In life science, advanced techniques like fluorescence imaging are widely known. For the unversed, George Gabriel Stokes defined fluorescence in 1852. He discovered that fluorite shines after coming into contact with UV light. So, fluorescence refers to the event of photon emission caused by the illumination of a material by light. The wavelength of the emitted photons or light can be longer than that of exciting light or Stokes shift. A higher level of energy marks the excited state.
Nevertheless, fluorescence has become integral to imaging and microscopy today. This tool helps trace targeted molecule movement. Many cellular molecules don't shine without being labeled by fluorescing molecules, which is part of immunostaining. Fluorescence microscopy also helps capture live tissue and cells at different intervals when proteins are marked with a genetically programmed green fluorescent protein or other fluorescing molecules. This technique has many medical uses, so let's learn more about it.
- Fluorescence imaging
This microscopic method facilitates visualizing tiny biological structures and processes with high-resolution light sources and fluorescent labeling and removes the problems faced in biological imaging. Researchers resort to this tool to target specific features at a smaller scale. Think of different brain cells, for example. You can also imagine tracking a cell's genetic material. How does a fluorescent imager work? All these processes rely on fluorescence mechanisms to target and stimulate specific molecules. The phenomenon occurs when high-intensity light with the correct wavelength comes into contact with the fluorescently labeled molecules. The ground state electrons become more energetic but remain unstable. After the excited state, they return to the lower levels. It is seen as fluorescence.
Earlier, researchers used brightfield microscopy to capture biomolecular samples with enhanced contrast. Today, advanced fluorescence imaging mechanisms offer better 3D resolution and precision, empowering bioengineering and life sciences fields. Fluorimetry, Molecule localization microscopy, Total internal reflection microscopy, Fluorescence-lifetime imaging microscopy, and Fluorescent widefield microscopy are examples of this imaging system.
- Imaging system setups
The success of a fluorescence imaging system depends on the use of the right light wavelength and fluorescent dyes. Many options are available to acquire low-noise fluorescent signals. For example, a fluorometer can apply a multi-wavelength light source with 200 to 750 nm wavelength and a monochromator to send select, well-adjusted thin wavebands. Due to numerous excitation wavelengths, this process can measure different components during a scan. However, laser light is considered more efficient and quicker in targeting fluorophores.
- Fluorescence imaging advancements
The use of fluorescence microscopes in research and science is a century-old practice. However, the technology evolves rapidly, allowing experts to apply it widely. Here is a small glimpse of this:
- Live cell observation
Scientists study live cells during different imaging experiments. In live-cell imaging, fluorescent dyes contribute immensely. These have reduced scientists’ dependence on phototoxic ultraviolet dyes. Markers have also improved. Some systems use Halo-Tags for imaging live cells in NIR, red, and far-red. These tags can be combined with artificial fluorescent dyes. This system's use of red-shifted light is greatly admired for its reduced reliance on phototoxic effects and for providing in-depth imaging. Another extremely fluorogenic dye is silicon rhodamine dye or the far-red. When interacting with the target, the process produces excellent fluorescence output.
- Single-cell measuring mechanism
Fluorescence microscopy can also help observe molecular interactions because of its fluorescence lifetime quality. It can obtain any biological sample’s functional property. For example, fluorescence imaging can classify calcium fluctuations in cellular bodies containing a calcium sensor, such as Oregon Green 488-BAPTA. It can do this outside the interaction between the cells and dye. Interestingly, fluorescent dyes perform much better today because of their stability and brightness.
- Common issues with fluorescence imaging and their solution
Microscopes must have good focus to capture images for critical experimental results because poor focus can affect the quality of work. If you face any such problem with this equipment, check that the lens type is suitable for observation. The numerical aperture (NA) is an essential ingredient in fluorescent observation. A large NA can offer high-resolution figures with more details. The field depth is reduced to bring the target into the spotlight.
Another thing you can check is the lens glass. Lenses require magnification adjustment—lower magnification works with a broader vision field, and higher magnification helps with the targeted area. One must take care of this, even if it means additional effort. Furthermore, correct imaging also requires the lens to be placed closer to the sample before moving away. Typically, microscopes with collision prevention control can be helpful in this area. Some people also encounter the issue of poor contrast or dark signals. Images received from this method must exhibit accurate fluorescent protein signals. This result can be achieved when all the units, such as the light source, filter, camera, etc, work perfectly. Otherwise, it's common to encounter those problems, and images will lack clarity.
- Pros of fluorescence imaging
As observed, many problems can be fixed when working with this technology. Clinical scientists use this tool in materials science, medicine, and biology. They depend on it to see and multiply the targeted molecular structures of a sample. The tool is preferred for its high sensitivity levels, which can track even the sparse molecules. By binding fluorescent tags to targets, this imaging technique fetches precise details of the molecules, signaling their location and concentration in the sample. This type of imaging is also unharmful, which enables scientists to observe live cells and their processes effortlessly. Multiple other benefits are also there.
Like any other method, fluorescence microscopy can have some downsides. One of them is the expense of the equipment and process. However, these complex studies need meticulous and highly trusted solutions to make their findings beneficial for humankind and anything related. Therefore, choosing advanced methods or technologies should be the priority. The effectiveness of the research results and their comprehensive application must be emphasized over other things. Hence, you can use this tool well by tapping into its strengths and overcoming weaknesses. If you need support with these technologies, please visit only trusted life sciences enterprises or companies.
