Eggert Dennis, Gaertner David, Rühm Adrian, Sroka Ronald, Arens Christoph, Davaris Nikolaos, Birkmeier Konrad, Brodschelm Andreas, Leisching Patrick, Studier Hauke, Becker Wolfgang, König Karsten, Betz Christan S
Clinic and Polyclinic for Otolaryngology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Department of Urology, Laser-Forschungslabor, LIFE Center, University Hospital, Planegg, Germany.
Lasers Surg Med. 2025 Feb;57(2):147-153. doi: 10.1002/lsm.23870. Epub 2024 Dec 16.
With over 184,000 new cases and more than 99,000 deaths per year, malignancies of the larynx are a global health problem. Currently, a dedicated screening method enabling a direct onsite diagnosis is missing. This can lead to delayed diagnosis and worse outcomes of the patients. An endoscopic optical method enabling a direct distinction between healthy tissue, dysplastic tissue and cancerous tissue would be an ideal tool for the detection of tumors of the upper aerodigestive tract (UADT). Healthy and tumor cells differ significantly in their metabolic state due to the different metabolic pathways they use (more oxidative phosphorylation in healthy cells, more glycolysis in tumor cells). Optical metabolic imaging (OMI) measuring relative intracellular concentration of NAD(P)H and FAD redox pairs could be a promising approach for early tumor detection and differentiation of suspicious mucosal lesions.
In this study, a specially designed endoscopic two-beam two-photon fluorescence lifetime imaging (FLIM) system was used to perform two-photon two-beam FLIM of NAD(P)H and FAD to image the metabolic state in different tissue samples of the UADT. FLIM data sets of 27 tissue samples from 16 patients were recorded directly after surgery ex vivo in a special tissue culture medium at 37°C on a dedicated microscope using multiphoton excitation.
Based on the FLIM measurements of NAD(P)H and FAD, six of the most common indices for the characterization of the cells' metabolism were calculated. Three of them, the ratio of the exponential coefficients (amplitudes) of the short and long lifetime components both for NAD(P)H and FAD (NAD(P)H a1/a2 ratio and FAD a1/a2 ratio) and the fluorescence lifetime redox ratio (FLIRR) enabled differentiation between healthy tissue, benign lesions, dysplastic tissue, and cancer tissue with statistical significance.
We showed by measurements on freshly collected tissue samples that mucosal lesions of the UADT can be differentiated using our newly designed endoscopic FLIM device. In vivo measurements in healthy volunteers were also possible. By means of this technology, differentiation of cancerous, pre-cancerous, and healthy tissue in the UADT by OMI could be possible. Of six indices used to characterize cell metabolism we calculated, the FLIRR showed the most significant differences between tissue types.
喉恶性肿瘤每年有超过18.4万新发病例和超过9.9万例死亡，是一个全球性的健康问题。目前，缺少一种能实现直接现场诊断的专门筛查方法。这可能导致诊断延迟和患者预后变差。一种能够直接区分健康组织、发育异常组织和癌组织的内镜光学方法将是检测上消化道呼吸道肿瘤（UADT）的理想工具。由于健康细胞和肿瘤细胞所使用的代谢途径不同（健康细胞中氧化磷酸化更多，肿瘤细胞中糖酵解更多），它们的代谢状态存在显著差异。测量NAD(P)H和FAD氧化还原对的相对细胞内浓度的光学代谢成像（OMI）可能是早期肿瘤检测和可疑黏膜病变鉴别诊断的一种有前景的方法。
在本研究中，使用专门设计的内镜双光束双光子荧光寿命成像（FLIM）系统对NAD(P)H和FAD进行双光子双光束FLIM，以成像UADT不同组织样本中的代谢状态。在手术后，将来自16名患者的27个组织样本立即在37°C的特殊组织培养基中离体，在专用显微镜上使用多光子激发记录FLIM数据集。
基于对NAD(P)H和FAD的FLIM测量，计算了用于表征细胞代谢的六个最常见指标。其中三个指标，即NAD(P)H和FAD的短寿命和长寿命成分的指数系数（幅度）之比（NAD(P)H a1/a2比值和FAD a1/a2比值）以及荧光寿命氧化还原比（FLIRR）能够在健康组织、良性病变、发育异常组织和癌组织之间进行有统计学意义的区分。
通过对新鲜采集的组织样本进行测量，我们表明使用新设计的内镜FLIM设备可以区分UADT的黏膜病变。在健康志愿者中进行体内测量也是可行的。借助这项技术，通过OMI区分UADT中的癌组织、癌前组织和健康组织可能成为现实。在我们计算的用于表征细胞代谢的六个指标中，FLIRR在不同组织类型之间显示出最显著的差异。
