Nonlinear optical responses of specimens enable super-resolution microscopy by confining the excitation and detection volumes of fluorescence to spatial regions smaller than the diffraction limit. These responses arise under specific illumination conditions, such as increased light intensity or specific excitation wavelengths, which induce non-linear emission behaviors in fluorescent molecules. This review highlights laser-scanning and light-sheet microscopy as key platforms where nonlinear optical effects have been applied to significantly enhance spatial resolution. In addition to these platforms, structured illumination microscopy (SIM), an established super-resolution technique, can further broaden its capabilities by incorporating nonlinear optical responses—a direction also highlighted in this review. Selective-plane activation SIM (SPA-SIM), for instance, integrates light-sheet illumination and structured illumination using photoswitchable fluorescent proteins, enabling the selective observation of specific planes with high spatial resolution. This approach effectively suppresses background signals and enables three-dimensional super-resolution imaging even in thick or dense specimens, such as cell spheroids.

Apodized phase contrast microscopy is a microscopic technique which visualizes fine structure of, such as intact biological cells, colourless and transparent phase objects. It uses the apodized phase plate placed at the Fourier transfer plane conjugating to the illuminating aperture. The apodized phase plate has a quarter wave phase shift ring with absorption and two adjacent apodization areas with absorptions. It weakens selected diffracted light through phase objects. Although conventional phase contrast method produces the halo artifact, a phenomenon in the image of large phase objects, the apodized phase contrast method reduces these halos and provides inner detailed images. We have recently developed apodized phase contrast objectives for bright high contrast (ABH), with a 2%-transmittance phase ring and adjacent 8%-transmittance apodization areas. These provide high sensitivities and to detect wider range of phase differences. We applied these ABH objectives to biomedical applications such as identification of asbestos, motion of cellular organelles and early embryos. This paper also describes a pupil-projection apodized phase contrast microscopy with an inverted microscope. This microscopy works with high-NA bright field objective lenses.

Expansion Microscopy (ExM) is a super-resolution imaging technique that improves resolution by isotropic physical expansion of biological samples embedded in a swollen hydrogel. As a result, ExM enables nanoscale imaging using conventional fluorescence microscopy. A decade following the inception of ExM, comprehensive spatial analysis of proteins, nucleic acids (RNA and DNA), lipids, glycan chains, and other biomolecules is now applicable to a broad spectrum of cellular and biological contexts. Recent methodological advances, such as increased expansion factors, improved labeling efficiency, and more efficient and cost-effective sample preparation protocols, have accelerated their application and facilitated many scientific discoveries. This review article focuses on the basic principles underlying ExM, identifies practical protocols for its implementation and observation, highlights recent technological advances, and discusses remaining challenges. Ultimately, it aims to provide a comprehensive overview of ExM to encourage its broad application and methodological innovation.

Electron ptychography is a method for recovering sample phase information from 4D-STEM data, and it is becoming increasingly practical thanks to advancements in high-speed detectors. Ptychography is suitable for analyzing materials susceptible to electron beam damage, which are challenging to observe using conventional methods. Additionally, it can extract previously unobtainable information, such as sample depth details and the aberrations and modes of the incident probe, which is expected to play an increasingly crucial role in future materials science research. However, challenges regarding solution validity and quantitativeness remain, necessitating ongoing research progress. This article attempts to provide an accessible explanation of the fundamentals of ptychography, including its principles and the detailed calculations involved in direct and iterative methods.

Morphological technique is a robust tool to investigate metabolism in animals. Here, I introduce my research on the functional analysis of diacylglycerol kinase (DGK) in the adipose tissue. This article may help readers recognize a significance of the classical morphological technique to analyze metabolic organs. We have investigated that phenotypical alteration of DGKε-KO mice during the course of high fat diet (HFD) feeding regimen. At an early phase of feeding (40 days), DGKε-KO mice show obesity and insulin resistance. Histological observation revealed that DGKε-deficiency leads to adipocyte size enlargement, and induces inflammatory reactions in the visceral adipose tissue (AT). In brown AT, atypical unilocular cells were observed in DGKε-KO mice. Unexpectedly, under 180 days of HFD feeding conditions, obesity and insulin resistance were improved in DGKε-KO mice. Multilocular cells were observed in the visceral AT, and the unilocular cells were increased in the brown AT of DGKε-KO mice. Morphological techniques can provide us useful information on local cellular or tissue responses against metabolic change in the adipose tissues.

A new method for electron wave phase measurement has been developed using structured illumination with non-uniform amplitude and phase distributions combined with ptychographic wavefield reconstruction. The proposed method utilizes full-field illumination of non-uniformly structured electron beams generated by a conductive film with random openings. It reconstructs the complex transmission function of the specimen from a series of in-line holograms acquired with different illumination positions. A simulation study under practical conditions with a total of 2.1 × 10⁸ electrons demonstrated phase reconstruction with a standard deviation error of 0.024 rad. The method was further validated using experimental holograms obtained from MgO particles. The reconstructed phase was consistent with the results expected from the particle shape and was equivalent to a mean inner potential close to previously reported values.