Ahola, A. | Search bioRxiv

bioRxiv2026

Quantitative 3D imaging of intact engineered heart tissues using inverted selective plane illumination microscopy

Abstract Background Advanced in vitro models of the human heart have become promising tools for studying cardiac diseases and drug discovery. Three-dimensional (3D) engineered heart tissues (EHTs) provide more adult cardiac tissue-like structural organization compared to traditional two-dimensional (2D) in vitro models with straightforward contractile readouts. However, the assessment of the architecture of EHTs, especially the quantification of cellular and tissue structural components, has remained poorly studied. Here we demonstrate the use of light-sheet microscopy for structural imaging of intact EHTs and provide quantification of the EHT cellular and subcellular structures. Methods We developed an in-house inverted selective plane illumination microscopy (iSPIM) platform and combined it with iterative indirect immunofluorescence (IT-IF) staining for better signal acquisition. We developed an image-processing and analysis pipeline dedicated to volumetric EHT analysis. Results With this integrated platform, we achieved volumetric imaging of intact EHTs and quantification of 3D tissue architecture, revealing aligned cardiomyocytes within the imaged volume and increasing cardiac nuclei content towards the core of the tissue. The developed methods allowed the detection of heterogeneities between tissue sections, demonstrating the sensitivity required for future disease modeling or maturation studies. Conclusions The developed integrated platform opens new possibilities for volumetric analysis and characterization of 3D tissue structure using iSPIM, providing new insights into EHT architecture and the use of EHTs as disease modeling tools.

bioengineering↗

bioRxiv2026

Defining ecologically realistic biodiversity offset multipliers with the Response-based Habitat Hectare Assessment of Biodiversity Gains (REHAB)

In biodiversity offsetting, balancing biodiversity losses with gains can be achieved by using multipliers that define the ratio between the magnitude of biodiversity loss and the area required to deliver equivalent biodiversity gains. Although there is broad scientific consensus that multipliers should be calibrated to deliver no net loss or a net gain for biodiversity, they are often applied without quantitative assessment of the ecological outcomes of offset actions. Here we operationalise the Response-based Habitat Hectare Assessment of Biodiversity Gains (REHAB), a framework where multipliers are informed by an understanding of habitat-specific ecological responses to offset action. To support Finlands national biodiversity offsetting scheme, we harnessed the knowledge of 111 experts to compile ecological attributes and condition matrices for all 388 Finnish habitat types and derive 346 offset action multipliers that represent ecological response functions for 216 habitat type-specific offset actions including restoration, management and passive recovery. Our analysis reveals substantial variation in response-functions, resulting in offset multipliers between 1.3-4,000 across offset actions and habitat types. We find that the fixed multipliers commonly used in offset schemes would result in net loss in 60% of the cases if action- habitat specific responses were not considered. This variability underscores that fixed multipliers cannot deliver reliable biodiversity outcomes and should be avoided in offsetting schemes. The REHAB framework has already been integrated into Finlands national offsetting policy. Other potential areas of application include informing ecosystem restoration planning and assessing biodiversity gains linked to credit issuance in emerging nature-credit markets.

ecology↗