CONFOCAL MICROSCOPE

CONFOCAL MICROSCOPE

·       A Confocal microscope is a type of microscope that uses laser light to produce high-resolution images of samples at different depths within the sample.

·       The basic principle of Confocal Microscopy is that the Illumination and Detection optics are focused on the same Diffraction-limited spot, which is moved over the sample to build the complete image on the detector.

·       The modern Confocal microscope has all the possible integration of technology and mechanical components including optical components, which perform the primary function of the configuration by use of electronic detectors, a computer, and laser systems.

·       Over the past three decades, Confocal microscopy has evolved as a useful, non-invasive, imaging technology that has diagnostic and prognostic implications.

·       Confocal microscopes are widely used in many different fields, including Biology, Medicine, and Materials science, to study the structure and function of cells, tissues, and molecules at the microscopic level.

·       Currently, the Laser Scanning Confocal Microscope (LSCM) is the most used confocal version for biomedical research.

Advantages of Confocal microscopy over conventional Optical microscopy

·       One of the main advantages of Confocal microscopy is its ability to produce high-resolution, three-dimensional images of samples. This is achieved by scanning the sample in a series of layers or optical sections and reconstructing the image using computer algorithms.

·       Confocal microscopy can also be used to visualize multiple different Fluorophores within a single sample, allowing for the simultaneous visualization of multiple processes or pathways.

·       Some of the advantages of Confocal microscopy over conventional Optical microscopy are

a)     Short depth of field.

b)    Elimination of out-of-focus glare.

c)     Ability to collect optical slices serially from thick specimens.

d)    Ability to produce high-resolution, three-dimensional images of samples.

e)     Used to visualize multiple different Fluorophores within a single sample.

History of Confocal microscope

·       The concept of Confocal microscopy was initially developed by Marvin Minsky in the 1955 at Harvard University with an aim of viewing the Neural network without staining the tissues but it did not bear fruit due to lack of enough light source and a computerized system to store the large data.

·       The work of Marvin Minsky was later adapted by David Egger and Mojmir Petran, forming a Multiple-beam Confocal microscope in the late 1960s. They used a Spinning disk known as Nipkow which they used to examine brain tissues and ganglion cells that were unstained. The technique was later modified and published by Egger forming a mechanical Scanned Confocal Laser Microscope, that was able to visualize images of cells.

·       The first practical Confocal microscope was developed by Marvin Minsky and a team of researchers in the 1970s. The first Confocal microscope used a Helium-neon laser and a Photomultiplier tube to detect the Fluorescence emitted by the sample.

·       The first commercial Confocal microscope was developed in 1987 with improved optics and electronics, powerful lasers with high scanning efficiency. The development of Solid-State Lasers and Charge-Coupled Device (CCD) cameras led to the widespread adoption of Confocal microscopy as a research tool.

·       In the 1990s, the development of Multi-photon excitation techniques and other advanced imaging techniques further expanded the capabilities of Confocal microscopy.

Parts of Confocal microscope

a)     Objective lens: The objective lens is a high-resolution lens that is used to collect and focus light from the sample.

b)    Laser light source: The Laser light source is used to produce a focused beam of light that is used to scan the sample and excite the Fluorophores within the sample. It can be chosen via a selection device and is matched with the fluorophores used in your experiment.

c)     Scanning system: The scanning system is responsible for moving the laser beam over the sample in a raster pattern to produce an image. It is typically based on a Galvanometer or Acousto-optic modulator.

d)    Beam splitter: It separates the excitation from the emitted light in the Fluorescence beam path of the microscope.

e)     Detectors: The detectors are used to detect the Fluorescence emitted by the sample at different depths. There are typically multiple detectors in a Confocal microscope, including Detectors for different wavelengths of Fluorescence.

f)      Control and data acquisition system: The control and data acquisition system is responsible for controlling the various components of the microscope and acquiring and storing the data from the detectors. It typically includes a computer and specialized software for controlling the microscope and analyzing the data.

g)    Stage: The stage is a platform that holds the sample and allows it to be moved in order to scan the sample.

h)    Z-control: 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.

i)      Eyepieces: The eyepieces are used to view the image produced by the microscope.

j)      Pinhole: 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.

k)    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.

Working principle of Confocal microscope

·       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, the Fluorescent light 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 Photomultiplier tube (PMT) releases an electrical signal, which is then converted into an image by using a Computer.

Types of Confocal microscope

a)     Single-photon confocal microscopes: These microscopes use a single photon detector and a laser to produce high-resolution images of a sample.

b)    Two-photon confocal microscopes: These microscopes use a two-photon excitation process to produce high-resolution images of a sample. They are typically used to image deep within tissues, as the two-photon excitation process allows for deeper penetration of the sample.

c)     Multiphoton confocal microscopes: These microscopes use a multiphoton excitation process to produce high-resolution images of a sample. They are similar to two-photon confocal microscopes, but they are able to produce even higher-resolution images and can be used to image even deeper within tissues.

d)    Laser scanning confocal microscopes: These microscopes use a laser beam to scan over the sample and produce a high-resolution image. They are commonly used to image live cells and tissues.

e)     Fluorescence lifetime imaging microscopes (FLIM): These microscopes use the fluorescence lifetime of a sample to produce images. They are often used to study biochemical processes within cells and tissues.

f)      Stimulated emission depletion (STED) microscopes: These microscopes use stimulated emission to selectively deplete the fluorescence of a sample, resulting in high-resolution images. They are able to achieve a higher level of spatial resolution than other confocal microscopes.

g)    Super resolution microscopes: These microscopes use techniques such as STED, structured illumination microscopy, or single-molecule localization microscopy to achieve a spatial resolution that is beyond the diffraction limit of light. They are able to produce images with a higher level of detail than other confocal microscopes.

h)    Hybrid Scanning Confocal Microscopes: The slit-scanning confocal, which replaces the circular aperture with a rectangular slit to reject out-of-focus light, is an intermediate approach between single and multi-point scanning confocal microscopes. Slit-scanning devices cover a larger portion of the sample in a single field of view and greatly increase imaging speed at the expense of quick photobleaching and reduced resolution.

i)      Swept field confocal microscope (SFC) - The SFC can be operated in either a pinhole or slit scanning mode, in which the apertures stay immobile while galvanometer- and piezo-controlled mirrors sweep the image of the illuminated apertures across the sample. The emitted photons are directed to a Charge-coupled device (CCD) camera via a set of complementary pinholes or slits. The primary benefits of this method are its speed, increase in light collection efficiency, and decrease in artefacts caused by the apertures’ movement, as in a spinning disc system.

j)      Programmable array microscope (PAM): This confocal microscope type employs a Spatial Light Modulator (SLM) (SLM – an object that imposes some form of spatially-varying modulation on a beam of light). The SLM is equipped with a series of moveable apertures (pinholes) and arrays of pixels with opacity, reflectivity, or optical rotation. In addition to microelectrochemical mirrors, the SLM is equipped with a Charge-coupled device (CCD) camera for image capturing.

k)    Spinning disk: Spinning disc, also known as the Nipkow disc, is a form of confocal microscope that uses several moveable apertures (Pinholes) on a disc to scan for light spots in a parallel way over a given plane for an extended length of time. Compared to a Confocal laser scanning microscope, the longer the exposure period, the less excitation energy is required for illumination. Reduced excitation energy minimizes phototoxicity and photobleaching; hence, live cell imaging is its primary application.

l)      Dual spinning Disk: Dual spinning Disk or Microlens enhanced confocal Microscope. It functions similarly to the spinning disc, with the exception that it has a second spinning disc with micro-lenses that is located before the spinning disc with pinholes. The microlenses capture a broad spectrum of light and focus it into each pinhole, so increasing the quantity of light directed into each pinhole and decreasing the amount of light obstructed by the spinning disc. These Confocal Microscopes with improved Microlenses are far more sensitive than spinning discs.

Comparison between Confocal Microscopy and Conventional Fluorescence Microscopy

Parameters	Confocal Microscopy	Conventional Fluorescence Microscopy
Light source	Monochromatic laser	Mercury arc lamp-white light
Pinholes	Two pinholes – one each at conjugate focal planes	Absent
Filters	Not required	Emission and Excitation filters required
Scanning	The specimen is scanned to create an image mosaic.	Widefield microscopy – whole specimen is illuminated.
Resolution	Superior – as focused and point source of light illuminates a tiny bit of tissue at a time, eliminating background illumination	Blurring effect due to background illumination
In vitro/In vivo utility	Confocal microscope can be used to examine both Live (Reflectance Confocal Microscopy) and Excised tissues (Reflectance Confocal Microscopy and Fluorescence Mode Confocal Microscopy)	Tissues have to be excised, processed, fixed, and stained before viewing
Time requirement	Few minutes to hours.	Hours to day.

Applications of Confocal microscope

a)     Biology and Medicine: Confocal microscopes are often used to study cells and tissues, including live cells. They are commonly used to study the structure and function of cells and tissues, as well as to identify and characterize specific proteins or other molecules within cells. 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.

b)    Materials science: Confocal microscopes are used to study the structure and properties of materials, including polymers, ceramics, and metals.

c)     Microelectronics: Confocal microscopes are used to study the structure and properties of microelectronic devices, including transistors and integrated circuits.

d)    Art conservation: Confocal microscopes are used to study the structure and composition of artwork, including paintings and sculptures. It is also used for optical scanning and recovery of damaged historical audio.

e)     Geology: Confocal microscopes are used to study the structure and composition of geological samples, including minerals and rocks.

f)      Agriculture: Confocal microscopes are used to study the structure and function of plants, including their cells and tissues.

g)    Industrial inspection: Confocal microscopes are used to inspect the quality and consistency of industrial products, including food, pharmaceuticals, and consumer goods. It also widely used in the pharmaceutical industry to control the quality and uniformity of the drug distribution.

h)    3D Optical data: Used in 3D optical data storage systems.

Overall, confocal microscopes are an important tool for researchers and scientists who need high-resolution images of small samples and structures.

Advantages of Confocal microscope

·       High spatial resolution: Confocal microscopes are able to produce high-resolution images of small samples and structures, making them ideal for studying cells, tissues, and other small samples.

·       Increased depth of field: Confocal microscopes use a pinhole to block out-of-focus light, which increases the depth of field of the image. This allows for a clearer image of samples with varying depths, such as tissues.

·       Reduced photobleaching: Confocal microscopes use a laser to excite the sample, which reduces the amount of light needed to produce an image. This reduces the photobleaching of samples, which can occur when samples are exposed to high levels of light for extended periods of time.

·       Reduced background noise: The pinhole in a confocal microscope blocks out-of-focus light, which reduces the background noise in the image. This allows for a clearer, more detailed image of the sample.

·       Live cell imaging: Confocal microscopes are often used to image live cells, as they do not produce a lot of heat and do not damage the sample.

·       Multiplexing capabilities: Confocal microscopes can be used to study multiple samples or multiple fluorescence labels at the same time, allowing researchers to study multiple aspects of a sample simultaneously.

·       Accuracy: Because the Confocal microscope analyses the image from one optical point to the next, there is no interference from scattered light from other portions of the specimen, resulting in a more accurate image.

·       Living and Dead Cell: It can be used to examine both living and dead cells

·       Confocal microscope can be used to collect Optical portions in series.

·       Confocal microscope illuminates the focus points consistently.

·       Using a factor known as the Zoom factor, Confocal microscope electronically adjust the magnification without changing the objectives.

·       Confocal microscope produces 3D picture sets.

 

Overall, Confocal microscopy is a powerful tool for producing high-resolution, detailed images of small samples and structures, and is widely used in a range of research and industrial applications.

Limitations of Confocal microscope

·       Complexity and cost: Confocal microscopes are more complex and expensive than other types of microscopes, such as light microscopes. This can make them difficult for some researchers to access or afford. It is costly to create the UV light that Confocal Microscopes employ. They are also expensive to produce and acquire.

·       Limited penetration depth: Confocal microscopes are not able to image very deep within tissues, as the laser beam used to excite the sample cannot penetrate very far into the sample.

·       Low light sensitivity: Confocal microscopes are less sensitive to low levels of light than other types of microscopes, such as widefield microscopes. This can make it difficult to image samples with low levels of fluorescence.

·       Sample preparation: Confocal microscopy requires the sample to be labeled with a fluorescent dye or protein, which can be time-consuming and may alter the sample’s properties.

Limited field of view: Confocal microscopes have a small field of view compared to other types of microscopes, which can make it difficult to study large samples or structures.