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Imaging Biological Samples with Optical Microscopy

Imaging Biological Samples with Optical Microscopy

Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...

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Updated: Aug 24, 2025

Automation of Mode Locking in a Nonlinear Polarization Rotation Fiber Laser through Output Polarization Measurements

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Published on: February 28, 2016

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Matthew G Clark¹, Gil A Gonzalez¹, Chi Zhang^1,2,3

¹Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana47907, United States.

Analytical Chemistry

|October 25, 2022

Summary

A new pulse-picking strategy enhances nonlinear optical microscopy signals, enabling multimodal imaging with coherent Raman scattering (CRS) and multiphoton excitation fluorescence (MPEF) or second harmonic generation (SHG). This method also assesses laser-induced phototoxicity for optimized imaging.

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Last Updated: Aug 24, 2025

Automation of Mode Locking in a Nonlinear Polarization Rotation Fiber Laser through Output Polarization Measurements

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Published on: February 28, 2016

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

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Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Published on: July 25, 2022

Area of Science:

Biophotonics
Chemical Imaging
Microscopy

Background:

Nonlinear optical microscopy offers label-free chemical mapping of biological samples.
Common techniques include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS).
Integrating CRS with MPEF/SHG is challenging due to differing laser requirements (femtosecond for MPEF/SHG, picosecond for CRS).

Purpose of the Study:

To develop a method for integrating coherent anti-Stokes Raman scattering (CARS) microscopy with MPEF and SHG for multimodal chemical imaging.
To enhance nonlinear optical signals and enable hyperspectral CARS microscopy at low laser average power.
To evaluate and compare laser-induced phototoxicity for optimizing imaging parameters.

Main Methods:

A pulse-picking strategy using an acousto-optic modulator was developed to program laser pulse train duty cycles.
This strategy increases pulse peak power at low input average power, enhancing nonlinear optical signals.
Cellular dynamics and morphological changes were used to assess phototoxicity.

Main Results:

The pulse-picking strategy significantly enhanced nonlinear optical signals.
Hyperspectral CARS microscopy was made compatible with MPEF and SHG for multimodal imaging at low laser average power.
The method allowed for the evaluation of phototoxicity, enabling the selection of optimal laser power for sensitivity and minimal sample damage.

Conclusions:

The developed pulse-picking strategy overcomes the challenge of integrating CRS with MPEF/SHG for multimodal chemical imaging.
This approach enables sensitive, label-free chemical imaging with reduced phototoxicity in biological samples.
The method provides a valuable tool for optimizing laser parameters in nonlinear optical microscopy.