A traditional simple microscope creates a murky and fuzzy image of 3-dimensional objects, because light from all areas of the object, not just the plane of focus, enters the microscope. This problem has been solved by the confocal scanning laser microscope (CSLM), or simply, confocal microscope.
- Confocal Microscope also called confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM).
- A confocal microscope creates a high optical resolution and high contrast image with a spatial pinhole, which blocks out-of-focus light in image formation.
- In an ordinary simple microscope, light passes through the sample, whereas in a confocal microscope focuses a smaller beam of light at one narrow depth level at a time.
- It creates a 3D picture of the specimen by using a technique, called optical sectioning. In this, a microscope captures multiple 2d pictures of the sample and reconstructs a three-dimensional structure.
- This microscope is used in life sciences, semiconductors,and materials science.
- In the 1950s, Marvin Minsky first developed the confocal microscope at Harvard University.
Principle of Confocal Microscope
Confocal Microscope uses fluorescence lights to create micrographs of specimens. In a confocal microscope, the laser light is focused onto a defined spot at a specific depth within the sample.
As a result, fluorescent lights are started to emit from the exact point. A pinhole located inside the optical pathway, it only allows the fluorescence signals from the illuminated spot to enter the light detector and cuts off signals that are out of focus. It scans the specimen in a raster pattern and creates a 3D picture of the specimen.
Mechanism of Confocal Microscope
- A confocal microscope uses laser beams instead of lights. The laser beams are released from their source and then focused onto a fluorescent stained sample.
- Neutral density filters and a set of scanning mirrors control the intensity of the laser light by moving them very precisely and quickly.
- One mirror tilts the beam within the X route, the opposite within the Y route. Together, they tilt the beam in a raster style.
- Then an objective lens focuses it onto the sample.
- The fluorochrome stained sample will be excited and then it will emit fluorescent lights.
- These fluorescent lights will travel back into the objective lens through the same path that the laser travels.
- The main effects of these scanning mirrors are on this light is to generate a spot of light which is not scanning, but standing still.
- Then a semi-transparent mirror reflects this fluorescent light away from the laser and toward the detection system.
- Before entering into the detection system, it passes through a pinhole. This pinhole allows only a small central portion of the light through to the light detectors.
- Confocal microscope produces a very low-intensity light, so the light is amplified by a photomultiplier tube (PMT).
- Photomultipliers have the ability to amplify a faint signal around one million times without introducing a single noise.
- After that, the PMT releases an electrical signal, which is then converted into an image by using a computer.
Parts of Confocal Microscope
A. The leaser
It can be chosen via a selection device and is matched with the fluorophores used in your experiment.
B. Beam splitter
It separates the excitation from the emitted light in the fluorescence beam path of the microscope.
It guides them focussed laser beam across the specimen, pixel-by-pixel, and line-by-line.
D. Objective lens
It determines the optical image formation and the resolution of the system.
- This allows the user to focus the laser beam on any focal plane within the specimen.
- The motorized Z-stepper allows us to move around the axial direction in small step sizes (approximately >10 nm) with high precision.
- Pinhole is a type of adjustable iris.
- Pinhole allows the exclusion of most of the out-of-focus light from the acquired image and thus provides optical sectioning capacity.
- The size of the pinhole can be set by using the software on the user’s computer.
G. Photomultiplier tube (PMT)
It converts the photons into an electrical signal which is then used up by the computer to create an image of the specimen.
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Application of Confocal Microscope
- Used for the examination of various eye diseases.
- Used for qualitative analysis, and quantification of endothelial cells of the cornea.
- Used for localizing of filamentary fungal elements in the corneal stroma in cases of keratomycosis.
- It also widely used in the pharmaceutical industry to control the quality and uniformity of the drug distribution.
- Used to determine the age of the Magdalen papyrus.
- Used in 3D optical data storage systems.
- It is also used for optical scanning and recovery of damaged historical audio.
Advantages of Confocal Microscopy
- It creates a high-resolution image.
- It creates 3D image of the specimen.
- Living and fixed cells can be used.
- Used in the collection of serial optical sections.
- Confocal Microscope illuminates uniformly across the focus points.
Disadvantages of Confocal Microscopy
- Confocal Microscopes are very expensive.
- It contains a limited number of excitation wavelengths, with very narrow bands.