Label-free Nonlinear Optical Microscopy for Biomedical Research
Nirmal Mazumder
Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104
Email ID- nirmal.mazumder@manipal.edu
Abstract
Non-linear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. Here, we discuss NLO microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples within the context of label-free non-invasive molecular imaging. A compact multimodal NLO microscope based on a laser scanning microscope, a femtosecond laser, a time-correlated single-photon counting system, and a photonic crystal fiber are introduced for biomedical applications. By integrating TPF, FLIM, SHG, and CARS microscopy, the proposed scheme provides profound insights into the physicochemical properties related to 3D molecular orientation distribution, inter- and intra-molecular interactions, and disease progression in biological systems and organs. The non-linear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-sensitive NLO microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-sensitive SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The high peak power and the low average intensity of near-infrared laser pulses allow for deep-penetration imaging without compromising sample vitality. Linking nonlinear optical phenomena with time/spectral/polarization-resolved imaging also makes it possible to obtain multidimensional information to address complex biomedical questions.
Link to file with lecture
https://disk.yandex.ru/i/upZdXOOYHBrYiA
Speaker
Nirmal Mazumder
Manipal Academy of Higher Education, India
India
Report
File with report
Discussion
Valery Tuchin
Dear Nirmal, excellent talk.
May I ask you to clarify your research on monitoring wound healing. You have demonstrated that this is possible, but what is the achievements and limitations of SHG imaging. What you will need to further improve technology.
Thank you.
Valery
Nirmal Mazumder
Dear Prof. Valery,
Thank you very much for your comment.
We can monitor the wound healing progression using SHG microscopy. However
signal to noise ratio is the main limitation in this case of in vivo imaging. I feel that
using adaptive optics, we can improve the imaging depth and get better SHG
signal. Also polarization resolved SHG can be added in it.
best regards,
Nirmal
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