Enhanced Multiplexity And Image Scale In Optical Tissue Clearing-Based 3D Immunofluorescence Microscopy

In recent times, cancer has come to be recognized as a multifaceted, three-dimensional ecosystem encompassed by a supportive microenvironment (TME) comprising diverse components such as stromal and immune cells, vasculature, and extracellular matrix (ECM). To comprehensively study such a complex system, a multiplexed technique becomes imperative. Conventional assays often lack critical spatial information as they dissociate the tissue for analysis. Traditional immunofluorescence techniques and microscopy retain X-Y spatial information, offering multiplexing or super-resolution, however, only within the confines of a 2D framework. Advancement in three-dimensional (3D) optical microscopy techniques and tissue clearing methods allowed tumor tissue to be interrogated and studied at varying levels of sample size and spatial resolution in 3D. High-dimensional information can be extracted in both pharmacokinetic and pharmacodynamic contexts, like 3D vasculature structure, drug disposition or immune cell infiltration for biological study or pathological diagnosis in clinic. However, limitation still exists for the current 3D microscopy in aspects of multiplexity and spatial image scale. Due to the limits in available fluorophores for simultaneous detections, only 3 to 4 biomarkers can be detected in a single tissue. The trade-off of a single microscopy in between resolution and compatible sample size (macrosection or whole tissue) limits a holistic analysis to the sample in disparate resolution. Here, we first present a novel method aimed at enhancing the multiplexity of current 3D microscopy. Our approach involves the use of an LED-photobleaching device, which effectively eliminates the existing fluorescence of an immuno-stained tissue macrosction. This process enables the subsequent restaining of the tissue with another round of fluorescent-labeled antibodies, thereby expanding the range of biomarkers that can be simultaneously visualized within the sample. Secondly, we implemented a correlation between LSFM and confocal microscopy to extend the image scale of a sample. Initially, whole tissue images are acquired using LSFM to identify regions of interest (ROI). These ROIs can then be separated out and subjected to immunofluorescence staining for cellular resolution imaging by confocal microscopy. This method facilitates accurate pinpointing of ROIs exhibiting pathological distortions or abnormal morphology and imaging them at both macroscopic and microscopic scales for analysis at both the whole tissue and cellular levels, offering detailed insights into the overall sample morphology as well as specific cellular information such as immune cell compositions or vasculature networks. Our novel microscopy technique offers extensive spatial data on tumor tissues, contributing significantly to various aspects of cancer research, including the identification of new tumor biomarkers, investigation of novel biological mechanisms, pathological examination, and the advancement of effective cancer therapies.