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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

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High dynamic range structured illumination microscopy based on multi-channel fusion and dual-phase binary fringe.

Yuzhu Zhao, Xiaojie Zhang, Xiaowei Peng

|July 2, 2026

Summary

This study introduces a novel high dynamic range structured illumination microscopy (SIM) method using multi-channel fusion and dual-phase binary fringe (MFDBF-SIM) to overcome limitations in 3D measurements. MFDBF-SIM significantly enhances reconstruction integrity on challenging surfaces.

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Area of Science:

Optics
Microscopy
Image Reconstruction

Background:

Binary fringe projection is efficient for 3D structured illumination microscopy (SIM).
Limitations include fringe edge blurring and exposure issues on reflective surfaces, causing measurement failures.
Existing methods struggle with high dynamic range reconstruction.

Purpose of the Study:

To develop a high dynamic range SIM (MFDBF-SIM) overcoming limitations of binary fringe projection.
To improve 3D reconstruction integrity and accuracy, especially on challenging reflective surfaces.
To address issues like fringe edge blurring and localized over/underexposure.

Main Methods:

Multi-channel fusion and dual-phase binary fringe (MFDBF-SIM) approach.
Phase shifting between adjacent binary fringes to distribute zero-modulation regions.
Adaptive selection of modulation curves using RGB channel intensity responses.
Development of horizontal and scanning-step calibration models for error compensation.

Main Results:

MFDBF-SIM effectively distributes zero-modulation regions, preventing local information loss.
High dynamic range reconstruction is achieved by adaptively selecting modulation curves.
Horizontal and axial focal-plane errors are compensated.
Reconstruction integrity improved by 121.5% (green channel) and 224.6% (blue channel) on silicon wafers and PCBs.

Conclusions:

MFDBF-SIM significantly enhances 3D reconstruction integrity and accuracy.
The method overcomes key limitations of binary fringe projection SIM on reflective surfaces.
MFDBF-SIM offers a robust solution for high dynamic range 3D measurements in microscopy.