Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters.

Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and confirmed the resolution improvement at a depth of 40 µm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens.

The molecular chaperone Hsp47 is essential for cartilage and endochondral bone formation.

Heat shock protein 47 kDa (Hsp47) is considered as a molecular chaperone essential for the correct folding of type I and type IV procollagen in the ER. However, the function of Hsp47 for other types of procollagen and its importance for chondrogenesis have never been elucidated. To examine the function of Hsp47 in cartilage formation and endochondral ossification, we conditionally inactivated the Hsp47 gene in chondrocytes using Hsp47 floxed mice and mice carrying a chondrocyte-specific Col2a1-Cre transgene. Hsp47 conditional null mutant mice died just before or shortly after birth, and exhibited severe generalized chondrodysplasia and bone deformities with lower levels of type II and type XI collagen. Second-harmonic generation (SHG) analysis and electron microscopy revealed the accumulation of misaligned type I collagen molecules in the intervertebral discs and a substantial decrease in type II collagen fibers, respectively. Whole-mount skeletal staining showed no calcified region in the vertebral bodies of sacral vertebrae, and revealed that the endochondral bones were severely twisted and shortened. These results demonstrate that Hsp47 is indispensable for well-organized cartilage and normal endochondral bone formation.

SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging.

We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy. We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light. Using FNDs, we quantified the spatial resolution improvement inherent in SAX microscopy, and experimentally demonstrated the scalability of the spatial resolution of SAX microscopy. The photostability of the FNDs allowed us to perform nanoparticle imaging of a multicolor-stained macrophage cell with a spatial resolution beyond the diffraction limit.

Beyond the diffraction-limit biological imaging by saturated excitation microscopy.

We demonstrate high-resolution fluorescence imaging in biological samples by saturated excitation (SAX) microscopy. In this technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity to induce strong nonlinear fluorescence responses in the center of laser focus, which contributes the improvement of the spatial resolution in three dimensions. Using SAX microscopy, we observed stained microtubules in HeLa cells with improved spatial resolution. We also measured the relation of the fluorescence and excitation intensity with several kinds of fluorescence dyes and, in the results, confirmed that SAX microscopy has the potential to observe any kind of fluorescence samples in current usage.

High-resolution confocal microscopy by saturated excitation of fluorescence.

We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.