Why Can Fluorescence Microscopes "See" Proteins Inside Cells? Principle Revealed
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I. Core Principle: 4 Steps to Make Proteins "Glow"
The imaging logic of fluorescence microscopes is similar to "lighting a glow stick in the dark," divided into 4 key steps:
1. Fluorescent Labeling: "Dressing Proteins in Fluorescent Coats"
Proteins are inherently transparent, so they need to be labeled in two ways:
- Fluorescent Dye Labeling: Incubate cells with dyes such as FITC or DAPI; the dyes attach to target proteins through chemical interactions (e.g., FITC makes proteins glow green);
- Fluorescent Protein Labeling: Use genetic engineering to introduce genes for fluorescent proteins (e.g., GFP, RFP) into cells, allowing cells to synthesize fluorescent-tagged proteins themselves. This method is suitable for long-term observation of dynamic processes in living cells (a 2008 Nobel Prize in Chemistry achievement).
2. Excitation Light Irradiation: "Activating" Fluorescent Molecules
Irradiate the sample with excitation light of a specific wavelength (similar to shining UV light on a glow stick). Fluorescent molecules only absorb light of a specific wavelength (excitation wavelength) and transition to an excited state. Different molecules have different excitation/emission wavelengths:
- FITC: Excited by 488nm blue light, emits 525nm green light;
- DAPI: Excited by 358nm UV light, emits 461nm blue light;
- Cy3: Excited by 550nm yellow light, emits 570nm orange light.
Thus, multi-wavelength excitation light sources (mercury lamps, LEDs) are required to match different molecules.
3. Emission Light Generation: Fluorescent Molecules "Releasing Light"
Excited fluorescent molecules release energy and return to the ground state in an extremely short time (10^-8 seconds), emitting light with a longer wavelength (lower energy) — this is the "fluorescent signal" to be captured.
4. Signal Filtration and Imaging: Separating and Capturing Signals
The intensity of excitation light is 1000 times that of emission light, so a filter system is needed for filtration:
- Excitation Filter: Only allows excitation light of a specific wavelength to pass (e.g., 488nm blue light);
- Dichroic Mirror: Reflects excitation light to the sample and transmits emission light (e.g., 525nm green light), achieving separation;
- Emission Filter: Only allows emission light of a specific wavelength to pass, filtering out stray light.
The pure signal is captured by the objective lens and imaged through an eyepiece or camera. Bright spots/areas correspond to the location of proteins.
II. Core Components: 4 Key Parts Determining Imaging Quality
1. Excitation Light Source: "Energy Source" for Fluorescent Signals
- Mercury Lamp: Wide wavelength coverage (300-700nm) and high light intensity, but short lifespan (2000 hours) and mercury pollution; gradually being replaced;
- LED Light Source: Precise wavelength (e.g., 488nm, 358nm), long lifespan (50,000 hours), and pollution-free. Multi-color modules (4-5 colors) match multiple fluorescent molecules, making it the first choice for most clients;
- Laser Light Source: Extremely high light intensity and good directionality, suitable for confocal microscopes (3D imaging), but high cost (5-10 times that of LEDs); only used in high-end research.
Foreign Trade Adaptation: Choose multi-color LED light sources, which comply with EU RoHS standards and avoid customs clearance risks.
2. Filter System: "Filter" for Signals
- Bandwidth: Narrower bandwidth means less stray light (e.g., 10nm bandwidth is more precise than 20nm);
- Transmittance: ≥90% transmittance reduces signal loss (high-quality filters reach 95%).
Foreign Trade Adaptation: Request a wavelength matching report from the supplier to ensure compatibility with fluorescent molecules (e.g., FITC matches 488nm excitation + 525nm emission filters).
3. Objective Lens: "Captor" for Signals
- High NA Value: An oil-immersion objective with NA ≥1.3 has strong light-gathering ability, capturing over 3 times more signals and producing brighter images;
- Low Autofluorescence: Choose objectives made of special materials (e.g., Plan-Fluor) to avoid background interference from autofluorescence.
Foreign Trade Adaptation: For living cells/weak signals, select oil-immersion objectives with NA ≥1.3; for thick samples, choose long-working-distance objectives (≥1mm).
4. Imaging System: "Recorder" for Signals
- CCD Camera: High sensitivity, suitable for weak signals (low-expression proteins), but low frame rate (several frames per second);
- CMOS Camera: High frame rate (tens of frames per second), suitable for dynamic observation. High-end sCMOS cameras have sensitivity close to CCD and high resolution (tens of millions of pixels), making them mainstream.
Foreign Trade Adaptation: Choose sCMOS cameras for research, high-frame-rate CMOS cameras for medical diagnosis, and entry-level CCD cameras for teaching.
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