Focal modulation microscopy for molecular imaging of thick biological tissue


Optical based microscopes for use in clinical applications such as non-invasive imaging of human skin, eyes and hollow organs; research in drug discovery such as 3D visualization of drug kinetics in vivo; longitudinal monitoring therapeutic processes or biomedical research applications such as imaging cancer angiogenesis in mouse models in vivo.


Patents granted in Japan and China


Partnership in commercial development


NUS Ref: Chen NG 02

Dr. Timothy Phua (

Industry Liaison Office, NUS Enterprise

Inventors: Assoc. Prof. Chen Nanguang, Prof Colin James Richard Sheppard, Mr. Wong Chee Howe


  • Imaging depth larger than conventional confocal microscopes.

Technology Overview

The imaging depth of confocal microscopy is up to 200 microns in biological tissues. To achieve a deeper penetration depth than confocal microscopy, the existing techniques include multi-photon microscopy (MPM) and optical coherence tomography (OCT). However, MPM requires the use of femto-second lasers, which are very expensive. MPM is also limited by available fluorophores and nonlinear photodamage. OCT is not compatible with fluorescence and cannot readily provide molecular information.

Technology Features

A novel microscopy method has been developed to achieve a deeper penetration depth than confocal microscopy. Equivalently selective excitation is achieved by modulating the light intensity at the focal point only. Fluorescence emission or backscattered light are collected and demodulated. Such a modulation and demodulation scheme significantly reduces the background signal caused by multiple scattering and effectively picks up the high resolution signal related to the ballistic excitation light. Consequently, the signal to background ratio and the spatial resolution can be maintained to a penetration depth comparable to optical coherence tomography (OCT), say, up to a few millimeters in biological tissues. Unlike OCT, the focal modulation microscopy is compatible with fluorescence and can readily provide molecular information.

Development Status

The proposed technology is lab scale proven and a prototype system has been developed (see Fig. 1).

Preliminary imaging experiments have been performed to verify the performance of such a prototype. Shown in Fig. 2 are autofluorescence images acquired from a plant leaf. The imaging depth is around 90 microns where autofluorescence from mesophyll cells is visible. The confocal image quality (Fig. 2(a)) is deteriorated by multiple-scattering light and appears blurred. Focal modulation microscopy image (Fig. 2(b)), however, shows much finer structures, well-defined boundaries, and significantly improved contrast. The image quality is similar or even better than that of two-photon microscopy (TPM) image of the same sample (Fig. 2 (c)).

Fig. 1. Schematic of a FMM system upgraded from an Olympus FV300 confocal microscope.

Fig. 2. (a) CM, (b) TPM, and (c) FMM images of mesophyll autofluorescence.

Fig. 3. (a) CM and (b) FMM images of chicken chondrocytes labeled with a lipid tracer.

Shown in Fig. 3 are animal tissue imaging results. A piece of chicken cartilage was prepared so that the chondrocytes inside the cartilage have their membrane fluorescence labeled with a lipid tracer. Fig. 3 (a) and (b) are CM and FMM images acquired simultaneously at an imaging depth of 500 microns. It is evident that FMM is capable of providing high-resolution information while CM image is seriously contaminated by background emissions.

Data is available for demonstration to interested parties.

About the Inventors

Assoc. Prof. Chen Nanguang, is currently an Associate Professor of Bioengineering at the National University of Singapore. He received his PhD in Biomedical Engineering in 2000 from Tsinghua University, MS in Physics in 1994 from Peking University, and BS in Electrical Engineering in 1988 from Hunan University. He joined the University of Connecticut in 2000 as a postdoctoral fellow and then became an Assistant Research Professor in 2002. He has been a faculty member with NUS since 2004. His research interests include various biomedical optical imaging methods (e.g., diffuse optical tomography, optical coherence tomography, and focal modulation microscopy) and their applications in biomedicine.

Prof. Colin Sheppard was a Professor and Head of the Division of Bioengineering in National University of Singapore, and was also Professor in the Department of Diagnostic Radiology in the School of Medicine until his retirement in 2012. His main area of research is in confocal microscopy, including instrumental development and investigation of novel techniques with bio-medical and industrial applications. He was a pioneer in this area and developed one of the world’s first confocal microscopes in 1975.

Mr. Wong Chee Howe was a PhD candidate co-supervised by A/P Chen Nanguang and Prof. Colin Sheppard. He is currently working in Singapore Technologies Engineering Ltd.

Link to Technical Brief


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