American Journal of Respiratory and Critical Care Medicine

A 61-year-old man without a relevant past medical history presented with cough and dyspnea and tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by PCR testing. Twelve days after disease onset, his condition deteriorated clinically, with respiratory failure requiring invasive mechanical ventilation. Chest computed tomography showed bilateral pulmonary embolism, diffuse ground-glass opacities, consolidations, and subpleural reticulation (Figure 1A). Infection other than COVID-19 was excluded with BAL. After 16 days of mechanical ventilation, he died of COVID-19 acute respiratory distress syndrome.

Twenty hours after the time of death, an autopsy was performed. Resected unprepared lung tissue (1 × 2 cm2) from the right upper lobe (Figure 1B), excised perpendicular to the bronchial tree, was immediately placed on a mobile higher harmonic generation microscope (HHGM) (Figure 1C). HHGM is a laser-based imaging technique that provides on-site images of unprocessed samples as large as 2 cm. Two-dimensional images and three-dimensional depth scans (up to 100 μm) can be acquired. HHGM images consist of three different signals visualizing cellular structures (green), collagen (red), and elastin fibers (blue). Earlier studies demonstrated the visualization of histological hallmarks of cancer in HHGM images (15).

Here, HHGM single images (0.5 × 0.5 mm2) were acquired within 1–3 seconds, and “overview” images (2.5 × 2.5 mm2) within 40 seconds to 2.5 minutes, depending on the image quality. HHGM images could be interpreted immediately and showed lung architecture including alveolar septa, alveolar spaces, interlobular septa, blood vessels, and fibrin networks; cellular structures including pneumocytes, lymphocytes, and macrophages; and squamous metaplasia, in agreement with standard histology (Figure 2). Pathologists were able to identify COVID-19 characteristics, including inflammation with septal leukocyte infiltration, alveolar architecture distortion, and fibrosis with alveolar septa thickening due to an increased amount of collagen.

This report shows that HHGM is able to rapidly visualize disease characteristics in fresh, unprepared lung tissue on site. Hence, HHGM has the potential to improve rapid diagnosis and understanding of lung diseases.

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This publication is part of the project Instant Pathology (with project number 15825) of the research programme Open Technology, by the domain which is (partly) financed by the Dutch Research Council (NWO- Applied and Engineering Sciences) and Horizon 2020 Framework Programme grants 654148 and 871124.

Originally Published in Press as DOI: 10.1164/rccm.202207-1259IM on March 8, 2023

Author disclosures are available with the text of this article at


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American Journal of Respiratory and Critical Care Medicine

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