Although confocal microscopy (CM) is effective for subcellular imaging to depths of tens of microns (and for imaging fluorescently tagged tissues, unlike optical-coherence tomography), attempts to image deeper molecular structures using CM are usually disrupted by scattered photons. But a new technique called focal-modulation microscopy (FMM) developed by researchers at the National University of Singapore achieves diffraction-limited spatial resolution and molecular specificity for imaging depths greater than 0.5 mm, without the complex setup and expensive ultrafast-laser sources required by competitive multiphoton-microscopy techniques.
The FMM setup is nearly identical to a CM setup, with the exception that a spatial phase modulator is inserted into the excitation path of the 25 mW, 640 nm single-frequency laser to ensure that only ballistic photons (those that contribute to a high-resolution image), and not scattered photons, contribute to the modulated signal detected by a photomultiplier tube. The signal is analyzed using a fast Fourier transform and the image information is retrieved. Fluorescence signals are further distinguished by special pinhole filtering techniques in the setup, which is also designed to maximize the modulated signal. An analysis of in vivo cellular and subcellular imaging of chondrocyte cells in chicken cartilage at depths of 280 µm revealed overlapping structures and blurred cellular shapes using the CM technique and conversely, detailed information with submicron spatial resolution and excellent contrast for the FMM technique at imaging depths up to 600 µm. Contact Nanguang Chen at [email protected]