Pathological tissue examination is of key importance in diagnosing diseases, for example for cancer diagnosis and assessment of complete surgical tumor resections. To date, extracted tissue samples are send to the pathology department and no direct on-site histological feedback on freshly excised tissue is available. Conventional histopathological assessment techniques require tissue processing, which is labor-intensive and time-consuming, taking at least 24 hours. An available technique to provide intraoperative histological feedback is frozen section analysis, however, this requires freezing and staining of the tissue which comes with freezing artefacts and still stakes 15–25 minutes.A promising technique is higher harmonic generation (HHG) microscopy, a laser-based imaging technique that acquires real-time images with a subcellular resolution of fresh unprocessed tissue. HHG microscopy includes third harmonic generation (THG) revealing all tissue interfaces showing cellular and cell nuclear morphology; second harmonic generation (SHG) mainly showing collagen fibers; and multiphoton excited autofluorescence (MPEF) visualizing autofluorescent molecules in cytoplasm as well as elastin fibers. In this thesis we validated a mobile HHG microscope in the clinical for instant on-site histological feedback on freshly excised human tissue, with as main focus lung tissue and diagnosis of lung cancer and interstitial lung disease (ILD). This thesis provides a HHG imaging atlas of both healthy and diseased human lung tissues, by comparing HHG images with corresponding standard histology images for diverse types of tissue samples. We showed that using HHG microscopy the tissue architecture could be revealed. In particular, the extracellular matrix components collagen and elastin could be distinguished by SHG and MPEF, respectively, and various types of cells were identified by THG microscopy. Specifically, the four main types of leukocytes (neutrophils, eosinophils, lymphocytes, macrophages) could be distinguished, and we showed that by using a deep learning network the percentages of different leukocyte types could be estimated. Based on the HHG images, pathologists were able to identify important histological characteristics. For cancer diagnosis this included increased cellularity, cellular pleomorphism, abnormal cell growth patterns, disrupted extracellular matrix, and tumor cell invasion. In ILD specimens, tissue architectural changes were identified, including different stages of fibrosis and inflammation, as well as specific histological ILD hallmarks such as honeycombing, masson bodies and granulomas. Fibrosis and inflammation were also shown to be present in HHG images of lung tissue affected by coronavirus disease (COVID-19).We reported for the first time on the feasibility of a HHG microscope in the clinic in terms of acquisition time, image quality and diagnostic accuracy. The HHG microscope acquires real-time images and is able to provide a first overview image of a biopsy in less than 7 minutes on average. The HHG image quality was according to two pulmonary pathologists sufficient for a malignancy versus non-malignancy diagnosis in 97% of the biopsies. The malignancy versus non-malignancy diagnosis based on HHG images was correct in on average 87%. This agreement is substantial, especially for a first study on assessing HHG images of bronchoscopy biopsies.In this thesis we brought HHG microscopy a step closer towards clinical use. Compared to conventional histology, HHG microscopy is fast, instant digitally available, it can acquire 3D images without the need for tissue slicing, and it enables identification and quantification of various specific histological structures without the need for tissue staining. In conclusion, HHG microscopy is a powerful technique to provide instant on-site histological feedback on fresh unprocessed tissue in the clinic, with a great potential to provide better disease understanding, as well as to improve and speed up the diagnostic workup, potentially resulting in increased diagnostic yield, reduced need for repeated or additional procedures and reduced associated costs and adverse effects for the patient
