As the starting point for human exploration of the microscopic world, the optical microscope has undergone a leapfrog development from simple magnification to super-resolution imaging since its invention by Leeuwenhoek in the 17th century. The core principle is based on the interaction between visible light and the sample, and the visualization of morphology and structure is achieved through a lens system. This article will explore the basic principles, technological evolution, and cutting-edge applications of optical microscopes.

1、 Bright and dark field microscopes: the cornerstone of traditional optical imaging

Bright field microscope illuminates the sample with transmitted light, which is the most fundamental optical imaging method. Its resolution is limited by diffraction of light (approximately 200 nanometers) and is suitable for observing stained cell or tissue sections. For example, in pathological diagnosis, hematoxylin and eosin (H&E) staining combined with bright field microscopy can identify morphological abnormalities in tumor cells.

The dark field microscope blocks the central beam through an annular aperture, allowing only oblique light to enter the objective lens, enhancing the contrast at the edges of the sample. In microbiology, dark field microscopy can clearly observe the movement trajectory of unstained bacteria, with a resolution 2-3 times higher than bright field microscopy.

2、 Fluorescence Microscopy: The Revolution of Molecular Labeling

Fluorescence microscopy achieves highly specific imaging by labeling specific molecules with fluorescent dyes or proteins. Its core components include excitation light source, filter, and detector. For example, the discovery of green fluorescent protein (GFP) has made it possible to localize proteins within living cells, driving the development of cell biology.

Multi color fluorescence imaging technology achieves simultaneous observation of multiple molecules by combining dyes with different emission wavelengths. For example, in neuroscience, four-color fluorescent labeling can distinguish neurons, glial cells, synapses, and blood vessels, providing multidimensional information for brain function research.

3、 Super resolution microscope: a milestone in breaking through the diffraction limit

The resolution of traditional optical microscopes is limited by the Abbe diffraction limit (λ/2NA). Super resolution technology breaks this limitation through the following strategies:

STED microscope: Utilizing the stimulated radiation loss effect, the excitation spot is reduced to below 50 nanometers. For example, when studying the distribution of mitochondrial membrane proteins, STED microscopy can resolve nanoscale clustered structures.

SIM (Structured Light Illumination Microscope): Increases resolution to 100 nanometers through periodic illumination mode. In live cell imaging, SIM can dynamically observe the dynamic assembly of microtubule networks.

PALM/STORM: Based on single-molecule localization technology, 20 nanometer resolution is achieved by randomly switching fluorescent molecules. For example, when analyzing the structure of nuclear pore complexes, PALM technology can locate the three-dimensional coordinates of hundreds of protein subunits.

4、 Technological Challenges and Future Directions

The current challenges include phototoxicity, labeling efficiency, and data analysis complexity. Future trends include:

Adaptive optics technology: compensates for aberrations through deformable mirrors to further improve resolution.

Multimodal imaging system: integrates optical, electronic, and mechanical signals to achieve multidimensional representation.

Artificial intelligence assisted analysis: automatically identifying the spatiotemporal dynamics of fluorescent signals and revealing complex biological processes.