Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy.
Department of Physics and Astronomy "G. Galilei", University of Padua, 35131 Padua, Italy.
Int J Mol Sci. 2021 Mar 6;22(5):2657. doi: 10.3390/ijms22052657.
Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.
多光子显微镜技术在生物医学研究领域的应用已经有 30 多年的历史了。最近，该技术的应用领域不断扩展，从基础研究到临床实践，涉及多个方面。此外，这种技术还具有无需染色和专用系统即可进行无标记多光子成像的优势。本文综述了无标记技术的最新进展；然后，我们重点介绍双光子自发荧光以及二次和三次谐波产生，并描述了它们的物理和技术特性。我们总结了该技术在许多生物医学研究领域和样本中的成功应用，突出了该技术的多功能性。本文还专门讨论了样本制备，这是每个显微镜实验中的关键步骤。接下来，我们对主要的定量方法进行了详细的综述分析，这些方法使用二次谐波产生从采集的图像中提取重要信息和参数。最后，我们讨论了无标记多光子显微镜技术的优势、限制和未来展望。
