Visualization of Blood-Brain Barrier Disruption in Septic Mice with the New Method Based on in Vivo Imaging Technology.

BACKGROUND: Dynamic monitoring of the blood-brain barrier (BBB) functional status in septic mice can help to explore the pathological mechanisms. Therefore, we proposed a new method for monitoring BBB permeability and applied it to the detection of sepsis models. METHODS: The new method involves the construction of an optical cranial window and in vivo imaging. We performed dynamic monitoring of BBB permeability and cerebral blood flow (CBF) in cecal ligation puncture (CLP) and endotoxemia (lipopolysaccharide [LPS]) mice. RESULTS: The sensitivity and accuracy of this method were higher than those of Evans blue evaluation. The increase of BBB permeability in the group of CLP mice was relatively mild and correlated with overall survival, and the damage was irreversible. Contrarily, BBB damage in the LPS group was more acute and severe, unrelated to overall survival, but recoverable. The CBF decreased significantly in both model mouse groups 24 h after modeling, but only the CBF proportion decrease in the LPS group was significantly correlated with an increase in BBB permeability. Within 24 h after both models were established, the decrease in blood flow in the digestive organs occurred earlier than in the brain and kidneys, and the decrease in small intestine blood flow in the LPS group progressed faster. CONCLUSIONS: We have successfully demonstrated the feasibility of our novel method to detect BBB permeability in mice. Our results revealed a significant difference in the BBB permeability change trend between the CLP and LPS model mice when survival curves were consistent. Notably, the CLP-model mice demonstrated a closer resemblance to clinical patients. Our findings suggest that early-stage brain tissue hypoperfusion has a greater impact on BBB function damage in endotoxemia mice, which is related to the faster progression of blood flow redistribution.

Half-curcumin-based chemiluminescence probes and their applications in detecting quasi-stable oxidized protein.

Numerous methods have been reported for detecting ROS/RNS in vitro and in vivo; however, detecting methods for the secondary products of the ROS/RNS reactions, particularly quasi-stable oxidized products, have been much less explored. In this report, we observed that half-curcumins could generate chemiluminescence. In contrast to other chemiluminescence scaffolds, the distinguishing feature of a half-curcumin is the formation of a carbanion intermediate of its acetylacetone moiety, opening unique avenues for applications. In this study, we designed a series of half-curcumins CRANAD-Xs and found that CRANAD-164 could be used to detect quasi-stable oxidized proteins (QSOP) in vivo and in patient serum samples. We illustrated that CRANAD-164 could be used to monitor the responses of taurine, an amino acid with newly reported anti-aging capacity, in an inflammatory mouse model. Remarkably, we further demonstrated that the QSOP levels were much higher in the disease serum samples, including Alzheimer's disease, compared to the samples from healthy controls. Moreover, our results revealed that the sera chemiluminescence intensities were higher in aged healthy controls compared to young healthy subjects, suggesting that CRANAD-164 can be used to monitor the increase of QSOP during aging.

Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.

Inflammatory response toward a Mg-based metallic biomaterial implanted in a rat femur fracture model.

The immune system plays an important role in fracture healing, by modulating the pro-inflammatory and anti-inflammatory responses occurring instantly upon injury. An imbalance in these responses can lead to adverse outcomes, such as non-union of fractures. Implants are used to support and stabilize complex fractures. Biodegradable metallic implants offer the potential to avoid a second surgery for implant removal, unlike non-degradable implants. However, considering our dynamic immune system it is important to conduct in-depth studies on the immune response to these implants in living systems. In this study, we investigated the immune response to Mg and Mg-10Gd in vivo in a rat femur fracture model with external fixation. In vivo imaging using liposomal formulations was used to monitor the fluorescence-related inflammation over time. We combine ex vivo methods with our in vivo study to evaluate and understand the systemic and local effects of the implants on the immune response. We observed no significant local or systemic effects in the Mg-10Gd implanted group compared to the SHAM and Mg implanted groups over time. Our findings suggest that Mg-10Gd is a more compatible implant material than Mg, with no adverse effects observed in the early phase of fracture healing during our 4-week study. STATEMENT OF SIGNIFICANCE: Degradable metallic implants in form of Mg and Mg-10Gd intramedullary pins were assessed in a rat femur fracture model, alongside a non-implanted SHAM group with special respect to the potential to induce an inflammatory response. This pre-clinical study combines innovative non-invasive in vivo imaging techniques associated with multimodal, ex vivo cellular and molecular analytics. The study contributes to the development and evaluation of degradable biometals and their clinical application potential. The study results indicate that Mg-10Gd did not exhibit any significant harmful effects compared to the SHAM and Mg groups.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

Intravital Microscopy for Imaging and Live Cell Tracking of Alveolar Macrophages in Real Time.

Classic in vitro coculture assays of pathogens with host cells have contributed significantly to our understanding of the intracellular lifestyle of several pathogens. Coculture assays with pathogens and eukaryotic cells can be analyzed through various techniques including plating for colony-forming units (CFU), confocal microscopy, and flow cytometry. However, findings from in vitro assays require validation in an in vivo model. Several physiological conditions can influence host-pathogen interactions, which cannot easily be mimicked in vitro. Intravital microscopy (IVM) is emerging as a powerful tool for studying host-pathogen interactions by enabling in vivo imaging of living organisms. As a result, IVM has significantly enhanced the understanding of infection mediated by diverse pathogens. The versatility of IVM has also allowed for the imaging of various organs as sites of local infection. This chapter specifically focuses on IVM conducted on the lung for elucidating pulmonary immune response, primarily involving alveolar macrophages, to pathogens. Additionally, in this chapter we outline the protocol for lung IVM that utilizes a thoracic suction window to stabilize the lung for acquiring stable images.

The influence of various polymer coatings on the in vitro and in vivo properties of PLGA nanoparticles: Comprehensive study.

Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) with various surface chemistry are widely used in biomedicine for theranostic applications. The nature of the external coating of nanoparticles has a significant influence on their efficiency as drug carriers or visualization agents. However, information about the mechanisms of nanoparticle accumulation in tumors and the influence of their surface properties on biodistribution is scarce due to the lack of systematic evaluation. Here we investigate the effect of different polymer coatings of the surface on in vitro and in vivo properties of PLGA nanoparticles. Namely, cell binding efficiency, cytotoxicity, efficiency of fluorescent bioimaging, and tumor accumulation were tested. The highest binding efficiency in vitro and cytotoxicity were observed for positively charged polymers. Interestingly, in vivo fluorescent visualization of tumor-bearing mice and quantitative measurements of biodistribution of magnetite-loaded nanoparticles indicated different dependences of accumulation in tumors on the coating of PLGA nanoparticles. This means that nanoparticle surface properties can simultaneously enhance imaging efficiency and decrease quantitative accumulation in tumors. The obtained data demonstrate the complexity of the dependence of nanoparticles' effectiveness for theranostic applications on surface features. We believe that this study will contribute to the rational design of nanoparticles for effective cancer diagnostics and therapy.

Assessment of tissue response in vivo: PET-CT imaging of titanium and biodegradable magnesium implants.

To study in vivo the bioactivity of biodegradable magnesium implants and other possible biomaterials, we are proposing a previously unexplored application of PET-CT imaging, using available tracers to follow soft tissue and bone remodelling and immune response in the presence of orthopaedic implants. Female Wistar rats received either implants (Ti6Al7Nb titanium or WE43 magnesium) or corresponding transcortical sham defects into the diaphyseal area of the femurs. Inflammatory response was followed with [18F]FDG and osteogenesis with [18F]NaF, over the period of 1.5 months after surgery. An additional pilot study with [68Ga]NODAGA-RGD tracer specific to αvß3 integrin expression was performed to follow the angiogenesis for one month. [18F]FDG tracer uptake peaked on day 3 before declining in all groups, with Mg and Ti groups exhibiting overall higher uptake compared to sham. This suggests increased cellular activity and tissue response in the presence of Mg during the initial weeks, with Ti showing a subsequent increase in tracer uptake on day 45, indicating a foreign body reaction. [18F]NaF uptake demonstrated the superior osteogenic potential of Mg compared to Ti, with peak uptake on day 7 for all groups. [68Ga]NODAGA-RGD pilot study revealed differences in tracer uptake trends between groups, particularly the prolonged expression of αvß3 integrin in the presence of implants. Based on the observed differences in the uptake trends of radiotracers depending on implant material, we suggest that PET-CT is a suitable modality for long-term in vivo assessment of orthopaedic biomaterial biocompatibility and underlying tissue reactions. STATEMENT OF SIGNIFICANCE: The study explores the novel use of positron emission tomography for the assessment of the influence that biomaterials have on the surrounding tissues. Previous related studies have mostly focused on material-related effects such as implant-associated infections or to follow the osseointegration in prosthetics, but the use of PET to evaluate the materials has not been reported before. The approach tests the feasibility of using repeated PET-CT imaging to follow the tissue response over time, potentially improving the methodology for adopting new biomaterials for clinical use.

Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo. Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics. Approach: The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator. Results: Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a 13 µm by 13 µm field-of-view at over 10 kHz frame rate with ∼0.5 µm lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution. Conclusions: We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.

Probing neuronal activity with genetically encoded calcium and voltage fluorescent indicators.

Monitoring neural activity in individual neurons is crucial for understanding neural circuits and brain functions. The emergence of optical imaging technologies has dramatically transformed the field of neuroscience, enabling detailed observation of large-scale neuronal populations with both cellular and subcellular resolution. This transformation will be further accelerated by the integration of these imaging technologies and advanced big data analysis. Genetically encoded fluorescent indicators to detect neural activity with high signal-to-noise ratios are pivotal in this advancement. In recent years, these indicators have undergone significant developments, greatly enhancing the understanding of neural dynamics and networks. This review highlights the recent progress in genetically encoded calcium and voltage indicators and discusses the future direction of imaging techniques with big data analysis that deepens our understanding of the complexities of the brain.

A transformable and self-oxygenated smart probe for enhanced tumor sonodynamic therapy.

Sonodynamic therapy (SDT) is emerging as a promising modality for cancer treatment. However, improving the tumor bioavailability and anti-hypoxia capability of sonosensitizers faces a big challenge. In this work, we present a tumor microenvironment (TME)-mediated nanomorphology transformation and oxygen (O2) self-production strategy to enhance the sonodynamic therapeutic efficacy of tumors. A smart probe Ce6-Leu@Mn2+ that consists of a glutathione (GSH) and leucine amino peptidase (LAP) dual-responsive unit, a 2-cyanobenzothiazole (CBT) group, and a Mn2+-chelated Ce6 as sonosensitizer for tumor SDT was synthesized, and its SDT potential for liver tumor HepG2 in living mice was systematically studied. It was found that the probes could self-assemble into large nanoparticles in physiological condition and spontaneously transformed into small particles under the dual stimulation of GSH and LAP in TME resulting in enhanced tumor accumulation and deep penetration. More notably, Ce6-Leu@Mn2+ could convert endogenous hydrogen peroxide to O2, thereby alleviating the hypoxia and achieving effective SDT against hypoxic tumors under the excitation of ultrasound. We thus believe this smart TME-responsive probe may provide a noninvasive and efficient means for malignant tumor treatment. STATEMENT OF SIGNIFICANCE: Sonodynamic therapy (SDT) is emerging as a promising therapeutic modality for cancer treatment. However, how to improve the tumor bioavailability and anti-hypoxia capability of sonosensitizers remains a huge challenge. Herein, we rationally developed a theranostic probe Ce6-Leu@Mn2+ that can transform into small-size nanoparticles from initial large particles under the dual stimulation of LAP and GSH in tumor microenvironment (TME) resulting in enhanced tumor accumulation, deep tissue penetration as well as remarkable O2 self-production for enhanced sonodynamic therapy of human liver HepG2 tumor in living mice. This smart TME-responsive probe may provide a noninvasive and efficient means for hypoxic tumor treatment.

Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Activity-dependent dendrite patterning in the postnatal barrel cortex.

For neural circuit construction in the brain, coarse neuronal connections are assembled prenatally following genetic programs, being reorganized postnatally by activity-dependent mechanisms to implement area-specific computational functions. Activity-dependent dendrite patterning is a critical component of neural circuit reorganization, whereby individual neurons rearrange and optimize their presynaptic partners. In the rodent primary somatosensory cortex (barrel cortex), driven by thalamocortical inputs, layer 4 (L4) excitatory neurons extensively remodel their basal dendrites at neonatal stages to ensure specific responses of barrels to the corresponding individual whiskers. This feature of barrel cortex L4 neurons makes them an excellent model, significantly contributing to unveiling the activity-dependent nature of dendrite patterning and circuit reorganization. In this review, we summarize recent advances in our understanding of the activity-dependent mechanisms underlying dendrite patterning. Our focus lays on the mechanisms revealed by in vivo time-lapse imaging, and the role of activity-dependent Golgi apparatus polarity regulation in dendrite patterning. We also discuss the type of neuronal activity that could contribute to dendrite patterning and hence connectivity.

A new GRAB sensor reveals differences in the dynamics and molecular regulation between neuropeptide and neurotransmitter release.

The co-existence and co-transmission of neuropeptides and small molecule neurotransmitters in the same neuron is a fundamental aspect of almost all neurons across various species. However, the differences regarding their in vivo spatiotemporal dynamics and underlying molecular regulation remain poorly understood. Here, we developed a GPCR-activation-based (GRAB) sensor for detecting short neuropeptide F (sNPF) with high sensitivity and spatiotemporal resolution. Furthermore, we explore the differences of in vivo dynamics and molecular regulation between sNPF and acetylcholine (ACh) from the same neurons. Interestingly, the release of sNPF and ACh shows different spatiotemporal dynamics. Notably, we found that distinct synaptotagmins (Syt) are involved in these two processes, as Syt7 and Sytα for sNPF release, while Syt1 for ACh release. Thus, this new GRAB sensor provides a powerful tool for studying neuropeptide release and providing new insights into the distinct release dynamics and molecular regulation between neuropeptides and small molecule neurotransmitters.

Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

[In vivo tumor imaging and therapy based on near-infrared cadmium-free quantum dots].

Near-infrared fluorescence imaging technology, which possesses superior advantages including real-time and fast imaging, high spatial and temporal resolution, and deep tissue penetration, shows great potential for tumor imaging in vivo and therapy. Ⅰ-Ⅲ-Ⅵ quantum dots exhibit high brightness, broad excitation, easily tunable emission wavelength and superior stability, and do not contain highly toxic heavy metal elements such as cadmium or lead. These advantages make Ⅰ-Ⅲ-Ⅵ quantum dots attract widespread attention in biomedical field. This review summarizes the recent advances in the controlled synthesis of Ⅰ-Ⅲ-Ⅵ quantum dots and their applications in tumor imaging in vivo and therapy. Firstly, the organic-phase and aqueous-phase synthesis of Ⅰ-Ⅲ-Ⅵ quantum dots as well as the strategies for regulating the near-infrared photoluminescence are briefly introduced; secondly, representative biomedical applications of near-infrared-emitting cadmium-free quantum dots including early diagnosis of tumor, lymphatic imaging, drug delivery, photothermal and photodynamic therapy are emphatically discussed; lastly, perspectives on the future directions of developing quantum dots for biomedical application and the faced challenges are discussed. This paper may provide guidance and reference for further research and clinical translation of cadmium-free quantum dots in tumor diagnosis and treatment.

Serum Amyloid A3 Promoter-Luciferase Reporter Mice Are Useful for Early Drug-Induced Nephrotoxicity Detection.

Early detection of drug-induced kidney injury is essential for drug development. In this study, multiple low-dose aristolochic acid (AA) and cisplatin (Cis) injections increased renal mRNA levels of inflammation, fibrosis, and renal tubule injury markers. We applied a serum amyloid A3 (Saa3) promoter-driven luciferase reporter (Saa3 promoter-luc mice) to these two tubulointerstitial nephritis models and performed in vivo bioluminescence imaging to monitor early renal pathologies. The bioluminescent signals from renal tissues with AA or CIS injections were stronger than those from normal kidney tissues obtained from normal mice. To verify whether the visualized bioluminescence signal was specifically generated by the injured kidney, we performed in vivo bioluminescence analysis after opening the stomachs of Saa3 promoter-luc mice, and the Saa3-mediated bioluminescent signal was specifically detected in the injured kidney. This study showed that Saa3 promoter activity is a potent non-invasive indicator for the early detection of drug-induced nephrotoxicity.

Link Brain-Wide Projectome to Neuronal Dynamics in the Mouse Brain.

Knowledge about the neuronal dynamics and the projectome are both essential for understanding how the neuronal network functions in concert. However, it remains challenging to obtain the neural activity and the brain-wide projectome for the same neurons, especially for neurons in subcortical brain regions. Here, by combining in vivo microscopy and high-definition fluorescence micro-optical sectioning tomography, we have developed strategies for mapping the brain-wide projectome of functionally relevant neurons in the somatosensory cortex, the dorsal hippocampus, and the substantia nigra pars compacta. More importantly, we also developed a strategy to achieve acquiring the neural dynamic and brain-wide projectome of the molecularly defined neuronal subtype. The strategies developed in this study solved the essential problem of linking brain-wide projectome to neuronal dynamics for neurons in subcortical structures and provided valuable approaches for understanding how the brain is functionally organized via intricate connectivity patterns.

Dendrite architecture determines mitochondrial distribution patterns in vivo.

Neuronal morphology influences synaptic connectivity and neuronal signal processing. However, it remains unclear how neuronal shape affects steady-state distributions of organelles like mitochondria. In this work, we investigated the link between mitochondrial transport and dendrite branching patterns by combining mathematical modeling with in vivo measurements of dendrite architecture, mitochondrial motility, and mitochondrial localization patterns in Drosophila HS (horizontal system) neurons. In our model, different forms of morphological and transport scaling rules-which set the relative thicknesses of parent and daughter branches at each junction in the dendritic arbor and link mitochondrial motility to branch thickness-predict dramatically different global mitochondrial localization patterns. We show that HS dendrites obey the specific subset of scaling rules that, in our model, lead to realistic mitochondrial distributions. Moreover, we demonstrate that neuronal activity does not affect mitochondrial transport or localization, indicating that steady-state mitochondrial distributions are hard-wired by the architecture of the neuron.