Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples

Area of Science:

• Biomedical Optics
• Microscopy and Spectroscopic Imaging
• Pathological Assessment

Background:

• Label-free optical techniques offer promising alternatives for biological tissue analysis.
• Current single optical modalities cannot provide the comprehensive morphological and biochemical data obtained through histology and immunohistochemistry.
• Integrated multimodal imaging aims to combine morphological and functional-chemical information in a label-free manner.

Purpose of the Study:

• To develop and demonstrate a custom laser-scanning microscopic platform integrating multiple label-free optical modalities.
• To achieve high-resolution morphological imaging alongside specific molecular and functional information.
• To validate the platform's utility in analyzing complex biological specimens.

Main Methods:

• Development of a custom laser-scanning microscopy platform.
• Integration of confocal Raman spectroscopy with multimodal non-linear imaging techniques: Coherent Anti-Stokes Raman Scattering (CARS), Second-Harmonic Generation (SHG), Two-Photon Excited Fluorescence (TPEF), and Fluorescence Lifetime Imaging Microscopy (FLIM).
• Application of the platform for ex vivo analysis of urothelial carcinoma and atherosclerosis tissues.

Main Results:

• The platform successfully acquired high-resolution morphological images of biological specimens.
• It provided specific information on molecular organization, functional behavior, and molecular fingerprints.
• Multimodal characterization of ex vivo tissues affected by urothelial carcinoma and atherosclerosis was achieved.

Conclusions:

• The developed multimodal imaging platform demonstrates a proof-of-principle for comprehensive label-free tissue analysis.
• This integrated approach offers a powerful tool for biological and biomedical research.
• The system has the potential to complement traditional histological and immunohistochemical methods.