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Updated: Nov 17, 2025

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

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Published on: August 29, 2025

Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond.

Stephen A Boppart¹, Sixian You¹, Lianhuang Li²

¹Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

APL Photonics

|February 15, 2021

Summary

New laser technology enables simultaneous, real-time imaging of multiple molecular contrasts in nonlinear optical microscopy. This breakthrough avoids sample labeling and motion artifacts for advanced in vivo molecular imaging.

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Area of Science:

Biomedical Optics
Microscopy
Molecular Imaging

Background:

Nonlinear optical microscopy offers subcellular resolution and depth using endogenous contrasts.
Previous methods required sequential imaging or complex algorithms.
Limitations included sample pretreatment and motion artifacts.

Purpose of the Study:

To develop a method for simultaneous, real-time acquisition of multiple endogenous molecular contrasts.
To overcome limitations of existing nonlinear optical imaging techniques.

Main Methods:

Engineered a novel laser source for single-beam, single-shot excitation.
Implemented simultaneous multicontrast epidirected signal detection.
Utilized endogenous molecular signals for contrast generation.

Main Results:

Achieved real-time simultaneous acquisition of multiple endogenous contrasts.
Enabled segmentation of diverse cellular and extracellular components.
Avoided perturbative sample pretreatments like labeling and sectioning.

Conclusions:

The developed platform significantly advances in vivo nonlinear optical molecular imaging.
It offers a label-free, artifact-free approach for studying biological and pathological states.
This technology facilitates deeper understanding of cellular and tissue dynamics.