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Updated: Dec 21, 2025

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Published on: August 29, 2025

Multi-photon attenuation-compensated light-sheet fluorescence microscopy.

Madhu Veettikazhy¹, Jonathan Nylk^2,3, Federico Gasparoli²

¹DTU Health Tech, Technical University of Denmark, Frederiksborgvej 399, 4000, Roskilde, Denmark. madve@dtu.dk.

Scientific Reports

|May 17, 2020

Summary

This summary is machine-generated.

This study demonstrates how attenuation-compensation can enhance imaging depth in two-photon light-sheet fluorescence microscopy. A simple graded neutral density filter effectively increases penetration, making advanced imaging accessible.

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Area of Science:

Biophotonics
Microscopy
Optical Imaging

Background:

Optical field attenuation from scattering and absorption limits imaging depth.
Aberration correction is challenging over large fields of view in heterogeneous tissues.
Propagation-invariant light fields offer potential for increased imaging depth.

Purpose of the Study:

To implement and validate attenuation-compensation in two-photon light-sheet fluorescence microscopy.
To demonstrate a facile method for achieving attenuation-compensation without complex apparatus.
To benchmark the proposed method against a gold standard system.

Main Methods:

Utilized a graded neutral density filter for attenuation-compensation.
Implemented the approach in a two-photon light-sheet fluorescence microscope.
Benchmarked against a spatial light modulator-based beam shaping system.

Main Results:

Successfully implemented attenuation-compensation in two-photon light-sheet microscopy.
Demonstrated facile implementation using a graded neutral density filter.
Achieved comparable performance to complex beam shaping systems.

Conclusions:

Attenuation-compensation is effectively implemented in two-photon light-sheet imaging.
A simple graded neutral density filter provides a practical solution for enhanced depth penetration.
This approach democratizes advanced light-sheet imaging capabilities for broader applications.