Research Progress and Clinical Translational Application of Ultrasound Localization Microscopy in Microcirculation Imaging

doi:10.26599/AUDT.2026.260040

Abstract

Abstract:

As an emerging super-resolution ultrasound imaging technology, ultrasound localization microscopy (ULM) breaks the acoustic diffraction limit of conventional ultrasound. By localizing and tracking individual microbubble contrast agents at the sub-wavelength scale, ULM achieves high-precision visualization and quantitative evaluation of micron-level microvascular structures and hemodynamic parameters. Compared with traditional color Doppler flow imaging and contrast-enhanced ultrasound, ULM possesses superior spatial resolution and microcirculation quantification capability, enabling in-depth analysis of microvascular morphological remodeling and functional perfusion abnormalities. This review summarizes the core technical principles, optimized algorithms, and key technical bottlenecks of ULM, including microbubble signal separation, single-bubble localization accuracy, physiological motion artifacts, and prolonged acquisition duration. Furthermore, we comprehensively elaborate the preclinical and clinical application progress of ULM in multiple systems, covering neurovascular, renal, tumor, cardiovascular, endocrine and cardiac microcirculation diseases, with a specific focus on its novel clinical value in identifying vulnerable carotid atherosclerotic plaques. We also discuss the current limitations restricting large-scale clinical translation, standardized protocol establishment, and deep-tissue imaging performance of ULM, noting that the majority of existing human ULM evidence is still at the feasibility stage and large-scale prospective multi-center clinical validations are still needed. Targeting the above-mentioned translational bottlenecks, finally, this review prospects the future development directions of ULM, including artificial intelligence optimization, three-dimensional whole-organ imaging, multi-modal and multi-omics fusion. With continuous technical iteration, ULM is expected to become a novel non-invasive imaging modality for early diagnosis, risk stratification and therapeutic efficacy evaluation of microcirculation-related disorders.

Key words: Ultrasound localization microscopy; Super-resolution ultrasound; Microbubble contrast agent; Microcirculation; Microvascular imaging; Clinical application

Han Xuedi, Cui Ligang. Research Progress and Clinical Translational Application of Ultrasound Localization Microscopy in Microcirculation Imaging. Advanced Ultrasound in Diagnosis and Therapy, 2026, 10(4): 298-316.

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Figures/Tables 6

Figure 1

Table 1

Comparison of representative microbubble signal extraction and localization algorithms for ULM."

Algorithm	Paper	Computational complexity	Localization accuracy	Capacity for high-density overlapping microbubbles	Dependence on SNR	Real-time performance	Main merits and limitations
note: “-” indicates this metric is not applicable the corresponding algorithm.
Clutter-suppression algorithms
SVD filtering	Brown 2019	High	−	Poor	High	Poor	Delivers effective tissue clutter suppression. Suffers heavy matrix-decomposition computational cost; fails for stationary microbubbles and may introduce hundreds-of-microns localization errors under 0-2 mm/s flow; mostly limited to offline processing.
Pulse inversion	Brown 2019	Moderate	−	Moderate	Moderate	Moderate	Insensitive to flow direction and velocity, suitable for slow-flow and tortuous microvasculature. Requires multiple transmit pulses and cannot separate overlapping microbubbles inherently.
Differential imaging (DI)	Brown 2019	Low-Moderate	−	Moderate	High	Good	Low computational overhead. Only functional for flow rates > 0.5 mm/s under axial-dominated blood flow; performance deteriorates for slow or lateral-oriented perfusion.
AF-UNet	Zhang 2026	Low	−	Moderate	Moderate	Good (GPU- dependent)	Lightweight multi-angle-fusion network achieving > 20-fold speedup relative to SVD with favourable cross-organ generalization. Better preserves microbubble point-spread function (PSF) than 3D U-Net variants but cannot intrinsically resolve spatially-overlapping microbubbles; faint microbubble signals may be attenuated. Real-time capability depends on GPU hardware, without native scanner-side implementation. relies on high-quality training datasets
Localization algorithms
Gaussian fit (GF)	Sobolewski 2024, Heiles 2022	High	High	Limited-test	Moderate-high	Poor	Iterative nonlinear least-squares fitting. Reaches precision close to the Cramér-Rao lower bound with properly selected patch size and sampling parameters and yields the lowest standard deviation among classical approaches. Performance degrades under Rician noise and oversized patches; bimodal error occurs with undersampling; computationally expensive. Only single-microbubble simulations in cited literature; overlapping-bubble scenarios remain unevaluated.
YOLO-modified object-detection-based CNN	Liu 2023	Moderate-High	High	Limited-test	Moderate	Moderate	Modified YOLO-style convolutional-neural-network detector outputting microbubble coordinates and confidence scores. Achieves 77% Jaccard index and 20 μm localization error with 7-fold speed-up against Gaussian fitting on ULTRASR-Challenge simulated datasets; confidence filtering suppresses false detections. Performance heavily relies on training-data distribution; lacks systematic overlapping-microbubble and in-vivo validations and requires GPU acceleration. Only simulated flow-like dataset in original publication; dedicated overlapping-microbubble tests are missing.
Tracking-association algorithms
Modified MCMCDA	Ackermann 2016	High	−	Good	Moderate	Poor	Designed for high-density microbubble track linking, accounting for false alarms and track birth-death events; validated via phantom and in-vivo mouse-tumor measurements. Heavy computational overhead prevents real-time deployment.
Hierarchical Kalman tracker (HK)	Taghavi 2022	High	−	Good	Moderate	Poor	Hierarchical Kalman framework adopting velocity-stratified initialization parameters. Delivers superior reconstruction accuracy under high microbubble density and large localization uncertainty. Multi-level iteration imposes high computational cost and supports only offline workflows (3 million MB positions, 5-level HK, with 35.79 min runtime).

Table 1

Table 2

Summary of preclinical and clinical application studies of ULM."

Paper	Study design	Cohort size	Application field	Research model	Research focus	Disease background	Reference standard	Core research findings	Evidence maturity
Lowerison 2020	Simulation assisted pre-clinical observational	5	chicken embryos	renal cell carcinoma xenograft model grown in the chorioallantoic membrane of chicken embryos	To provide a quantitative measures of tissue hypoxic status of ULM	Tumor	fluorescent histology	ULM derived intervessel distance strongly correlates with histological microvascular density, and vascular tortuosity reliably reflects tissue hypoxia levels	Pre-clinical animal
Lowerison 2022	Pre-clinical observational	99	chicken embryos	human colorectal carcinoma tumor xenograft model engrafted into the CAM of chicken embryos	To indicate the functional microvascular status of chemotherapy sensitivity	Tumor	histology	Elevated vascular fractal dimension on ULM images is closely associated with tumor therapeutic resistance	Pre-clinical animal
Huang 2020	phantom + in vivo chicken embryo proof of concept study	biological replicate number not reported, representative datasets shown	chick chorioallantoic membrane, embryo tumour xenograft	chicken embryo tumor xenograft model and chicken brain	To resolve the inherent trade-off between microbubbles concentration and acquisition duration	Tumor	optical imaging	This novel algorithm separates overlapping microbubbles based on flow velocity and direction differences, enabling robust high-resolution microvascular imaging within seconds under high microbubble dosage	Pre-clinical animal
Chen 2020	Pre-clinical observational	15	mice	mouse acute kidney injury model	To evaluate early acute kidney injury	Kidney disease	histology and immunohistochemistry (CD31)	ULM identifies early renal microcirculatory disorders prior to serum creatinine elevation, supporting early warning of acute kidney injury	Pre-clinical animal
Lowerison 2022	Pre-clinical observational	27	mice	aging mouse model	To varify differences of aged brain and normal brain	Aging	not appliable	Aged brains exhibit reduced blood velocity, increased vascular tortuosity, and decreased cortical blood volume across whole-brain regions on ULM	Pre-clinical animal
Ma 2022	Pre-clinical observational	8	rat	stroke events and Alzheimer's disease	To assess microvascular features of cerebral small vessel disease	Cerebral small vessel disease	H&E staining, PTAH staining, TB staining, MRI	A controllable cerebral small vessel disease model exhibits reduced microvascular number, decreased flow velocity and diminished blood volume in sonicated lesions	Pre-clinical animal
Andersen 2020	Pre-clinical interventional	10	rat	Sprague-Dawley rat	To identify early ischemic damage to the renal microcirculation	Kidney disease	Hematoxylin and eosin staining, Ex-vivo MRI	ULM reliably resolves 15–20 μm ascending vasa recta and distinguishes microvascular alterations after renal ischemia-reperfusion injury	Pre-clinical animal
Andersen 2022	Pre-clinical interventional	7	rat	normal rat	To assess renal vascular velocity responses to pharmacological intervention	Kidney disease	not appliable	Pharmacological intervention with prazosin induces velocity reduction specifically in cortical arterioles and outer-medullary vasa recta, enabling compartment-specific renal hemodynamic evaluation by ULM	Pre-clinical animal
Beliard 2022	Pre-clinical interventional	10, 5 per group (4 week post contusion SCI, 8 week post contusion SCI)	rat	chronic spinal cord injury	to explore changes in spinal cord blood vessels	Spinal cord injury	immunohistochemistry (laminin and for SMI-71), BBB locomotor behavioural scoring	Spinal cord injury presents reduced perfusion velocity and asymmetric hypoperfusion, with ULM-derived vascular biomarkers closely correlating with injury severity and supporting longitudinal vascular function assessment	Pre-clinical animal
Qiu 2022	Pre-clinical observational	11	rat	spontaneously hypertensive rats and normal Wistar-Kyoto rats	To detect features of CKD in early stage	Kidney disease	phantom experiments, Pathology (H&E staining)	ULM detects altered interlobular arterial flow velocity, allowing early diagnosis of hypertensive nephrosclerosis.	Pre-clinical animal
Søgaard 2022	Pre-clinical observational	8 male Zucker rats: 4 obese Zucker rats + 4 age matched lean Zucker rats	rat	obese Zucker rats	To quantify renal vascular density and tortuosity in vivo	Kidney disease	renal functional markers,,Histopathology (H&E, periodic acid-Schiff, Sirius Red, and Jones Silver staining)	Obese rats show increased renal vascular density and subclinical renal dysfunction without apparent histological damage, which can be non-invasively identified by ULM	Pre-clinical animal
Demeulenaere 2022	Pre-clinical observational ex-vivo + in-vivo animal study	9 Sprague Dawley male rats: 6 isolated Langendorff-perfused beating hearts (ex-vivo), 3 closed chest in-vivo anaesthetized rats	rat	isolated beating rat hearts	To visualize whole heart coronary microvasculature beyond the acoustic diffraction limit (sub-20 μm resolution)	Heart	Ex-vivo micro computed tomography of fixed hearts; aortic perfusion flow measurements from Langendorff perfusion system	ULM enables sub-20 μm three-dimensional visualization of beating-heart coronary microvasculature and accurately quantifies adenosine induced coronary flow augmentation.	Pre-clinical animal
Qian 2022	Pre-clinical observational：in-vitro phantom + single animal in-vivo proof of concept study	Custom built flow channel phantoms; 1 New Zealand white rabbit imaged under three IOP levels (10 mmHg, 30 mmHg, 50 mmHg)	rat	pre-designed flow phantoms in vitro; New Zealand white rabbit eye in vivo	To monitor alterations of ocular blood flow of the deep eye vasculature	Retinal and optic nerve diseases	Phantom: preset pump driven ground truth flow speeds; in-vivo: calibrated manometer controlled intraocular pressure; literature reported OCT-A performance as comparative benchmark	Elevated intraocular pressure reduces ocular vascular density and flow velocity, and ULM provides high-resolution deep ocular imaging comparable to optical coherence tomography angiography	Pre-clinical animal
Zhang 2021	Pre-clinical interventional longitudinal in-vivo animal feasibility study	8 male New Zealand white rabbits; 6 rabbits with successfully implanted VX2 liver tumors for longitudinal baseline/post sorafenib therapy imaging	Rabbit	liver tumor model	To present a novel anesthesia protocol to enable the long image acquisition times	Tumor	Post mortem histopathology including H&E staining, CD31 immunohistochemistry for microvascular endothelium, Hypoxyprobe pimonidazole staining for intratumoral hypoxia; in-vivo physiological vital sign monitoring	Optimized apneic imaging protocols eliminate motion artifacts, enabling clear visualization of hypovascular tumor cores and intact tumoral microvascular architecture	Pre-clinical animal
Lei 2022	Pre-clinical observational longitudinal in-vivo animal feasibility study (endoscopic ULM)	6 female New Zealand white rabbits; longitudinal imaging at baseline, 7, 14 and 21 days after rectal VX2 tumour implantation	rabbit	gastrointestinal tract model	Feasibility assessment of endoscopic ULM for gastrointestinal tumour microvascular evaluation	Tumor	In-vitro flow phantom ground truth tube dimensions; ex-vivo micro-CT angiography of resected rectal tumour tissue for vascular pattern validation	Endoscopic ULM reveals significant microvascular pattern and density differences across distinct tumor growth stages	Pre-clinical animal
Yu 2018	Pre-clinical observational proof of concept in-vivo animal study	1 New Zealand white rabbit; balloon injured femoral artery atherosclerosis model, with contralateral non-injured artery as internal control	New Zealand white rabbit	atherosclerosis model	To develop deconvolution combined ULM with STIC synchronization to tolerate cardiac related physiological motion	Atherosclerosis	In-vitro synthetic phantom for algorithm validation; post mortem H&E histology and vWF immunofluorescence staining for vasa vasorum assessment	A customized deconvolution and spatiotemporal correlation workflow achieves 41 μm microvascular visualization in rabbit atherosclerotic models under cardiac motion interference	Pre-clinical animal
Zhu 2019	Pre-clinical observational feasibility in-vivo animal study	3, 2 male New Zealand white rabbits for in-vivo popliteal LN imaging; one extra rabbit for ex-vivo micro-CT vascular corrosion cast reference	rabbit	lymph node	Feasibility of 3D ULM for in-vivo lymph node microvascular and flow assessment	Lymph node disease	Ex-vivo micro-CT vascular corrosion cast (separate animal) for vessel size distribution benchmark; in-vivo ASAP enhanced power doppler as imaging comparator	3D-ULM resolves vessels separated by 30 μm (~15-fold resolution improvement vs Doppler); most ULM detected vessels < 80 μm (PD mostly > 100-250 μm); predominant intranodal blood-flow velocity < 5 mm/s	Pre-clinical animal
Zhu 2022	Prospective human feasibility pilot clinical study	10 lymph nodes from 10 patients (4 reactive benign LNs, 6 metastatic LNs); screened from 44 patients	human	metastatic and reactive LNs	To detect and distinguish metastatic lymph nodes from benign lymphadenopathy	LNs disease	Ultrasound guided core needle biopsy histopathology	LFDI, a metric reflecting local blood flow direction disorder, is a promising ULM derived biomarker for identifying metastatic lymph nodes. This pilot study highlights major practical challenges including patient motion and scanner frame rate constraints	Human feasibility
Bodard 2023	Prospective human feasibility pilot study including method optimization and proof of concept	49 kidney transplant recipients; 35 for imaging protocol optimization; final analysis on 7 kidney allograft patients	human	human kidney allografts	Evaluate feasibility of ULM implemented on conventional low frame rate clinical ultrasound scanner for renal microvascular assessment	Kidney disease	Comparative imaging benchmark versus SMI, advanced dynamic flow (ADF) and CDFI	ULM resolved vessels 2-4-fold thinner than Doppler modes, with the minimum analyzable vessel cross-section averaging 0.3 ± 0.2 mm, and enabled quantitative cortical blood velocity measurement	Human feasibility
Huang 2021	Prospective multi-organ human feasibility proof of concept study	Multiple human subjects; imaging of healthy liver, healthy kidney, pancreatic tumour and DCIS breast tumour; exact individual subject count not explicitly reported	human	a healthy liver and a diseased liver with acute-on-chronic liver failure, a kidney, a pancreatic tumor	To study a robust in human ULM on a high frame-rate clinical ultrasound scanner to achieve short acquisition time	Human multiple-organ in-vivo model (Liver disease, kidney disease, and tumor)	Contrast enhanced power doppler reconstructed from identical microbubble dataset for imaging performance comparison	Clinical high frame rate ULM enables ultrafast human microvascular imaging across multiple tissues and shows broad clinical potential, though rigorous gold-standard validation for tiny vascular structures remains necessary	Human feasibility
Zhang 2022	Prospective human clinical cohort study	46 breast masses from 46 female patients (20 benign, 26 malignant)	human	different breast masses	To evaluate its feasibility and value for the differentiation of breast masses	Breast lesions	Ultrasound guided core needle biopsy or surgical resection	ULM provides supplementary morphological and functional microvascular information for breast lesions, and MVD serves as a reliable biomarker for differentiating breast malignancy	Human feasibility
Li 2025	Prospective human clinical cohort study	31 breast lesions (14 benign,17 malignant), confirmed by pathology	human	Human breast lesions	Evaluate qualitative and quantitative ULM parameters for differentiating benign and malignant breast lesions; assess intra and inter operator reproducibility of ULM quantitative indices	Benign and malignant breast lesions	Post biopsy surgical histopathology for global lesion diagnosis	Qualitative vascular morphology patterns and quantitative ULM metrics show high diagnostic performance to discriminate benign and malignant breast lesions; ULM-derived quantitative parameters exhibit excellent intra and inter operator reproducibility, supporting its diagnostic potential for breast lesion evaluation	clinical cohort
Zhang 2022	Prospective human feasibility pilot clinical study	24 thyroid nodules from 24 patients (12 benign, 12 malignant)	human	different thyroid nodules	Pilot evaluation of ULM derived vascular metrics for distinguishing benign and malignant thyroid nodules	Thyroid lesions	FNA or surgical resection histopathology for global nodule diagnosis	Benign thyroid nodules exhibit significantly higher MVD and MFR values than malignant thyroid lesions on ULM	Human feasibility
Zhang 2025	Prospective human clinical cohort study	135 cervical LNs from 135 patients (61 reactive hyperplastic LNs, 74 metastatic LNs)	human	metastatic and reactive lymph nodes	To evaluate ULM for discriminating reactive hyperplastic versus metastatic cervical lymph nodes	Lymph nodes	Ultrasound guided core needle biopsy histopathology	integrating ULM with routine ultrasound improves diagnostic accuracy for metastatic cervical lymph nodes	Human feasibility
Li 2024	Prospective human clinical cohort study	58 Patients with obstructive azoospermia (OA) and 36 non-obstructive azoospermia (NOA)	Human	Obstructive and non-obstructive azoospermia	To evaluate testicular microcirculation via ULM; to explore correlations between ULM haemodynamic metrics and spermatogenic function; construct combined diagnostic model for differentiating NOA and OA	Human testis of azoospermia patients	Clinical diagnosis of OA/NOA; no pixel wise spatial registration between ULM vascular indices and testicular immunohistochemistry	ULM enables non-invasive detailed assessment of human testicular microvascular haemodynamics. ULM-quantified vascular parameters reflect links between testicular microcirculation and spermatogenic function. Combining serum FSH with ULM derived mean vascular diameter greatly improves diagnostic performance for discriminating NOA from OA	clinical cohort
Papanikolaou 2026	Prospective case control study	Human cohorts: 10 healthy men, 9 HH patients; 11 HH patients receiving testosterone, 9 gonadotrophins, 12 untreated; 7 HH paients for 12 month fertility response follow-up; 5 rodent pubertal blockade model	human	HH and male infertility related testicular disorders	To assess ULM as a treatment responsive biomarker for testicular function in hypogonadotrophic HH; to evaluate its capability for early detection of therapeutic response	Human testis and rodent pubertal blockade testis patients	Clinical endocrinological laboratory markers	ULM yields biologically meaningful, treatment responsive vascular biomarkers for testicular function. It enables patient stratification and earlier identification of therapeutic response in HH compared with conventional clinical markers, with supporting pre-clinical evidence from rodent pubertal blockade experiments.	clinical cohort
Li 2026	Prospective observational human clinical study	20 traumatic brain injury patients after decompressive craniectomy	human	Human brain of traumatic brain injury patients after decompressive craniectomy	To evaluate bedside ULM cerebral microvascular imaging performance using a multihypothesis multiframe global optimization algorithm; investigate post-injury cerebral microvascular remodelling and prognostic value of ULM composite metrics	Traumatic brain injury following decompressive craniectomy	Invasive intracranial pressure(ICP) monitoring and 6 month clinical functional outcome assessment	A multihypothesis multiframe global optimization algorithm enables bedside cerebral ULM on clinical grade systems without strict ultrafast sequence prerequisites. Bedside ULM can depict post traumatic cerebral microvascular remodelling, correlate with invasive ICP, and stratify long term clinical outcomes for neurocritical care patients

Table 2

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