• Molecules and Cells
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• v.45 no.1
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• pp.41-50
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• 2022

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.

• Applied Microscopy
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• v.51
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• pp.12.1-12.12
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• 2021

Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which combines bright-field and fluorescence microscopy in a standard light microscope. The technique enables simultaneous real-time observation of fluorescently-labeled structures in relation to their direct physical surroundings. The advancement provides context for the orientation, movement, and function of labeled structures in the microcirculation.

• Molecules and Cells
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• v.45 no.2
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• pp.84-92
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• 2022

To understand the microcircuitry of the brain, the anatomical and functional connectivity among neurons must be resolved. One of the technical hurdles to achieving this goal is that the anatomical connections, or synapses, are often smaller than the diffraction limit of light and thus are difficult to resolve by conventional microscopy, while the microcircuitry of the brain is on the scale of 1 mm or larger. To date, the gold standard method for microcircuit reconstruction has been electron microscopy (EM). However, despite its rapid development, EM has clear shortcomings as a method for microcircuit reconstruction. The greatest weakness of this method is arguably its incompatibility with functional and molecular analysis. Fluorescence microscopy, on the other hand, is readily compatible with numerous physiological and molecular analyses. We believe that recent advances in various fluorescence microscopy techniques offer a new possibility for reliable synapse detection in large volumes of neural circuits. In this minireview, we summarize recent advances in fluorescence-based microcircuit reconstruction. In the same vein as these studies, we introduce our recent efforts to analyze the long-range connectivity among brain areas and the subcellular distribution of synapses of interest in relatively large volumes of cortical tissue with array tomography and superresolution microscopy.

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2008.06a
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• pp.697-698
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• 2008

• Applied Microscopy
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• v.46 no.1
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• pp.6-13
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• 2016

Fluorescence lifetime imaging microscopy (FLIM) has been considered an effective technique to investigate chemical properties of the specimens, especially of biological samples. Despite of this advantageous trait, researchers in this field have had difficulties applying FLIM to their systems because acquiring an image using FLIM consumes too much time. Although analog mean-delay (AMD) method was introduced to enhance the imaging speed of commonly used FLIM based on time-correlated single photon counting (TCSPC), a real-time image reconstruction using AMD method has not been implemented due to its data processing obstacles. In this paper, we introduce a real-time image restoration of AMD-FLIM through fast parallel data processing by using Threading Building Blocks (TBB; Intel) and octa-core processor (i7-5960x; Intel). Frame rate of 3.8 frames per second was achieved in $1,024{\times}1,024$ resolution with over 4 million lifetime determinations per second and measurement error within 10%. This image acquisition speed is 184 times faster than that of single-channel TCSPC and 9.2 times faster than that of 8-channel TCSPC (state-of-art photon counting rate of 80 million counts per second) with the same lifetime accuracy of 10% and the same pixel resolution.

• Journal of the Korean Society of Visualization
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• v.21 no.3
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• pp.65-74
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• 2023

Compared to epifluorescence(EPI) microscopy which captures fluorescence from the entire depth of sample, total internal reflection fluorescence(TIRF) can selectively visualize only a single surface of it. TIRF uses a thin evanescent field generated by the total internal reflection of laser light on surface. However, conventional TIRF system are designed for total internal reflection to occur at the upper surface of sample, making them unsuitable for sessile droplet imaging. We designed a TIRF system suitable for a sessile droplet imaging by utilizing slide glass as a lightguide. We presented the details for constructing the TIRF system using a prism, slide glass, air slit, and optical trap. Then, we compared the TIRF with EPI by imaging the droplet with fluorescent particles during its drying process. As a result, TIRF allows us to distinctly visualize the drying pattern on the bottom surface of droplet.

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2002.05a
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• pp.187-190
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• 2002

Confocal microscopy is very popular technology in bio-medical inspection due to its ability to reject background signals and to measure very thin slide of thick specimens, which is called optical sectioning. But intensity of detected signal in fluorescence type confocal microscopy is so small that only 0.2% of emitted fluorescence light can be detected in the best case. In this paper, we proposed the reflecting optical system to improve the detection intensity and designed the optical system by optimal design method. At the end of the paper, we analyzed the characteristics of the proposed reflecting optical system.

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2009.06a
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• pp.493-494
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• 2009

• 한국정보디스플레이학회:학술대회논문집
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• 2005.07a
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• pp.493-495
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• 2005

The director profiles of the Bloch walls are directly visualized using fluorescence confocal polarizing microscopy. Both pure twist Bloch walls and diffuse Bloch walls are analyzed. Polar anchoring energy was measured from optical simulation of the transmitted light interference pattern or the fluorescence intensity profile of a pure twist wall..

• Journal of the Korean Society of Visualization
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• v.11 no.2
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• pp.41-45
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• 2013

Two-photon microscopy (TPM) is minimally-invasive 3D fluorescence microscopy based on nonlinear excitation, and TPM can visualize cellular structures based on auto-fluorescence. Line-scanning TPM is one of high-speed TPM methods without sacrificing the image resolution by using spatial and temporal focusing. In this paper, we developed line-scanning TPM based on spatial and temporal focusing for auto-fluorescence imaging by exciting the tryptophan. Laser source for this system was an optical parametric oscillator (OPO) and it made near 570 nm femtosecond pulse laser. It had 200fs pulse width and 1.72 nm bandwidth, so that the achievable depth resolution was 2.41um and field of view (FOV) is 10.8um. From the characterization, our system has 3.0 um depth resolution and 12.3 um FOV. We visualized fixed leukocyte cell sample and compared with point scanning system.