An optical microscope called a fluorescence microscope employs the fluorescence principle to image biological specimens. A molecule can absorb light of one wavelength and then fluoresce, which is the process by which it emits light of a longer wavelength. Fluorescence is the term for this released light. By being aware of the variables that may impact the quality of your images, you may take action to reduce the effects and get better outcomes. For crucial research or applications in medicine, this may be extremely important. Knowing what factors may reduce the quality of the fluorescence microscope images can help you locate and address any issues. For example, an incorrect microscope or a poor-quality objective lens may be to blame for hazy images. The following factors can reduce the quality of fluorescence microscope images:
Factor 1: Fluorescence signal
The fluorescence signal is the intensity of the light emitted by the fluorescent sample. This signal can be reduced by a number of factors, including:
- Fluorescence microscope sample preparation: A strong fluorescence signal can only be produced by a properly prepared sample. This could involve mounting the sample in a suitable medium, staining, or fixing it.
- Photobleaching: Fluorescent molecules can be photobleached, or destroyed, by exposure to light. This is particularly problematic when using high-intensity fluorescence microscope light sources like lasers. Fluorescent molecules are destroyed through a process known as photobleaching when they are exposed to light. This is particularly problematic when using high-intensity fluorescence microscope light sources like lasers. A fluorescent molecule loses its ability to emit light when it is photobleached. This may lessen the image’s contrast and make it challenging to discern the fluorescent signal. Loss of sample data, such as the distribution of fluorescent molecules, can also result from photobleaching.
- Background fluorescence: Fluorescence that comes from unidentified sources, such as the mounting material or the microscope itself, is known as background fluorescence. This could reduce the image’s contrast and make it difficult to figure out the fluorescent signal.
Factor 2: Fluorescence microscope camera noise
Because it might be challenging to distinguish between the fluorescence signal and the background, fluorescence microscope camera noise is one of the reasons that lowers the quality of fluorescence microscope images. Camera noise is errant noise that the camera sensor adds to the image. Many things, including the type of sensor, the temperature, and the exposure period, might contribute to it. There are two main types of camera noise that are relevant to fluorescence microscope:
- Readout noise: The noise that is added to the fluorescence microscope image during readout occurs when the camera sensor reads the signal from every pixel in the image. When compared to sCMOS cameras, it is often higher in CCD cameras.
- Shot noise: The statistical characteristics of light result in a particular sort of noise called shot noise. Images with low light levels, like fluorescence images, usually have greater values.
Factor 3: Background light
Any light that reaches the camera sensor and is not produced by the fluorescent sample is known as background light. This could be light coming from the environment, the microscope, or other sources. The fluorescence signal may be difficult to discern due to the background light’s ability to lower the contrast of the image. One of the factors that lowers the quality of fluorescence microscope images is background light, which can make it harder to discern the fluorescence signal and lower the contrast of the image. A fluorescence microscope light source of a specific wavelength is used for illuminating the specimen in a fluorescence microscope, and the light that is emitted is then collected. To properly notice the fluorescence signal, background light must be kept to a minimum because the emitted light is often much weaker than the excitation light. Background light can come from a variety of sources, such as:
- Microscope light: The fluorescence microscope light source and objective lens of the microscope in particular can emit some light.
- Sample fluorescence: When exposed to the excitation light, some non-fluorescent molecules in the sample can produce light. The term for this is autofluorescence.
Factor 4: Microscope alignment
One of the factors that reduce the quality of fluorescence microscope images is microscope alignment since it might result in hazy images and uneven lighting. Producing clean and clear images requires proper microscope alignment. Aligning the fluorescence microscope light source, the condenser, the objective lens, and the eyepieces are part of this process. The image will be damaged if any of these elements are out of alignment. The production of even illumination also depends on the proper alignment of the microscope. The image will be more brightly lit in certain parts than in others if the fluorescence microscope light source is not correctly positioned. The fluorescence signal may be difficult to observe clearly as a result.
Factor 5: Objective lens quality
Objective lens quality is one of the factors that can reduce the quality of fluorescence microscope images because it can lead to blurred images, chromatic aberration, and spherical aberration. Images produced by a high-quality objective lens will be more clear and precise. Particularly at high magnifications, a poor objective lens may result in images that are blurry. The type of aberration known as chromatic arises when light with various wavelengths is focused at different distances from the objective lens. This may result in vibrant fringes around the edges of picture objects. When light from various areas of the objective lens is focused at various distances from the objective lens, a type of aberration known as spherical aberration results. Images that are blurry may result from this.
Factor 6: Fluorescence microscope camera settings
One of the factors that may reduce the quality of fluorescence microscope images is the fluorescence camera settings, which can change the image’s exposure, gain, and white balance. The image may be overly bright, too dark, too noisy, or have the wrong color temperature if the camera settings are not ideal for a fluorescence microscope. The fluorescence signal may be difficult to observe clearly as a result.
- Exposure is the amount of time that the fluorescence microscope camera sensor is exposed to light. More light will be captured with a longer exposure time, but the image’s noise level will increase as well. Even though a shorter exposure period will collect less light, the resulting image will be less noisy.
- Gain is the amplification of the signal from the fluorescence microscope camera sensor. The signal will be amplified more with a greater gain, but the picture noise will also be amplified. A lower gain will result in less signal amplification, but it will also result in a cleaner image.
- White balance is the adjustment of the color temperature of the image. White balance can be utilized to give the image’s hues a more natural appearance.
A fluorescence microscope is a powerful tool used to visualize fluorescently labeled molecules and structures in biological and non-biological samples. Fluorescence microscope applications are in a wide range of fields, including cell biology, developmental biology, immunology, neuroscience, materials science, and chemistry. Researchers and doctors can create high-quality images that give significant insight into the structure and function of cells and materials by knowing the basic principles of fluorescence microscopes and the factors that can affect image quality.