Keywords: fluorescence microscope, fluorescent dyes, multi-color imaging, super-resolution technology

Fluorescence microscopy, through fluorescence labeling technology, visualizes biomolecules or structures and has become a core tool in cell biology and medical diagnosis. The core principle is based on the emission of fluorescent signals by fluorescent proteins or dyes under specific wavelength excitation, combined with techniques such as confocal microscopy and multiphoton imaging to achieve high-resolution imaging.

1、 Fluorescent labeling technology: from single dye to quantum dots

The core of fluorescent labeling is the selection of specific dyes. Traditional organic dyes such as FITC and Cy3 have high fluorescence quantum yields, but are prone to photobleaching; Quantum dots have become the preferred choice for long-term observations due to their wide excitation spectrum, narrow emission spectrum, and anti photobleaching properties. For example, in live cell imaging, membrane proteins labeled with quantum dots can be continuously tracked for several hours, revealing the mechanism of cell migration.

Multi color imaging technology achieves simultaneous observation of multiple molecules by combining dyes with different emission wavelengths. For example, the four-color imaging system can distinguish between the nucleus (DAPI), microtubules (Alexa Fluor 488), mitochondria (MitoTracker), and endoplasmic reticulum (ER Tracker), providing multidimensional information for subcellular structural analysis.

2、 Super resolution fluorescence microscope: breaking through the diffraction limit

The resolution of traditional fluorescence microscopes is limited by the diffraction of light (approximately 200 nanometers). Super resolution technology breaks through this limitation through the following strategies:

STED microscope: Utilizing the stimulated radiation loss effect, the excitation spot is reduced to below 50 nanometers.

PALM/STORM: Based on single-molecule localization technology, 20 nanometer resolution is achieved by randomly switching fluorescent molecules.

In neuroscience, STED microscopy has successfully resolved the distribution of neurotransmitter receptors in synaptic cleft, revealing the molecular basis of learning and memory.

3、 Clinical application: from pathological diagnosis to drug screening

Fluorescence microscopy is used in pathological diagnosis to detect tumor markers. For example, immunofluorescence staining can mark HER2 protein and help breast cancer typing; FISH technology uses fluorescence in situ hybridization to detect gene amplification and guide targeted therapy.

In the field of drug screening, high-throughput fluorescence imaging platforms can simultaneously monitor the response of thousands of cells to drugs. For example, the calcium ion indicator Fluo-4 combined with a microscope can observe in real-time the effect of drugs on calcium signaling in myocardial cells, accelerating the development of new drugs.

4、 Technological bottlenecks 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.

Photogenetics combination: utilizing light controlled ion channels to achieve precise regulation and imaging of neural activity.

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