The core pathological feature of immune complex-mediated glomerular diseases is the abnormal deposition of electron-dense deposits (EDD) in different regions of the glomerulus, and their distribution pattern is a key marker for disease classification [3]. Electron microscopy can clearly distinguish deposition patterns in subepithelial, subendothelial, mesangial, and intramembranous locations, providing decisive evidence for disease diagnosis [4]. Acute post-streptococcal glomerulonephritis is characterized by pathognomonic subepithelial “hump-shaped” electron-dense deposits [5]. Active lupus nephritis often shows massive subendothelial electron-dense deposits, which may be accompanied by tubuloreticular inclusions, significant for assessing disease activity [6]. IgA nephropathy is diagnosed based on predominantly mesangial electron-dense deposits; electron microscopy can simultaneously assess the extent and degree of foot process effacement. Foot process effacement is clearly associated with the clinical prognosis of IgA nephropathy. Extensive foot process effacement (>50% of capillary loops) is independently associated with more severe proteinuria and faster decline in renal function [7]. Membranous nephropathy is characterized by typical subepithelial spike-like deposits; electron microscopy is an important supplement for distinguishing primary from secondary membranous lesions. By evaluating the ultrastructural distribution pattern of deposits and, in antigen-negative cases, assessing GBM structural changes and deposit morphology, electron microscopy can provide crucial clues for differential diagnosis [8], thereby avoiding the simplistic treatment of MN as a single disease and guiding the development of more targeted therapeutic strategies.

Hereditary nephropathies primarily involve structural abnormalities of the basement membrane or podocyte cytoskeleton. Light microscopy often lacks specific findings, making electron microscopy the only means to provide definitive diagnostic evidence. Alport syndrome is characterized by typical irregular thickening, lamellation, splitting, and basket-weave changes of the glomerular basement membrane [9][10], with basement membrane thickness significantly deviating from the normal range, allowing precise differentiation from the diffuse thinning of the basement membrane seen in thin basement membrane nephropathy (TBMD). Such diseases are difficult to diagnose solely by light microscopy and immunofluorescence; ultrastructural changes are the most important morphological evidence prior to genetic testing.

The causes of transplant kidney injury are complex and diverse, primarily including antibody-mediated rejection (ABMR), cellular rejection, recurrent glomerular disease, and drug toxicity. Recurrence of glomerulonephritis after transplantation is a significant clinical challenge leading to graft loss [11]. For example, the recurrence of focal segmental glomerulosclerosis (FSGS) is closely related to various circulating factors and molecular biomarkers. Membranous nephropathy (MN) can either recur or de novo occur after transplantation, and both conditions are often accompanied by a similar risk of antibody-mediated rejection. In such cases, electron microscopy (EM) can accurately determine the nature of the lesion by observing the degree of foot process effacement, the location of electron-dense deposits, and ultrastructural changes in the basement membrane [12]-[14], providing key evidence for adjusting clinical treatment strategies. Transplant glomerulopathy is primarily caused by chronic antibody-mediated rejection [15]. Electron microscopy can clearly reveal characteristic ultrastructural changes that are difficult to discern under light microscopy, such as double contours of the basement membrane, basement membrane duplication, and widening of the subendothelial space.

These technologies are directly applied to the ultrastructural analysis of *ex vivo* renal biopsy tissue, aiming to simplify sample preparation, shorten diagnostic turnaround time, or provide three-dimensional structural information unattainable by traditional TEM.

5.1.1. Low-Vacuum Scanning Electron Microscopy (LVSEM)

LVSEM allows direct observation of resin-embedded blocks under low-vacuum conditions [18], eliminating the need for ultrathin sectioning. This significantly simplifies sample preparation and markedly shortens diagnostic time, making it more suitable for rapid clinical diagnosis. Studies have shown that LVSEM can clearly visualize glomerular basement membrane duplication, lamellation, and mesangial changes. It demonstrates high sensitivity for detecting early ultrastructural changes associated with antibody-mediated rejection, offering the potential for rapid early warning of subclinical transplant injury [19] and showing good potential for clinical translation.

5.1.2. Structured Illumination Microscopy (SIM)

SIM, a super-resolution optical microscopy technique, overcomes the diffraction limit, enabling nanoscale structural observation. Its application in renal pathology has extended beyond single disease types. Liuet al. [20] applied dual-color fluorescence SIM to renal mass biopsies. By performing rapid, non-destructive optical sectioning imaging of fresh biopsy tissue while maintaining tissue integrity, they achieved high sensitivity and specificity in diagnosing renal tumors, providing a new clinical translation pathway for real-time pathological assessment of renal masses. Furthermore, SIM has demonstrated unique advantages in evaluating foot process effacement, measuring basement membrane thickness, and quantifying autophagosomes in non-neoplastic renal diseases [21][22], indicating its potential as a versatile analytical tool for renal ultrastructural pathology.

These technologies require minimal or no invasive procedures, allowing real-time observation of kidney structure and function *in vivo*, extending ultrastructural pathology from *ex vivo* diagnosis to *in vivo* functional assessment.

5.2.1. Multiphoton Microscopy (MPM)

MPM offers advantages such as deep tissue imaging, low phototoxicity, and the ability for long-term *in vivo* observation. It enables real-time visualization of tubular blood flow, organelle dynamics, immune cell migration, reactive oxygen species (ROS) generation, and mitochondrial functional changes in living kidneys. This provides novel tools for elucidating the pathophysiological processes of diabetic nephropathy, ischemia-reperfusion injury, hypertensive nephropathy, and renal inflammation [23]. Compared to traditional static electron microscopy, MPM truly achieves simultaneous assessment of structure and function, propelling renal pathology from “morphological diagnosis” towards “mechanistic analysis.”

5.2.2. Super-Resolution Ultrasound Localization Microscopy (sULM)

sULM overcomes the resolution limitations of traditional ultrasound, enabling non-invasive visualization of microvasculature. It can precisely quantify renal microvascular hemodynamics, representing a breakthrough technology for early non-invasive diagnosis of kidney diseases. In early diabetic kidney disease (DKD), glomerular microvascular injury is the core initiating factor. Traditional ultrasound cannot detect subtle blood flow abnormalities, whereas sULM can clearly visualize glomerular microvascular distribution, blood flow velocity, and perfusion, capturing the characteristics of early DKD microvascular lesions and providing a new avenue for non-invasive, early, and precise diagnosis [24][25]. Furthermore, sULM can directly visualize glomerular structures in both native and transplanted kidneys in humans without invasive procedures, holding broad application prospects for non-invasive monitoring of transplant kidneys and screening high-risk populations, extending ultrastructural pathology from invasive biopsy to non-invasive screening [26].

Virtual microscopy (VM) technology itself is not a novel microscopic imaging technique. Instead, it involves the whole-slide digital scanning of traditional pathology slides (including light microscopy, immunofluorescence, and electron microscopy slides) to generate high-resolution digital images that can be viewed, annotated, and shared on computers. The rationale for including it within the framework of ultrastructural pathology technological innovation is as follows: 1) **Technological Bridge: **VM serves as the bridge connecting ultrastructural pathology images with AI analysis, teleconsultation, and multi-center collaboration. Without the digital foundation provided by VM, AI models cannot obtain training data, and remote pathological evaluation cannot be realized. 2) **Process Restructuring: **VM transforms the workflow of ultrastructural pathology from a linear model of “sectioning → microscopy → reporting” to a parallel model of “sectioning → digitization → AI-assisted analysis → remote review → reporting,” significantly improving diagnostic efficiency and accessibility. 3) **Prerequisite for Standardization: **VM enables the storage, retrieval, and quantitative analysis of ultrastructural pathology images, serving as the foundational infrastructure for advancing ultrastructural pathology from empirical interpretation towards standardized, quantitative, and traceable diagnosis.

Therefore, although VM does not directly enhance imaging resolution or functional imaging capabilities, as the core supporting technology of digital pathology, it is an indispensable component of the ultrastructural pathology technology system. Through digital slides, experts can perform remote pathological evaluation and achieve rapid sharing of donor kidney biopsy results, breaking down geographical barriers and significantly optimizing the efficiency of donor kidney assessment for transplantation, multi-center pathological review, and consultation for difficult cases [27], driving the transformation of the renal pathology workflow towards digitization and networking.