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Related Concept Videos

Imaging Biological Samples with Optical Microscopy01:18

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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
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Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...
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Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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A versatile miniature two-photon microscope enabling multicolor deep-brain imaging.

Runlong Wu1,2,3, Chunzhu Zhao4,5, Shan Qiu6

  • 1National Biomedical Imaging Center, State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, College of Future Technology, Peking University, Beijing, China. rlwu@bistu.edu.cn.

Nature Methods
|August 21, 2025
PubMed
Summary
This summary is machine-generated.

We developed FHIRM-TPM 3.0, a miniature microscope for deep-brain imaging in mice. This advanced two-photon microscopy system enables multicolor imaging of neuronal activity and cellular structures in vivo.

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Area of Science:

  • Neuroscience
  • Biomedical Engineering
  • Microscopy Technology

Background:

  • Deep-brain imaging in freely behaving animals is crucial for understanding neural circuits.
  • Existing two-photon microscopy systems face limitations in size, flexibility, and imaging depth.
  • Multicolor imaging is essential for dissecting complex cellular and molecular processes in the brain.

Purpose of the Study:

  • To introduce FHIRM-TPM 3.0, a novel, compact two-photon microscope for advanced in vivo brain imaging.
  • To demonstrate the system's capability for multicolor deep-brain imaging in freely behaving mice.
  • To showcase the system's versatility and high resolution for various neuroscience research applications.

Main Methods:

  • Integration of a miniature two-photon microscope with a broadband anti-resonant hollow-core fiber.
  • Correction of optical aberrations and optimization of fluorescence collection for deep tissue penetration.
  • Engineering of interchangeable objectives to achieve scalable fields of view and high lateral resolution.
  • Utilizing multicolor excitation wavelengths (780, 920, 1030 nm) for simultaneous cellular activity monitoring.

Main Results:

  • Achieved cortical neuronal imaging at depths exceeding 820 μm.
  • Enabled hippocampal Ca2+ imaging at single dendritic spine resolution using a GRIN lens.
  • Provided a tenfold scalable field of view (up to 1 × 0.8 mm²) with resolutions from 0.68 μm to 1.46 μm.
  • Successfully investigated mitochondrial and cytosolic Ca2+ activities relative to amyloid plaques in APP/PS1 mice.

Conclusions:

  • FHIRM-TPM 3.0 is a versatile and powerful tool for multicolor deep-brain imaging in neuroscience research.
  • The system's miniaturization and advanced optical design facilitate in vivo studies in freely behaving subjects.
  • It enables high-resolution, deep-tissue imaging, advancing the study of neurological diseases and brain function.