## Special issue-Fluorescence microscopy: techniques and applications

2022, 51(11): 20220735. doi: 10.3788/IRLA20220735
[Abstract](324) [FullText HTML] (45) [PDF 6184KB](117)
Spatial resolution is a key specific parameter of the optical microscopic imaging system. According to the optical diffraction theory, the spatial resolution of imaging system is determined by the wavelength of illumination light and the numerical aperture of microscope objective. In the practical imaging process, the resolutions of microscopic imaging system obtained from different criteria are slightly different. It is necessary to select an appropriate criterion according to the coherence of light sources and the structural characteristics of obsversed targets to accurately calculate the resolutions of imaging system. In this paper, the calculation methods for spatial resolution under different conditions are provided via theoretical analysis and numerical simulation. Furthermore, we compare and discusse the difference of imaging resolutions under the illuminations of coherent and incoherent light sources for double points and double slits targets, respectively.
2022, 51(11): 20220550. doi: 10.3788/IRLA20220550
[Abstract](151) [FullText HTML] (26) [PDF 2977KB](69)
Two-photon microscopy (TPM) imaging has the characteristics of high resolution, natural chromatography capability and large penetration depth, and plays an important role in the imaging of living animals. How to enlarge the field-of-view (FOV) of TPM while maintaining the high resolution to monitor large-scale dynamic responses in biomedical applications especially brain science, however, remains challenging. In this paper, the recent progress of large-FOV two-photon imaging technology is reviewed. The theoretical basis of achieving large-FOV TPM is elaborated from the perspective of optical invariant. Large- FOV TPM methods can be divided into three categories: FOV-edge aberration calibration with scanning relay engines, the design and manufacture of high-throughput objectives and correcting aberrations with adaptive optics. These methods have highly strengthened the capability of TPM used in large scale biomedical imaging. If further improved especially the imaging speed, large-FOV TPM will have great potential to contribute the development of life science and broaden the cognitive of large-scale biological activities. Large-FOV TPM, based on its outstanding spatial and temporal resolution, will become a powerful tool for dynamic monitoring across large-area in some applications that requires high resolution and mesoscale imaging simultaneously.
2022, 51(11): 20220494. doi: 10.3788/IRLA20220494
[Abstract](157) [FullText HTML] (28) [PDF 5679KB](71)
Confocal microscopy has high spatial resolution and signal to background ratio, possessing the capability of three-dimensional tomography of biological samples, and thus has been widely used in medicine and biology areas. Light in near-infrared II (NIR-II, 900-1 880 nm) regions fulfils moderate absorption, low scattering in biological tissues, and weak autofluorescence of biological tissues. Therefore, NIR-II in vivo fluorescence imaging has the advantages of large depth and high contrast. Point-excitation and point-detection based NIR-II confocal microscopy combines the advantages of the two technologies mentioned above and features high spatial resolution and high signal to background ratio in large-depth biological imaging. Therefore, it has been widely used in the biomedical fields. This review summarizes the principle and the development progress of NIR-II confocal microscopy and the application of biological imaging based on it. The future improvement and development directions of NIR-II confocal microscopy are also discussed.
2022, 51(11): 20220527. doi: 10.3788/IRLA20220527
[Abstract](164) [FullText HTML] (29) [PDF 5808KB](55)
In recent years, due to the unique advantages such as excellent fluorescence properties, ultra-small size, precise chemical structure and good biocompatibility, gold nanoclusters (AuNCs) have become a kind of emerging fluorescent nanoprobe of great concern. In order to promote the application of AuNCs in fluorescence imaging, researchers have been devoted to designing preparation strategies for various high-performance fluorescent AuNCs. Based on the continuous understanding of the structure and luminescence mechanism of AuNCs, strategies such as enhancing the fluorescence quantum yield and cellular uptake of AuNCs have been proposed and applied to enhance the cellular imaging ability of AuNCs. These strategies greatly improve their potential as fluorescent imaging probes. Furthermore, fluorescent AuNCs are also utilized in advanced fluorescence imaging technologies such as fluorescence lifetime imaging and multi-photon fluorescence imaging. In addition, AuNCs with near-infrared II fluorescence have greatly promoted their application for in vivo imaging recently. This article summarizes the preparation methods of fluorescent AuNCs probes, reviews strategies to improve the fluorescence cellular imaging ability of AuNCs, and introduces the latest progress in the application of AuNCs in fluorescence imaging and also the challenges and future developments in the field.
2022, 51(11): 20220622. doi: 10.3788/IRLA20220622
[Abstract](106) [FullText HTML] (21) [PDF 4868KB](67)
Observing dynamic interaction between organelles and analyzing the law of action is of great significance for revealing the mechanism behind the phenomenon of physiological and pathological processes. Due to the limitation of the optical diffraction determined by wavelength and aperture, traditional optical microscopes cannot observe the nanoscale fine structure of organelles and the dynamic changes of interactions among them. The emergence of super-resolution microscopy imaging technology provides an important mean for the study of organelle interaction. This paper introduces the fluorescence microscopy (STED), structured illumination imaging (SIM), and single-molecule localization imaging (SMLM). The application of these super-resolution microscopy in the study of dynamic interaction between organelles provides the expansion of application ideas for super-resolution microscopy. Finally, the advantages and disadvantages of super-resolution microscopy in the study of organelle interactions are analyzed. In conclusion, the demand and development trend of super-resolution microscopy technology in the imaging of intracellular organelle interaction in living cells is prospected, which provides a certain reference for the cross-integration development of optics, medicine and biology.
2022, 51(11): 20220546. doi: 10.3788/IRLA20220546
[Abstract](116) [FullText HTML] (28) [PDF 3095KB](64)
With the advantages of high resolution, high sensitivity, high molecule specificity and non-invasive, fluorescence microscopy can characterize the morphological and molecular functional information of samples at the micron or even nanometer scale, making it an important tool for life science research. As microbiology research continues to advance, fluorescence microscopy is expected to provide dynamic and 3D observation of microscopic biological structures and molecular events. This paper systematically reviews the research progress of fast 3D fluorescence microscopy in recent years, including the main technical means, improvement strategies and representative research results of point-scan imaging, wide-field imaging, and projection tomography to improve the imaging speed, expand the imaging dimension and enhance the imaging quality. In the end, we look forward to the future challenges and prospects of fast 3D fluorescence microscopic imaging techonology.
2022, 51(11): 20220354. doi: 10.3788/IRLA20220354
[Abstract](127) [FullText HTML] (15) [PDF 2118KB](59)
Light-sheet microscopy is a biological imaging technology that has been studied a lot in recent years. Compared with traditional confocal laser scanning microscopy, light-sheet microscopy can achieve rapid volume imaging with low phototoxicity. The illumination beam of light-sheet microscope can choose Gaussian beam or other non-diffracting beams (such as Bessel beam, Airy beam, etc.). Airy light-sheet microscopy is the most researched technology at present, but there is a big problem with ordinary Airy light-sheet microscopy. Airy beam has the characteristic of self-bending, which causes Airy beam to exceed detection at both ends of the field of view. The depth of field of the objective lens cannot produce the best imaging effect. The Airy beam was rotated by 45° to form a planar-Airy light-sheet, so that the Airy light-sheet did not exceed the depth of field of the detection objective, so as to increase the imaging field of view of the light-sheet microscope. And using two-photon fluorescence excitation technology, the post-processing process of the image was eliminated, greatly improving the efficiency of imaging. In this study, Matlab was used for optical simulation, and the imaging field of view (~900 μm) of the planar-Airy light-sheet microscope was increased by 50% compared with the imaging field of view (~600 μm) of the ordinary Airy light-sheet microscope. The constructed planar-Airy light-sheet microscope was calibrated with fluorescent microspheres, and the lateral resolution of the imaging system was (1.93±0.17) μm and the axial resolution was (3.19±0.41) μm. In the real-time observation of the zebrafish intracerebral hemorrhage model, imaging results with a temporal resolution of x\begin{document}$\times$\end{document}y\begin{document}$\times$\end{document}z = 0.60 mm\begin{document}$\times$\end{document}0.60 mm \begin{document}$\times$\end{document}0.40 mm/60 s can be obtained, and the growth and development of local blood vessels can be monitored in real-time to explore the mechanism of cerebral hemorrhage disease.
2022, 51(11): 20220541. doi: 10.3788/IRLA20220541
[Abstract](125) [FullText HTML] (25) [PDF 6787KB](48)
Photoacoustic imaging technology based on laser-induced ultrasound mechanism combines the high contrast of optical imaging and the deep penetration of ultrasound imaging, which can reflect the distribution of endogenous absorbents in living organisms in a label-free and non-invasive way, especially suitable for real-time imaging of the whole brain of rodent models. In order to prove the application of photoacoustic imaging technology in brain science research and brain disease monitoring, a photoacoustic microscopic imaging system with spatial resolution of tens of microns and effective imaging depth of more than 1 mm was constructed. Taking APP/PS1 transgenic Alzheimer’s disease (AD) model mice and WT mice as research objects, the ability of photoacoustic imaging in characterizing the brain structure changes and vascular network of AD mice and WT mice was explored from three levels of brain tissue slices, in vitro whole brain and in vivo whole brain. It demonstrates the great potential of photoacoustic imaging technology in monitoring brain structural changes and cerebrovascular network characteristics during the development of brain diseases, which can provide deeper insights into many brain science studies and the development mechanism of neurodegenerative brain diseases.
2022, 51(11): 20220536. doi: 10.3788/IRLA20220536
[Abstract](221) [FullText HTML] (25) [PDF 8102KB](83)
Fluorescence microscopy has the advantage of minimal invasion to bio-samples and visualization of specific structures, and therefore, it has been acting as one of mainstream imaging tools in biomedical research. With the rapid development of artificial intelligence technology, deep learning that has outstanding performance in solving sorts of inverse problems has been widely used in many fields. In recent years, the applications of deep learning in fluorescence microscopy have sprung up, bringing breakthroughs and new insights in the development of fluorescence microscopy. Based on the above, this paper first introduces the basic networks of deep learning, and reviews the applications of deep learning in fluorescence microscopy for improvement of spatial resolution, image acquisition and reconstruction speed, imaging throughput, and imaging quality. Finally, we summarize the research on deep learning in fluorescence microscopy, discuss the remaining challenges, and prospect the future work.
2022, 51(11): 20220402. doi: 10.3788/IRLA20220402
[Abstract](422) [FullText HTML] (59) [PDF 5522KB](191)
Because the phase contains more information about the field in contrast to the amplitude, phase measurement has always been a hot topic in many branches of modern science and engineering. Within the visible range of electromagnetic wave, it is quite difficult to directly obtain phase information by the existing photodetectors. Phase retrieval provides an effective method to “figure out” the phase information from the captured intensity information, and has achieved successful applications in several scientific fields including astronomical observation, biomedical imaging and digital signal restoration. Algorithm is not only the core of phase retrieval, but is also the key to its development and applications. This paper demonstrates the basic principles of phase retrieval algorithms in combination with physical principles and signal processing methods, summarizes the development of various kinds of algorithms as well as their advantages and disadvantages, and briefly lists some typical applications in the field of optics. Finally, the challenges are pointed out, and the future development directions are described as: better convergence performance and noise robustness, phase-retrieval ability for more complex objects, compatibility for integration of multiple objectives and tasks.
2022, 51(11): 20220597. doi: 10.3788/IRLA20220597
[Abstract](93) [FullText HTML] (6) [PDF 1427KB](34)
CRISPR/Cas9 system are widely used in gene editing due to its high efficiency, simple operation and wide species adaptability. The system consists of a guide RNA (sgRNA) that targets the target DNA series and Cas9 with cleavage enzyme activity. In recent years, researchers have developed a range of super-resolution live cell imaging techniques by combining nuclease-inactivated Cas9 mutants dCas9 (dead Cas9) or sgRNA with fluorescent proteins (FPs), organic dyes, and quantum dots (QDs). This technology helps researchers to study different genes, chromosomes and the spatio-temporal relationship between genes and chromosomes at higher resolutions, which is of great significance to promote the rapid development of genetics, cell biology and biomedicine. This paper summarizes the advances in live cell imaging technology based on CRISPR/Cas9 system, which is expected to further expand the wide application in the biomedical field.