Photoluminescence (red) and second-harmonic generation (grayscale) from pure cadmium-telluride solar cells, imaged simultaneously. Standard resolution is on the left, and enhanced resolution, on the right, was captured by the MP-SPIFI microscope. Credit: Jeff Field / CSU
Superresolution microscopy is a valuable tool for biological imaging, and most techniques require fluorescent molecules to “break” the diffraction limit in order to view structures smaller than a wavelength of light. This fluorescence, however, prevents further imaging of the sample using certain contrast mechanisms such as harmonic generation.
Now, U.S. scientists report developing a new multimodal superresolution microscopy technique based on spatial frequency-modulated imaging (SPIFI) that allows for simultaneous multiphoton fluorescence and second-harmonic-generation imaging (Proc. Nat. Acad. Sci. USA, doi: 10.1073/pnas.1602811113). The authors say that this technique could provide multimodal multiphoton imaging deep within scattering biological tissues and optically thin media at unprecedented resolutions.
The new multiphoton microscope, custom-built by OSA Fellow Randy Bartels and his team at Colorado State University, uses SPIFI to enable superresolved nonlinear microscopy with any contrast mechanism. The setup combines the “relative scattering immunity” offered by wavelengths of light in the near-infrared with single-pixel detection of signal light, giving it the potential, the authors say, to collect images from unlabeled specimens that exhibit harmonic generation.
Using HeLa cells (a common biological sample) and cadmium-telluride solar cells (an optically thin material), the team demonstrated their microscope’s multimodal superresolution imaging capabilities via two-photon excited fluorescence and second-harmonic generation. Bartels and his colleagues report that their recorded images have a spatial resolution of up to 2ƞ below the diffraction limit (with ƞ defined as twice the power of the nonlinear intensity response)—which is beyond what is achievable with traditional laser-scanning multiphoton microscope.
The authors say their MP-SPIFI microscope could eventually provide high-quality in vivo imaging of unlabeled, deeply-set biological tissue specimens, as well as imaging unlabeled structures in inorganic media for materials science and other disciplines.