Label-Free Multiphoton Microscopy: Much More Than Fancy Images

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.

Automated summary

Multiphoton microscopy has been actively used in biomedical research for the past 30 years, offering label-free imaging and a range of applications. This technique, including two-photon autofluorescence and harmonic generation, provides versatile and detailed sample analysis for various research fields. The future of label-free multiphoton microscopy holds promise for further advancements in the study of biological samples.