Confocal Microscopy

A confocal laser scanning microscope (CLSM) has gained popularity in recent years due to its ability to provide clear images of specimens that, when viewed through a traditional microscope, would otherwise appear blurry. Images with improved contrast and less haze have been made possible by excluding the majority of specimen light that is not in the microscope’s focal plane. Through the combination of a series of thin slices taken along the vertical axis, it is now possible to construct three-dimensional representations of a volume of the specimen in addition to improving the viewing of small details through thin cross-sections of the specimen. Let’s cover some important details of confocal microscopy.

History of CLSM

To begin with, Marvin Minsky created the confocal microscopy theory that underlies all contemporary confocal microscopes in 1955. Minsky wanted to visualize neural networks in brain cell preparations that hadn’t been labelled. Imaging biological events as they take place in living tissues was the motivation for the invention of the confocal technique. A zirconium arc was utilized as the point source of light in the first iteration of the confocal microscope, which was invented by Minsky. A point of light is successively focused across a specimen, and a point-by-point image is created by gathering part of the returning rays.

The specimen was scanned by moving the stage rather than the light rays in order to maintain a sensitive alignment of moving optics. Thus, Minsky came up with a frame rate of roughly one image every ten seconds. The majority of the unwanted light that obstructs an image might be avoided by simultaneously illuminating the entire sample. The light being returned from the specimen can travel through a second pinhole aperture, which rejects light rays that did not originate from the focal point. Moreover, a photomultiplier gradually reconstructs the image by gathering the remaining desirable light rays.

Confocal Microscopy

• A technique for optical imaging that improves the optical resolution and contrast of a micrograph
• Laser radiation causes the material to glow
• Particularly high-resolution images are produced using a pinhole screen

It is an improved form of fluorescence microscopy that eliminates out-of-focus, improves contrast, and reduces haziness in the images. A succession of thin slices of the specimen is integrated to provide a 3D image.

Working mechanism

A confocal microscope incorporates 2 ideas:

1. Point-by-point illumination of the specimen
2. Rejection of out of focus light.

In Minsky’s design, a zinc arc lamp was utilized as an extremely high-intensity light source, while in later designs,

1. A laser is employed as the light source.
2. The light bounces off a dichroic mirror, sending it to a group of mirrors that scan both vertically and horizontally.
3. The laser beam is scanned across the material by these motor-driven mirrors.
4. The optics are kept stationary while the stage is moved back and forth in a vertical and horizontal orientation to scan the material.

In confocal microscopy two pinholes are typically used:

• To allow transmission just through a narrow area, a pinhole is positioned in front of the illumination source.
• Only one spot of the specimen is lighted at a time because this illumination pinhole is imaged onto the specimen’s focal plane.
• Fluorescence generated within the specimen’s focus plane will pass through the detecting pinhole once the tiny segment has been scanned and put together for improved visibility.

Also, check out-Sample preparation for Electron microscopy – My Biology Dictionary

Application

1. Confocal microscopy enables the examination of thick fluorescently marked specimens without actual physical sectioning.
2. Reconstruction in three dimensions of the specimen
3. More colour options: Since a computer rather than the human eye is used to detect images, there are more colour variations that can be seen.
4. Increased clarity.

Drawbacks

• Resolving power: Diffraction results in an intrinsic resolution restriction. Confocal microscopy’s 200 nm maximum best resolution is usual.
• The size of the pinhole: The strength of optical sectioning is dependent on the size of the pinhole.
• The light’s intensity upon impact
• Fluorophores:

1. A) The fluorophores must identify the proper area of the material to tag.

Fluorophores need to be sensitive enough for the specified excitation wavelength, according to b).

1. c) The organism’s dynamics in the living specimen shouldn’t be drastically changed.

• Photobleaching: When a dye or fluorophore molecule is photochemically altered, it loses its ability to fluoresce permanently.

Benefits of confocal microscopy

• Reducing blurring of the image from light scattering
• Increased effective resolution
• Improved signal-to-noise ratio
• A clear examination of thick specimen
• Z-axis scanning
• Depth perception in Z-sectioned images
• Magnification can be adjusted electronically

Alternative methods to confocal microscopes

Laser scanning confocal microscopes (LSCM)

This technique uses a laser beam that is focused on the object to scan it point by point. It uses a pinhole (or other types of spatial filter) to block undesired fluorescence from above and below the focus plane of interest. Reasons to use the LSCM over traditional epifluorescence light microscopy.

• Glare from blurry images. The specimen has little structure.
• Resolution is particularly improved axially in the Z direction and laterally in the X and Y directions (0.14mm) (0.23mm).
• The LSCM can only be used to simulate thicker specimens like fluorescently labelled multicellular embryos.

Spinning Disk confocal microscopes

Multiple beams scan the specimen simultaneously rather than just one, allowing for real-time visualization of the optical section.

Applications-

• Used to track the dynamics of fluorescently labelled proteins in living cells;
• Used in research where high-quality images are generated at a rapid rate (high spatial and temporal resolution).

Multiple photon microscopes

It has changed since the CLSM. The high-energy pulsed laser with tunable wavelengths that serve as the light source makes a difference, though. As opposed to CLSM, these microscopes’ use of red light enables the collecting of optical sections from deeper within the specimen. Red light is typically preferred for imaging fluorescently labelled living cells since it is less harmful to them than shorter wavelengths (usually employed by confocal microscopes). Multiple photons rather than a single photon are used to stimulate fluorescent molecules. Since the fluorochrome excitation is limited to the point of focus in the specimen, there is less likelihood that it may overexcite (photobleach) the fluorescent probe and lead to the photodamage of the sample. Only when energy levels are sufficiently high and statistically limited to the objective lens’ point of focus is multiple photon excitation of a fluorophore achievable.

Thank you for reading!

Team MBD

Also, check out Confocal microscopy principle tutorial – YouTube