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Background and aims  Sampling size variability of liver biopsy remains a major limitation in the assessment of liver fibrosis. We aimed to evaluate the diagnostic value of a fully quantitative method (second harmonic generation/two-photon excitation fluorescence, SHG/TPEF based) in “short” liver biopsy samples.    Methods  Liver biopsy samples from chronic hepatitis B (CHB) patients were constructed into “virtual” biopsies with different lengths. The original and “virtual” samples were measured by SHG/TPEF-based technology to obtain qFibrosis score, respectively. Here, ΔqFibrosis was defined as difference of qFibrosis between original biopsy and “virtual” biopsy. Equivalence test was used to compare ΔqFibrosis with the clinically acceptable error (deviation of 0.50) in each group.    Results  In real-world practice, qFibrosis score increased significantly with fibrosis progression in ≥ 1.5-cm-, 1.0–1.5-cm-, and 0.5–1.0-cm-long specimens ( p  < 0.05), compared with ≤ 0.5-cm-long specimens ( p  > 0.05). In virtual biopsy samples with specified length, the equivalence was confirmed in 0.5–1.0-cm- and 1.0–1.5-cm-long specimens (0.27 vs. 0.22,  p  < 0.001), whereas not in ≤ 0.5-cm-long specimens (0.53,  p  > 0.05). The number of cross-linked collagen fibers, the total and aggregated collagen proportionate area, and the collagen strings in number, length, width and perimeter showed excellent consistency with original biopsy samples in 0.5–1.0-cm- and 1.0–1.5-cm-long specimens (ICC > 0.90).    Conclusions  The use of SHG/TPEF-based image technology may give useful suggestive information in evaluation of CHB-related liver fibrosis for the short sample (biopsy length > 0.5 cm).    Electronic supplementary material  The online version of this article (10.1007/s12072-019-09955-2) contains supplementary material, which is available to authorized users.

SHG/TPEF-based image technology improves liver fibrosis assessment of minimally sized needle biopsies