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Seeing dynamic processes in cells: How does fluorescence imaging help us understand gene expression?
Fluorescence imaging is a non-invasive imaging technique that helps us visualize biological processes occurring in living organisms. This technique uses a variety of methods including microscopy, imaging probes and spectroscopy to generate images. Fluorescence is essentially a luminescence phenomenon that occurs when a substance absorbs electromagnetic radiation and then releases light of a specific wavelength. Molecules that can emit light are called fluorophores. Fluorescence imaging uses fluorescent dyes and fluorescent proteins to label molecular machinery and structures, enabling experimental observation of the dynamic processes of gene expression, protein expression, and molecular interactions.
Fluorescence imaging provides a precise quantitative tool for biochemical applications.
There is often a misunderstanding between fluorescence and bioluminescence, the difference between the two being the protein process that produces the light. Bioluminescence is a chemical process involving enzymes breaking down substrates to produce light, whereas fluorescence is the physical excitation of electrons followed by their return to the ground state to release light.
Fluorescence Mechanism
When a molecule absorbs light, the energy of the molecule briefly rises to a more excited state. When it subsequently returns to its ground state, it emits fluorescent light, which can be detected and measured. The specific wavelength of the emitted light depends on the energy of the absorbed photons, so this wavelength needs to be known in advance in the experiment so that the measurement equipment can correctly detect the generation of light.
The formula for determining the fluorescence emission wavelength is: λ emission = hc / Energy emission
Here, h is Planck's constant and c is the speed of light. Typically, a large scanning device or CCD is used to measure the intensity and digitize the image.
Fluorescent dyes and proteins
Fluorescent dyes have higher photostability and brightness and do not require maturation time compared to fluorescent proteins. In terms of brightness, the extinction coefficient (the ability to absorb light) and quantum efficiency (how well it converts absorbed light into fluorescent light) of a fluorophore are closely related. The dye itself is not very fluorescent, but when it is bound to a protein, it becomes more detectable. For example, NanoOrange can bind to the coating and hydrophobic regions of proteins and is not affected by reducing agents.
Proteins can autofluoresce when they absorb incident light of a specific wavelength. For example, green fluorescent protein (GFP) emits green light when exposed to light in the blue to ultraviolet range. Fluorescent proteins are excellent reporter molecules that help locate proteins, observe protein binding, and quantify gene expression.
Imaging range
Because some fluorescence wavelengths are beyond the range of the human eye, CCD is used to accurately detect the light and form an image. This is typically done in the 300–800 nm range. One advantage of fluorescence signals is that the relationship between the intensity of the emitted light and the number of fluorescent molecules present is generally linear, essentially requiring that the incident light intensity and wavelength remain constant. The final image is usually rendered in 12-bit or 16-bit data format.
Imaging System
The main components of a fluorescence imaging system include: an excitation source (which can produce broad wavelength light or laser light), light display optics (used to illuminate the sample), and light collection optics (usually composed of lenses, mirrors, and filters). , and detection, amplification, and visualization devices (such as photomultiplier tubes or CCDs).Applications
Fluorescence imaging has been widely used in different scientific fields, including:
- SYBR Green is a common dye used to visualize DNA bands in PCR (agarose gel electrophoresis).
- Use fluorescence imaging to aid navigation in transplantation. For example, Indian Teal can be used in cancer patients to detect lymph nodes.
- In calcium imaging, fluorescent molecules are used to monitor the activity of living cells in the nervous system.
Advantages and Disadvantages
Although fluorescence imaging has some advantages, such as non-invasive operation and high sensitivity, it also has some challenges, such as fluorescence bleaching, environmental sensitivity and limited resolution capabilities.Future Directions
Scientists are working to develop more efficient fluorescent proteins to improve the performance of imaging probes. Through methods such as genetic engineering and environmental stabilization, future fluorescence imaging technology is expected to achieve breakthroughs in multiple dimensions.
Fluorescence imaging provides a wide range of opportunities to explore what's going on inside cells, so what new biological phenomena might future discoveries reveal?
