Definition
A fluorescence microscope is an optical instrument that uses a specific wavelength of excitation light to irradiate a sample, causing the fluorescent substances in the sample to be stimulated to emit fluorescence, thereby performing high-contrast imaging. It is widely used in life sciences, materials science and medical diagnostics and other fields, and is an important tool for studying subcellular structure, biomolecular localization and dynamic processes.
Principle
The working principle of fluorescence microscopy is based on the phenomenon of fluorescence. When a fluorescent substance (e.g., fluorescent dye, fluorescent protein) absorbs excitation light at a specific wavelength (shorter wavelength), its electrons transition to the excited state, and then releases energy in the form of light at a longer wavelength when it returns to the ground state, i.e., fluorescence. The instrument typically consists of a light source, excitation filter, dichroic mirror, objective lens, emission filter, and detector. The excitation light is screened by a filter, reflected from the dichroic mirror to the objective lens and focused on the sample, and the excited fluorescence is collected by the objective lens, passed through the dichroic mirror and the emission filter, and finally received by the camera or eyepiece for imaging. The core process can be described by a formula: fluorescence intensity is related to excitation intensity, fluorescent substance concentration, and quantum yield, and is approximately expressed as If ∝ I0 · C · Φ, where If is the fluorescence intensity, I0 For the excitation of light intensity, C is the concentration of fluorescent substances, and Φ is the quantum yield.
Measurement method
Fluorescence microscopy measurements mainly involve fluorescence imaging and quantitative analysis. During the imaging process, the matching excitation/emission filter combination is selected based on the characteristics of the fluorescent substance, and the appropriate light intensity and exposure time are set to avoid signal saturation or photobleaching. For static samples, 2D images can be acquired by widefield fluorescence microscopy; For dynamic processes or thick samples, confocal microscopy is often used for optical sectioning and 3D reconstruction. During quantitative analysis, molecular expression levels or interactions can be assessed by measuring parameters such as fluorescence intensity distribution, colocalization coefficient, or fluorescence resonance energy transfer efficiency. Follow relevant standards (e.g., ISO 10934 guidance on optical microscopy) and regularly calibrate the instrument with fluorescence standard samples to ensure measurement consistency.
Influencing factors
The imaging quality of fluorescence microscopes is influenced by several factors. In terms of optical system, the numerical aperture and light transmittance of the objective lens determine the resolution and signal collection efficiency. The bandwidth and barrier of the filter affect the signal-to-noise ratio. In sample preparation, fluorescent labeling efficiency, background autofluorescence, and refractive index of the mountant may cause signal interference or image distortion. Environmental conditions such as temperature fluctuations or mechanical vibrations can also reduce stability. In addition, phototoxicity and photobleaching can damage live samples or attenuate fluorescence signals, which need to be controlled by optimizing lighting strategies, such as using LED cold light sources. The focus accuracy of the user's operation and the setting of the image processing parameters also have a direct impact on the results.
Application
Fluorescence microscopy has a wide range of uses in several fields. In life sciences, it is used to observe organelle morphology, protein localization, gene expression, and cell-cell interactions, such as the detection of specific antigen distributions by immunofluorescence techniques. In medical diagnosis, it can be used for pathological section analysis or microbiological detection to assist in disease judgment. In materials science, the luminescence properties or polymer structures of nanomaterials can be studied. In addition, combined with fluorescence in situ hybridization, calcium ion imaging or photoactivation technology, genetic analysis and dynamic physiological process monitoring can also be realized. These applications benefit from the diversity of fluorescent labeling technologies and the continuous expansion of instrument capabilities.
Selection
When choosing a fluorescence microscope, it is necessary to consider the needs of the study and the characteristics of the system. For conventional microscopy, the structure of widefield fluorescence microscope is relatively simple, suitable for rapid two-dimensional imaging; For high-resolution 3D imaging or weak signal detection, confocal microscopy or total internal reflection fluorescence microscopy are more suitable. When configuring, attention should be paid to the stability and longevity of the light source type (e.g., mercury lamp, LED, or laser), the flexibility and accuracy of the filter set, and the sensitivity and dynamic range of the detector (e.g., CCD or sCMOS). In addition, system scalability (e.g., support for multiphoton and super-resolution modules) and software analysis capabilities need to be evaluated. It is recommended to refer to international standards such as ASTM E1242 on performance evaluation of fluorescence microscopy for comparison and test against actual samples to ensure that the instrument meets the requirements of long-term use.