Confocal Laser Scanning Microscope: Principle, Parts, Uses microbiologystudy

In 1952, Marvin Lee Minsky, an American computer scientist, patented confocal imaging based on the idea of producing a point source of light and rejecting out-of-focus light using illumination. Confocal microscopy is used for its ability to provide images with high resolution and 3-D reconstruction of images with optical sectioning.

Confocal Laser Scanning Microscope (CLSM) is based on the foundation of confocal imaging aimed at solving a major difficulty in observing and examining dense tissues conferred by conventional light microscope.

Since the 1980s, the development and advancements in instrumentation, computation, and hardware/software technology of CLSM have made it a powerful characterization tool in life science and materials science.

Today, it has become an important microscopic technique used widely for studying 3-D cells and tissue structures, co-localization of cell organelles, detailed analysis of tissue morphology, and dynamic activities in live cells. 

A laser scanning confocal microscope (LSCM) uses a laser beam and galvanometer mirrors to scan the sample. The laser beam is directed onto mirrors that sweep it in both horizontal and vertical directions to capture a single field of view. The process then repeats to cover the entire sample, producing an optical section or slice. To create a z-stack, the focal point is adjusted, and the scanning process is repeated for each new slice. Once all optical sections are collected, a 3D image of the sample can be reconstructed.

This technology has been implemented commercially and has a wide range of applications, mostly in research and imaging laboratories. Due to its noninvasiveness, it has been broadly used as dermatologic imaging technology to perform sectioning of skin, visualize cutaneous structures and dynamic behavior, monitor disease progression, and perform optical biopsies on skin. 

CLSM can be conducted in either fluorescence or reflectance mode, depending on the image contrast source. Fluorescence confocal microscopy (FCM) utilizes a fluorescent agent to create contrast with remarkable results in experimental studies involving both lesional and nonlesional skin. Reflectance confocal microscopy (RCM) depends on variances in the refractive indices of cellular structures and has been widely used for the noninvasive evaluation of both melanocytic and non-melanocytic skin tumors, along with dermoscopic and histologic examinations.

Principle of Confocal Laser Scanning Microscope

The operation of confocal imaging is based on the principle that both the illumination and detection optics are precisely focused on the same diffraction-limited spot, which is then scanned across the sample to construct the entire image on the detector. Although confocal imaging illuminates the entire field of view, anything beyond the focal plane has minimal impact on the image, reducing the blur often seen in standard light microscopy when dealing with thick and highly scattering samples and allowing for optical sectioning.

When a laser or any other light source illuminates a pinhole, the light that emerges from the pinhole travels through a beam splitter and is focused by an objective lens to a spot in the focal plane where the sample is positioned. The light reflected from this spot is partially directed by the beam splitter toward the pinhole in front of the detector. Any light reflected from other parts of the sample, including areas above or below the focal plane, reaches the edge of the detector pinhole and is not detected by the detector.

The objective lens creates an image of the detector pinhole and the illuminating pinhole at the same location in the focal plane, making them confocal. The selective rejection of light produces sharp images and enhances the depth resolution of confocal microscopes. This property of depth discrimination makes confocal microscopes advantageous over others. 

Parts of Confocal Laser Scanning Microscope

• Laser: The fluorophores in the sample are illuminated using laser photons in CLSM techniques. A selection device can be used to select laser lines and match them with the fluorophores.
• Beamsplitter: The separation of the excitation laser light and the emission light from the fluorescent sample is carried out using a filter, called beamsplitter. It ensures that the user detects light from the sample.
• Scanner: Consisting of two or more mirrors, this unit directs the focused beam of laser light across the specimen by raster scanning in pixels, lines, and direction.
• Objective Lens: The formation of the image along with the resolution limits of the microscope is determined by the objective lens.
• Z-control: In the microscope, the Z-control allows the construction of 3D images, enabling one to focus anywhere on the focal plane within the sample. Moreover, the Z-stepper which is motorized enables the monitoring of step movements precisely from the normal to the horizontal plane of the sample.
• Pinhole: One of the key components of confocal imaging, the pinhole is an adjustable iris in the intermediate image plane that controls the amount of out-of-focus light in the captured image, allowing for optical sectioning. It determines the thickness of the optical slice and is influenced by the objective lens properties. The pinhole size can be adjusted using computer software, and setting it to 1 Airy unit provides the best balance between light collection efficiency and optical sectioning.
•  Photomultiplier Tube: These are detectors that are highly sensitive and used to gather photons emitted by the sample. The transmitted light signals are transformed into electrical signals by the photomultiplier tube which is recorded by the computer. It is also responsible for amplifying the current generated by the incident light at different levels of magnitudes.

Sample Preparation for Confocal Laser Scanning Microscope

When the 3D structure of the specimen is to be studied using the confocal approach, the sample should be mounted carefully such that the structure is preserved. This can be obtained by placing a spacer between the slide and the coverslip. Similarly, the study of samples containing living cells is accompanied by mounting them in such a way that the essential requirements are provided. Moreover, the use of a chamber also enables the objective lens with adequate access to view the required area of the sample. 

It is crucial to understand that the properties of the ‌‌‍‍​‍sample affect the opacity, turbidity, and transmission of light. This, in turn, affects the way the laser beam penetrates the sample, influencing the image of the structures to be viewed. For example, the unstained and transparent sample of corneal epithelium will be penetrated by a depth of 200 micrometers by the laser beam while the unfixed and opaque sample skin limits the penetration of the laser to 10 micrometers. Therefore, various fixation procedures with clearing agents increasing the transparency of the samples are used. Moreover, to ensure adequate laser penetration, a microtome is used to cut the thick specimen.

Steps of using Confocal Laser Scanning Microscope

• The lights of the operating room are turned off. Turn on the microscope by switching the system switch and components switch on. 
• Log in to the computer to start the software and access the confocal imaging. The slide is calibrated through the stage focus option available in the program. 
• After proper calibration, the sample is loaded into the stage of the microscope. 
• The sample is located by the use of an eyepiece and fluorescent channels through the software program. 
• The confocal laser imaging begins by configuring the settings by opting the “Accusation” tab. Then, the emission is set using the “imaging setup” in the software. 
• The image of the sample is viewed by pressing the “Live” option. Adjust the laser intensity, master gain, and fine focus to obtain a clear and bright image. The image can be captured using the “Snap” option. 
• To turn off the microscope, the focus is set to 5x. The objectives are lowered to the minimum value. The components switch is turned off followed by the system switch. 

Applications of Confocal Laser Scanning Microscope

• In cell biology, it is used to observe and study cell organelles, membranes, and cytoskeleton. In neuroscience, it is used for studying neurons, dendrons, and synapses. Similarly, it is used in immunology to study immune cells and tissues and in developmental biology to study embryos and organs.
• This technique also examines the environmental samples to analyze the presence and distribution of pollutants.
• In materials science, it has been of great significance in observing and analyzing polymers, ceramics, the surface roughness of metals and cement, molecule dispersion in materials, and the growth process of inorganic crystals.
• Moreover, it is also used in investigating and analyzing skin structures and monitoring tumor progression in case of skin carcinogenesis. In cancer research, it is used to examine cancerous tissues and understand interactions between drugs and tumor cells.
• CLSM technique also allows the live viewing of unusual tissue organization, abnormal formations, and the blood circulation and vessels associated with skin growths. It is also used in the diagnosis of inflammatory skin diseases and dermatologic conditions. 

Examples of Confocal Laser Scanning Microscope

1. ZEISS LSM 980 with Airyscan 2

• The LSM 980 beam path design ensures imaging with the highest sensitivity, which is key to visualizing the lowest signal and resolving all structures, as well as spectral flexibility, allowing you to freely select fluorescent labels from 380 nm to the near-infrared (NIR) range.
• Airyscan 2 allows you to do more than any conventional LSM detector. Each of its 32 detector elements collects additional information while all of them together gather even more light, yielding super-resolution quantitative results.

2. FV4000 Confocal Laser Scanning Microscope

• Superior image quality with ultra-low-noise SilVIR™ detector and an industry-leading six channels, ten laser lines, and 400–900 nm dynamic range.
• Acquire confocal images up to 60x faster and super-resolution images up to 8x faster than the FV3000.
• Count the number of photons in each pixel and represent features as discrete histograms of photons captured at different wavelengths.
• The game-changing dynamic range enables one to count from a few photons to thousands with linearity—a first in confocal microscopy.
• Easy to use with minimal adjustments required to obtain high-impact images and data.



3. STELLARIS Confocal Microscope Platform

• It offers enhanced photon detection efficiency, minimal dark noise, and sensitive spectral detection across the range of 410 to 850 nm.
• It can maintain the integrity and appearance of the sample for longer durations by utilizing efficient signal acquisition at minimal illumination levels.
• Enhanced image quality: Ideal combination of brightness, resolution and contrast.
• Gentle live cell imaging.

4. Nikon AX / AX R Confocal Microscope System

• It has both upright and inverted microscopes with the availability of the widest field-of-view and scanning sizes. 
• Its rapid resonant scanning feature shortens the illumination duration, significantly minimizing biases arising from image acquisition.
• With a full 25mm FOV, up to 8192 x 8192 pixels, incredibly low noise, and the capability for supra video frame rates, the AX/AX R allows for spectacular imaging with high resolution at any magnification.
• Drive research and drug discovery with more data in every image, collected at higher speeds.

Advantages of Confocal Laser Scanning Microscope

• It allows both live imaging and visualization of fixed samples. It has a simple operation and allows imaging under wet conditions as well with high contrast. 
• It is a versatile instrument capable of optical sectioning, 3-D imaging, and resolution with limited diffraction. 
• It also allows multi-color visualization. 
• It allows a maximum magnification of 2000X. 
• During visualization, it highlights the individual cellular components in the sample. 
• As it allows the construction of 3-D images, it accurately analyzes the spatial structure of samples. 

Limitations of Confocal Laser Scanning Microscope

• In comparison to the electron microscope, it has a lower resolution. 
• It has a limited depth of penetration, allowing surface imaging, especially in skin samples. 
• Phototoxicity and low imaging speed are some of the limitations. 
• It produces artifacts due to fluorescence. 

Precautions

• Care must be taken while using the objective lenses. Avoid contact between oil and dry/water lenses. 
• A 10% bleach solution should be used to clean the area in case of a spill.
• The lens must be cleaned using 70% ethanol with the help of a cotton applicator. 
• Do not forget to warm up the laser before use to ensure stability in its power. 
• Once turned on, the mercury lamp should be in use for no less than 30 minutes. After use, it must remain turned off for no less than 30 minutes. 
• Avoid plugging anything in the ports of the confocal system unless the software has been turned off. 
• The system must be cooled down for 5 minutes before turning off the system switch. 

Conclusion

With rapid advancements in the scientific world, Confocal Laser Scanning Microscopy has emerged as a remarkable and indispensable technique widely used in fundamental and applied research across various disciplines, including chemistry, biology, life sciences, and engineering.

The wide range of applications and high-quality output offered by this microscopy has deepened the understanding of structural components, cellular dynamics, material visualization, and reaction processes among researchers, scientists, and medical practitioners. Its ability to produce high-resolution images with minimal distortion and quantitative analysis of images has contributed to significant development in medical and material science.

In conclusion, the introduction of advanced strategies along with this confocal laser scanning technique has the potential to open new opportunities and possibilities in the research arena.

References

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