Abstract
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.
Highlights
Light microscopy is a gold standard technique in biomedical research and clinical diagnosis [1]
The huge technical developments toward more and more sophisticated apparatus [2,3,4] enhanced the broad use of light microscopy, which in turn increased the need for innovative microscopy approaches
Confocal laser scanning microscopy is routinely used in biomedical research because it ensures high resolution and high contrast compared to epifluorescence microscopes
Summary
Light microscopy is a gold standard technique in biomedical research and clinical diagnosis [1]. Thanks to the use of longer wavelengths (800–1200 nm), multiphoton microscopy reduces sample damage and provides deeper penetration (250–500 μm) into the specimen, with some loss of resolution. This technique has found success as a non-invasive imaging tool for thick biological tissues and living animals. For the analysis of structural components within tissues, label-free multiphoton (LFM) microscopy can be successfully used to retrieve information in a staining-free fashion This results in a great advantage in terms of sample preparation ease where the absence of immunofluorescence or fluorescent protein expression simplifies benchwork protocols. The longer wavelengths used in MPM face less scattering and absorption by biological
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