500µm Slices of Live Right Ventricular Outflow Tract cut for SHGI-based fibrosis quantification

Abstract

(from Quantitative collagen assessment in right ventricular myectomies from patients with tetralogy of Fallot, Wülfers et al, 2021)

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Aims Patients with tetralogy of Fallot (TOF) are often affected by right ventricular fibrosis, which has been associated with arrhythmias. This study aimed to assess fibrosis distribution in right ventricular outflow tract (RVOT) myocardium of TOF patients to evaluate the utility of single histology-section analyses, and to explore the possibility of fibrosis quantification in unlabelled tissue by second harmonic generation imaging (SHGI) as an alternative to conventional histology-based assays.

Methods and results We quantified fibrosis in 11 TOF RVOT samples, using a tailor-made automated image analysis method on Picrosirius red-stained sections. In a subset of samples, histology- and SHGI-based fibrosis quantification approaches were compared. Fibrosis distribution was highly heterogeneous, with significant and comparable variability between and within samples. We found that, on average, 67.8 mm2 of 10 µm thick, histologically processed tissue per patient had to be analysed for accurate fibrosis quantification. SHGI provided data faster and on live tissue, additionally enabling quantification of collagen anisotropy.

Conclusion Given the high intra-individual heterogeneity, fibrosis quantification should not be conducted on single sections of TOF RVOT myectomies. We provide an analysis algorithm for fibrosis quantification in histological images, which enables the required extended volume analyses in these patients.

Method

Second harmonic generation imaging

Live RVOT tissue samples from five patients were cut into 500 μm thick tissue slices with a vibratome (7000smz, Campden Instruments, Loughborough, UK) and mounted on glass slides in phosphate buffered saline. Three randomly chosen regions were immediately imaged with SHGI for each tissue slice. SHGI was performed using an upright multiphoton microscope (TCS SP8 DIVE, Leica Microsystems, Wetzlar, Germany). The SHGI signal was generated using 920 nm excitation light from a pulsed laser (InSight X3 Dual, Spectra-Physics, Santa Clara, USA) and detected with a hybrid detector using a 25× water-immersion objective (IRAPO L 25x/1.00 W, Leica Microsystems). Autofluorescence was also recorded using a second detection channel. 3D volumes were acquired with a size of 442.86 μm × 442.86 μm in x–y-direction, and 90–110 μm in z-direction (x–y–z voxel size: 0.099 μm × 0.099 μm × 0.51 μm). Excitation compensation was applied linearly along the z-axis to compensate for intensity loss at increasing tissue depths. After live tissue imaging, slices were fixed for 12 h in 4% paraformaldehyde. Tissue landmarks were used to identify regions that had been imaged previously in live tissue, and SHGI of those areas was repeated.