Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues.

Steven Y Leigh,Ye Chen,Jonathan T.C Liu

doi:10.1364/boe.5.001709

Abstract

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively 'labels' in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

Highlights

To enable noninvasive real-time pathology of fresh tissues [1,2,3], improving the depth of optical sectioning is of high priority and would provide a broad clinical impact
In the modulatedalignment dual-axis (MAD) approach, we take advantage of the fact that the dual-axis confocal (DAC) microscope signal is highly sensitive to the precise spatial alignment between the illumination and collection paths while the background signal is much less sensitive to this alignment
We have presented a new strategy to enable optical-sectioning microscopy at large depths that benefits from the inherent strengths of dual-axis confocal (DAC) microscopy

Summary

Introduction

To enable noninvasive real-time pathology (optical biopsy) of fresh tissues [1,2,3], improving the depth of optical sectioning is of high priority and would provide a broad clinical impact. The need for high spatial resolution (super-resolution microscopy [5,6,7,8]) in clinical diagnoses is less immediate since the “gold standard” of histopathology relies on the observation of tissue sections under low magnification (e.g., 2x 10x is typical for Mohs surgical pathology) [9] Rather, for applications such as early cancer detection, guided biopsy (e.g., in oral cancer), or surgical guidance (e.g., in dermatology), the ability to visualize large fields of view in three dimensions with cellular resolution is valuable, especially if there is sufficient depth to enable assessment of the local invasion of cancer.

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Conclusion