Focal Modulation Microscopy For Molecular Imaging Of Thick Biological Tissues
Dr NG Chen (Division of Bioengineering & Department of Electrical & Computer Engineering)
ight microscopy has been an indispensible and powerful tool in various fields of biological research and medical diagnoses. Biological tissues are heterogeneous from the microscopic to macroscopic scales. Generally they appear opaque to visible and near infrared light as photons are
subject to strong scattering and absorption. Scattering, especially multiple scattering, is an undesirable phenomenon in imaging science that alters the propagation direction of photons. The scattering mean free path l s, the average distance between consecutive scattering events, has a typical value of around 100 microns in human soft tissues. Conventional wide field microscopy can only deal with very thin samples prepared on glass slides, while confocal microscopy (CM) is capable of optical sectioning and can provide an imaging depth up to 200 microns. Multi-photon microscopy (MPM) can achieve a penetration depth up to 700 microns by the use of nonlinear absorption. MPM has become an increasingly popular
alternative to confocal microscopy as a result of improved imaging depth and localized photochemistry. However, MPM is a very expensive technique that uses ultra short laser pulses as the source. The coherence gating mechanism in optical coherence tomography OCT) is very effective in picking up the desired signal. The imaging depth and speed have been further improved recently with Fourier domain techniques. A cross-sectional scanning rate over 30 frames per second is readily achievable with an imaging depth up to 3 mm. Unfortunately OCT is not compatible with fluorescence and itsmolecular imaging capability is rather limited.
Figure 1: Schematic diagram of the prototype focal modulation microscopy system.
We therefore developed focal modulation microscopy (FMM), a novel technique that targets an imaging depth greater than 0.5 mm combined with diffraction limited spatial resolution and molecular specificity. Shown in Figure 1 is the schematic of a prototype FMM system developed at the Optical Bioimaging Lab of NUS. By the use of a spatial phase modulator in the excitation light path, anintensity modulation is achieved mainly in the focal volume only, even when the focal point is located deep inside a turbid medium.The oscillatory component in the detected fluorescence signal can be readily differentiated from background signal caused by multipleand scattering.
Our implementation allows simultaneous acquisition of confocal and FMM images. We have demonstrated the advantagescoherence ( of FMM over CM with a series of image experiments using cartilage tissue from chicken. Chondrocytes are the only cells found in cartilage. The cells are usually of a rounded or bluntly angular form, lying in groups of two or more in a glandular or almost homogeneous matrix. Chicken cartilage was cut into slices around 1 mm in thickness and stained with DiD (DiIC18(5), Invitrogen Corp). Figure 2 is an FMM image acquired at an imaging depth of 500 microns, showing excellent contrast and spatial resolution.
To conclude, we have developed and experimentally demonstrated a novel microscopy method for molecular imaging of thick biological tissues with one photon excited fluorescence. We believe that FMM will find numerous applications in basic biological research and clinical diagnoses.
This work was done in collaboration with graduate student CH Wong.
Figure
2: Fluorescence images of chondrocytes obtained from chicken cartilage. Imaging depth: 500 microns.
Dr
Chen Nanguang received his PhD degree in Biomedical Engineering in 2000 from Tsinghua University. He also received his MS in Physics and BS in Electrical Engineering in 1994 (Peking University), and 1988 (Hunan University), respectively. He has been an Assistant Professor of Bioengineering and Electrical Engineering with the National University of Singapore since 2004. His research interests include diffuse optical tomography, optical coherence tomography, and novel microscopic imaging methods including focal modulation microscopy.
