identified as pansexual or queer; only 3% of non-binary individuals identified as straight. Over half of patients reported primary care, hormone management, or surgical services as the primary reason for contacting our program. Straight, transgender women were more likely to report surgical services as their primary reason for contacting our program, whereas gay transgender men were more likely to report primary care as their reason. CONCLUSIONS: Individuals contacting our gender health program to establish care were diverse in sexual orientation and gender-affirming care needs. Care needs varied with both gender identity and sexual orientation, but primary care, hormone management, and surgical services were high priorities across groups. Providers of gender-affirming care should inquire about sexual orientation and detailed treatment priorities, as trans and gender-diverse populations are not uniform in their treatment needs or goals.

Sodium zirconium cyclosilicate to increase lithium elimination.

Lithium is considered a mood stabilizer for bipolar affective disorders, but it has a narrow therapeutic index of 0.6-1.2 mEq/L. This can easily result in toxic levels after minimal changes in renal function or individual patient's pharmacokinetics. Lithium toxicity can arise with levels as low as 1.5 mEq, and there are limited therapeutic options to treat these patients presenting to the emergency department (ED). At therapeutic levels 95% of lithium is eliminated unchanged by the kidneys. However, previous literature has examined sodium polystyrene sulfonate (SPS) as an option to reduce lithium levels by binding the lithium cation and enhancing its excretion via the gastrointestinal tract. This suggests there may be an increased degree of non-renal clearance and altered toxicokinetics at supratherapeutic levels. However, SPS has been associated with intestinal necrosis and may cause treatment limiting hypokalemia, and is therefore not commonly recommended in treatment algorithms for lithium toxicity. A newer cation exchange resin, sodium zirconium cyclosilicate (SZC), may provide a safer alternative to SPS while also aiding in the clearance of lithium. We present a patient case where a patient with symptomatic acute-on-chronic lithium toxicity had increased clearance of lithium after a dose of SZC.

Spatio-temporal performance in an incoherent holography lattice light-sheet microscope (IHLLS).

We propose an Incoherent holography detection technique for lattice light-sheet (IHLLS) systems for 3D imaging without moving either the sample stage or the detection microscope objective, providing intrinsic instrumental simplicity and high accuracy when compared to the original LLS schemes. The approach is based on a modified dual-lens Fresnel Incoherent Correlation Holography technique to produce a complex hologram and to provide the focal distance needed for the hologram reconstruction. We report such an IHLLS microscope, including characterization of the sensor performance, and demonstrate a significant contrast improvement on beads and neuronal structures within a biological test sample as well as quantitative phase imaging. The IHLLS has similar or better transverse performances when compared to the LLS technique. In addition, the IHLLS allows for volume reconstruction from fewer z-galvo displacements, thus facilitating faster volume acquisition.

Do cognitive interventions for preschoolers improve executive functions and reduce ADHD and externalizing symptoms? A meta-analysis of randomized controlled trials.

Many interventions targeting executive function (EF) development in the preschool period, where malleability might be particularly high, have been created and evaluated. We conducted a meta-analysis of randomized controlled trials (RCTs) on the effects of these interventions on (a) EFs in preschool children from the general population as well as preschool children with (symptoms of) attention-deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD), and (b) ADHD and ODD symptoms in preschool children with ADHD/ODD (symptoms). Literature search yielded 35 RCTs. Risk of bias of the individual studies was assessed. A random-effects model was used. Moderator effects were tested using mixed model analyses. The overall effects on EFs were: d = 0.46 (95% CI 0.30-0.61) for working memory (WM), d = 0.30 (95% CI 0.21-0.38) for inhibitory control (IC), d = 0.33 (95% CI - 0.04 to 0.71) for reward-related IC, and d = 0.47 (95% CI 0.28-0.66) for flexibility. In children with ADHD/ODD, mean effects were d = 0.64 (95% CI 0.31-0.96) for WM and d = 0.46 (95% CI 0.07-0.84) for IC. Studies on reward-related IC and FL were lacking. Effects on ODD and ADHD symptoms were d = 0.40 (95% CI - 0.23 to 1.03) and d = 0.28 (95% CI - 0.08 to 0.64), respectively. Interventions targeting multiple EFs and using interpersonal cognitive scaffolding approaches showed large and statistically significant effects on ADHD and ODD symptoms. In preschool children of the general population and in those with ADHD/ODD (symptoms), interventions led to an improvement of EF performance. In children with ADHD and ODD, cognitive scaffolding interventions were most effective in terms of reducing ADHD and ODD symptoms. However, more well-controlled studies need to be conducted before any firm conclusions can be drawn.

Parental positive regard and expressed emotion-prediction of developing attention deficit, oppositional and callous unemotional problems between preschool and school age.

Parental expressed emotion and positive reinforcement are assumed to affect the development of oppositional and callous-unemotional behaviors in children at risk of attention deficit hyperactivity disorder (ADHD). As longitudinal research on this issue is scarce, we analyzed the respective links between preschool and school age. 138 five-year-old (m = 58.2, s = 6.2 months) children (59% boys) with elevated ADHD symptoms (according to screening) were assessed at the ages of five and eight years. At 5 years, maternal expressed emotion (using the Five Minute Speech Sample) and positive regard of child (using a standardized at-home observation procedure) were assessed. At 5 and 8 years, symptoms of ADHD, oppositional defiant disorder (ODD), and callous-unemotional (CU) behaviors were measured using a multi-informant approach. Multiple linear regression analyses revealed that positive regard specifically predicted a decrease in ODD symptoms between preschool and school age. The expression of high negative emotion specifically predicted an increase in CU behaviors. The development of ADHD symptoms was not predicted by parenting. Knowledge on these specific links can help to elaborate diagnostic and counseling processes in preschoolers with high ADHD symptoms. Underlying mechanisms and the role of neurocognitive deficits of the preschool child should be further analyzed.

Using lactate threshold to predict 5-km treadmill running performance in veteran athletes.

Measuring lactate threshold to predict endurance performance is difficult among veteran athletes, due to age-related decreases in net lactate concentration. The objective of this study was to determine whether lactate threshold, as assessed using the maximal deviation method (Dmax), which is not dependent on net values of lactate, could be used as a more valid measure of 5-km treadmill running performance than other methods of determining lactate threshold. Veteran runners (18 male and 18 female, aged 47.3±6.7 years) performed an incremental exercise test to establish mean treadmill velocity at lactate threshold using Dmax, a log-log method, a visual method, and a 4-mmol·L-1 method, and, on a separate occasion, completed a 5-km time trial. Mean treadmill velocity at Dmax was 12.2±1.8 km·h-1, not being significantly different to mean treadmill velocity (12.1±1.8 km·h-1) attained during the 5-km time trial (p>0.05); velocities were also significantly correlated (r=0.92, p<0.001), and limits of agreement narrow (-1.61 to 1.35 km·h-1). Correlations were weaker and limits of agreement wider for the other methods of lactate threshold determination. Using a two-way, mixed-methods ANOVA, there was no significant effect of sex when using the different methods of determining Tlac (F4,136=3.70, p=0.15). Mean treadmill velocity, when using Dmax for determining lactate threshold, can be used to predict 5-km running performance among male and female veteran athletes.

Circumferential resection margins and perineal complications after neoadjuvant long-course chemoradiotherapy followed by extralevator abdominoperineal excision of the rectum: Five years of activity at a single institution.

BACKGROUND: Prone extralevator abdominoperineal excision of the rectum (ELAPE) has been introduced to improve the circumferential resection margins (CRM) compared with traditional APER. OBJECTIVE: We present short-term results achieved with prone ELAPE preceded by neoadjuvant chemoradiotherapy during the last 5 years of activity. DESIGN: A retrospective review was conducted. SETTINGS AND PATIENTS: Prone ELAPE operations performed between September 2010 and August 2014 at Leicester Royal Infirmary preceded by neoadjuvant chemoradiotherapy. INTERVENTIONS AND MAIN OUTCOME MEASURES: Data regarding demographics, staging, neoadjuvant therapies, intraoperative perforations, and perineal complications were collected. RESULTS: Seventy-two patients were included. Pretreatment radiological T4 were 25.0%, histological T4 2.8%. Intraoperative perforations occurred in 2.8%, CRM was involved in 11.1%. Perineal complications consisted of superficial wound infections (20.8%), full thickness dehiscences (16.7%), hematomas (9.7%), pelvic collections (6.9%), and perineal hernias (5.6%). CONCLUSIONS: In our experience, prone ELAPE preceded by long-course chemoradiotherapy has been successfully used in the last 5 years to resect low rectal tumors. Perineal wound complications rates are similar to those presented in series using direct perineal closures. J. Surg. Oncol. 2016;114:86-90. © 2016 Wiley Periodicals, Inc.

Risk of future offense among probationers with co-occurring substance use and mental health disorders.

The criminal justice system is the primary service delivery system for many adults with drug and alcohol dependence, mental health, and other health service needs. The purpose of this study was to examine the relationship between risk of future offense, mental health status and co-occurring disorders in a large substance abuse diversion probationer population. A purposive sample of 2,077 probationers completed an assessment to screen for mental health disorders, substance use disorders, risk of future crime and violence, and several demographic characteristics. Probationers who screened positive for co-occurring substance use and mental health disorders were significantly more likely to be at higher risk of future crime and violence compared to probationers who screened positive for only substance use, only a mental health disorder, or no substance use or mental health disorder. Implications for substance use and mental health service delivery are discussed, and recommendations are made for further research.

Perceptions of structural and provider-based substance use stigma interventions among primary care professionals.

Background: Stigma enacted in primary care settings remains a barrier to care for people who use drugs (PWUD). Little is known about the acceptability of potential stigma interventions to target structural drivers of stigma affecting the organizational- or provider-level. Methods: In-depth interview data were collected from 21 individuals working in Michigan primary care facilities. Participants included clinical (e.g., physicians, nurses) and non-clinical (e.g., administrators, receptionists) staff. Interviews explored perceptions of stigma toward PWUD and the acceptability of interventions to mitigate such stigma. Thematic analysis was used to identify stigma themes. Results: Participants largely reported substance-use stigma as a matter of individual attitudes or knowledge limitations and described such stigma as rarely occurring during interpersonal interactions. Participants were still acutely aware of upstream societal and organizational factors creating structural barriers to care and/or worsening outcomes among PWUD, but seldom labeled these as stigma. Some provider and structural stigma reduction interventions were enthusiastically supported because they address participant ideas of substance-use stigma drivers (e.g., lack of knowledge) or provide resources that could improve care quality or provide resources for PWUD. Conversely, participants opposed some potential stigma interventions, e.g., less-frequent urine drug testing and increasing clinical visit time, deemed infeasible because of outside forces like insurers or regulators. Conclusions: Although most participants conceptualized substance-use stigma as an individual or interpersonal process best addressed with training, their awareness of social determinants of health seemed to fuel an openness to some structural interventions to reduce organizational and provider stigma toward PWUD in primary care settings.

Needs of transgender children and adolescents presenting to an urban gender health program.

BACKGROUND: Youth who identify as transgender and gender diverse (TGD) are increasingly presenting to pediatric providers. Gender-affirming surgery is often delayed until after a patient reaches the age of majority; however, patients may desire surgery at a younger age. OBJECTIVE: We explore the specific clinical needs of this vulnerable population, including surgical requests. STUDY DESIGN: We present a cross-sectional study of patient intake interviews at time of presentation to our gender health program from 2017 to 2020. We summarize patient demographics, medical histories, and gender-affirming care needs by gender identity and age of presentation. RESULTS: Of 92 patients analyzed, those included were 19 trans girls, 55 trans boys, and 18 non-binary individuals. The median age of our sample was 15 (range 5-17). The median age (IQR) while first questioning gender was 10 (7-12). Sexual orientation was variable with 28 (43%) not sure/unknown. The majority of patients present for primary care services (grade schoolers 75%, early teens 78%, and late teens 77%, p = 0.97) and hormone management (grade schoolers 42%, early teens 62%, and late teens 77%, p = 0.06). Late teens were more likely to present for surgical services (49%) compared to grade schoolers (25%) and early teens (11%), p = 0.001. Prior psychiatric diagnoses were common in all age groups. Trans girls were interested in a variety of affirming procedures whereas trans boys and non-binary individuals primarily sought chest surgery (see summary figure). CONCLUSION: Pediatric gender affirming care needs are varied and multidisciplinary within our center. By age 16, about half of TGD individuals are seeking surgical services. On average, there was a 4-5 year delay from age at first questioning one's gender and presenting to our gender health program. Primary care physicians in particular may prepare to serve this complex population by familiarizing themselves with treatment needs, including developing a network of competent surgical referrals.

High temperature measurements of martensitic transformations using digital holography.

During thermal cycling of nickel-aluminum-platinum (NiAlPt) and single crystal iron-chromium-nickel (FeCrNi) alloys, the structural changes associated with the martensite to austenite phase transformation were measured using dual-wavelength digital holography. Real-time in situ measurements reveal the formation of striations within the NiAlPt alloy at 70°C and the FeCrNi alloy at 520°C. The results demonstrate that digital holography is an effective technique for acquiring noncontact, high precision information of the surface evolution of alloys at high temperatures.

Macrophage plasticity and the role of inflammation in skeletal muscle repair.

Effective repair of damaged tissues and organs requires the coordinated action of several cell types, including infiltrating inflammatory cells and resident cells. Recent findings have uncovered a central role for macrophages in the repair of skeletal muscle after acute damage. If damage persists, as in skeletal muscle pathologies such as Duchenne muscular dystrophy (DMD), macrophage infiltration perpetuates and leads to progressive fibrosis, thus exacerbating disease severity. Here we discuss how dynamic changes in macrophage populations and activation states in the damaged muscle tissue contribute to its efficient regeneration. We describe how ordered changes in macrophage polarization, from M1 to M2 subtypes, can differently affect muscle stem cell (satellite cell) functions. Finally, we also highlight some of the new mechanisms underlying macrophage plasticity and briefly discuss the emerging implications of lymphocytes and other inflammatory cell types in normal versus pathological muscle repair.

Live Cell Light Sheet Imaging with Low- and High-Spatial-Coherence Detection Approaches Reveals Spatiotemporal Aspects of Neuronal Signaling.

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca2+ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca2+ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca2+ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.