Shogo Kawano

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.

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.

Determination of the Expanded Optical Transfer Function in Saturated Excitation Imaging and High Harmonic Demodulation

Most limits of a microscope can be understood in terms of its spatial frequency mapping ability, known as the optical transfer function (OTF). Here, we demonstrate the expansion of the confocal fluorescence microscopy OTF under conditions where the population of excitation states of the sample is saturated. We show how the OTF of saturated excitation can be determined by estimating fluorescence signals using a five-level molecular electronic state model. We then show how modulating excitation and demodulating fluorescence signals can extract high frequency components. We provide experimental validation by demonstrating that harmonic demodulation can extract high frequency OTF components.

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

Abstract. 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.

Nonlinear fluorescence imaging by photoinduced charge separation

Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.

High-resolution laser scanning microscopy with saturated excitation of fluorescence

We propose the use of saturated excitation to improve the spatial resolution of a laser scanning confocal fluorescence microscope. The saturated excitation induces nonlinear response in fluorescence emission that gives higher-order spatial frequency components in the point spread fuction of the microscope. To extract the nonlinear responses in fluorescence emission, we modulate the excitation intensity temporally at a single frequency (ω) and demodulate the fluorescence signal at the harmonic frequencies (2ω, 3ω, ...). We found that the fluorescence signal demodulated at nth harmonic frequency is proportional to nth power of the excitation intensity, where n-fold improvement of the spatial resolution can be achieved. We experimentally confirmed that the demodulated signal at the second harmonic frequency was proportional to the square of the excitation intensity. The improvement of spatial resolution was also confirmed by observing a fluorescent sample.

Imaging properties of saturated excitation (SAX) microscopy

We used nonlinear fluorescence emission under the condition of saturated excitation (SAX) of fluorescent molecules for high-resolution laser scanning microscopy. In the technique, SAX microscopy, we modulate the excitation intensity at a single frequency and demodulate the fluorescence signal at a harmonic frequency to extract a nonlinear fluorescence response that contributes to improvement of the spatial resolution. This nonlinear fluorescent response on saturated condition was analyzed by rate equations formulated from a five-level system Jablonski diagram. By calculating relationship between excitation intensity and fluorescence signal demodulated at harmonic frequencies for rhodamine-6G molecules with 532 nm excitaion, we found that the fluorescent signal exhibits high-order nonlinear dependence on the excitation intensity under conditions of saturated excitation. We also calculated effective point spread functions (ePSFs) of SAX microscopy. The result of the calculation shows that ePSFs given with the harmonic demodulation provides the spatial resolution beyond the resolution limit of conventional confocal microscopy. The optical transfer functions have also been calculated from the ePSFs. The result of the calculation shows that a higher spatial-resolution can be obtained by demodulating fluorescnece signal at a higher harmonic frequency without theoretical limitation.

Nonlinear fluorescence probe using photoinduced charge separation (Presentation Recording)

Two-photon excitation microscopy (TPEM) provides spatial resolution beyond the optical diffraction limit using the nonlinear response of fluorescent molecules. One of the strong advantages of TPEM is that it can be performed using a laser-scanning microscope without a complicated excitation method or computational post-processing. However, TPEM has not been recognized as a super-resolution microscopy due to the use of near-infrared light as excitation source, which provides lower resolution than visible light. In our research, we aimed for the realization of nonlinear fluorescence response with visible light excitation to perform super-resolution imaging using a laser-scanning microscope. The nonlinear fluorescence response with visible light excitation is achieved by developing a probe which provides stepwise two-photon excitation through photoinduced charge separation. The probe named nitro-bisBODIPY consists of two fluorescent molecules (electron donor: D) and one electron acceptor (A), resulting to the structure of D-A-D. Excited by an incident photon, nitro-bisBODIPY generates a charge-separated pair between one of the fluorescent molecules and the acceptor. Fluorescence emission is obtained only when one more incident photon is used to excite the other fluorescent molecule of the probe in the charge-separated state. This stepwise two-photon excitation by nitro-bisBODIPY was confirmed by detection of the 2nd order nonlinear fluorescence response using a confocal microscope with 488 nm CW excitation. The physical model of the stepwise two-photon excitation was investigated by building the energy diagram of nitro-bisBODIPY. Finally, we obtained the improvement of spatial resolution in fluorescence imaging of HeLa cells using nitro-bisBODIPY.

Biological imaging beyond the diffraction limit by saturated excitation (SAX) microscopy

We present an alternative high-resolution fluorescence imaging technique, saturated excitation (SAX) microscopy, for observations of biological samples. In the technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity. Under this condition, the fluorescence intensity is no longer proportional to the excitation intensity and the relation of the fluorescence and excitation intensity shows strong nonlinearity. In the centre of laser focus, the nonlinear responses induced by the saturation appear notably because of higher excitation intensity. By detecting fluorescence signals from the saturated area, we can push the spatial resolution beyond the diffraction barrier in three dimensions. SAX microscopy can be realized with a simple optics, where a laser intensity modulation sisytem and a lock-in amplifier are simply added to a conventional confocal microscope system. Using the SAX microscope, we demonstrated high-resolution imaging of a biological sample by observing mitochondria in HeLa cells. We also examined the nonlinear response of commercially available dyes under saturated excitation conditions.