What Is Confocal Microscopy? A Clear Explanation of Its Principle, History, and the Difference from Laser Microscopy

The name “confocal” microscopy comes from the fact that it has two focal points, one on the sample side and the other on the detector side. This article describes the classification and principles of confocal microscopy, a technique that uses laser confocal optics, as well as the history of its development. 

Classification of Microscopes

Microscopes are classified according to their use in research, the configuration for imaging and magnifying, and the way the sample is observed. This section explains microscopes classified into the following three categories according to principles of imaging.

・Optical Microscope
・Electron Microscope
・Scanning Probe Microscope

Optical Microscope

An optical microscope (also called a light microscope) uses visible light to observe an object. In general, the term “microscope” often refers to optical microscopes. Optical microscopes are often used in fields such as medicine and biology, with magnifications ranging from several tens to 2,000x, depending on the nature of the research. The confocal microscope, which is the focus of this article, is a type of optical microscope.

Electron Microscope

A microscope that uses electron lenses is called an electron microscope. It is characterized by observation using an electron beam rather than visible light. It has a higher magnification than an optical microscope and can image even extremely small objects such as viruses and DNA. Invented in 1932 and registered as a trademark in 1939, the electron microscope was a major breakthrough in research at the time. Since the object to be observed is irradiated with a high-speed electron beam, it must be handled in a vacuum and under high voltage. Therefore, it is a large-scale microscope equipped with devices for control. 

Scanning Probe Microscope

A scanning probe microscope observes the shape and properties of a sample by scanning it with a tiny needle called a probe. Unlike the optical and electron microscopes described above, it doesn’t use a beam or lens for imaging. Another feature is that it does not necessarily require a vacuum environment as when using an electron microscope, and allows observation of samples in air or immersion medium at high magnification. In recent years, scanning probe microscopes have made it possible not only to observe the shape of surface structures but also to image various physical properties of the sample surface.

 

History of Confocal Microscopy and Differences from Confocal Laser Scanning Microscopy

The history of confocal microscopy goes back over 60 years, as described below.

• 1957: Marvin Minsky, then a student at the Massachusetts Institute of Technology, invented the confocal microscope and applied for a patent. Although the basic principle of confocal microscopy had been completed at this point, it was difficult to put it to practical use with the technology at the time.
• 1970's: Research was initiated for practical application. The situation changed drastically especially with the use of lasers as a light source.
• 1982: Technical improvements such as scanning devices and sensors. The development of digital technology also boosted commercialization.
• Present: Confocal microscopy using a laser as a light source has become mainstream.

Although confocal microscopy without lasers was once considered, laser-based techniques have now become mainstream. Therefore, today, “laser microscopy” almost always refers to confocal laser scanning microscopy.
 

Principles and Features of Confocal Microscopy

In general microscopes that uniformly illuminate the entire field of view (wide-field microscopes), light generated out of focus is superimposed as so-called blur, whereas confocal microscopes form an image by scanning the entire field of view while eliminating blur one point at a time.

When a point light source is placed on the image plane (focal plane), its light is focused through the optical path of the microscope to a point on the focal plane, and the light generated there returns to the same image plane (focal plane) and is focused to a point on the point light source by the reversibility of light. If a pinhole is placed here, blurred light generated outside the focal plane that cannot be focused is blocked by the pinhole, allowing light from a single point on the focal plane to be selectively detected. This is the principle of confocal microscopy. In the actual configuration, the point light source and the pinhole are separated and positioned symmetrically by a dichroic mirror, and optical scanning is performed with two galvanometer mirrors or the like to obtain a two-dimensional image.
Although confocal microscopy takes a long time to scan, it produces cross-sectional images, and can also construct a three-dimensional image by moving the objective lens in the Z direction and acquiring multiple cross-sectional images.

 

Benefits of Confocal Microscopy

This section summarizes the main features and benefits of confocal microscopy.

High-contrast Observation Images

A great benefit of confocal microscopy over other optical microscopy is its ability to produce high-contrast and high-resolution images. By eliminating unnecessary scattered light from outside the focal plane and suppressing stray light, confocal microscopy improves lateral resolution by 30% compared to ordinary optical microscopy. This produces images that are easy to see with less blurring.

MIP Images

Maximum Intensity Projection (MIP) images can be obtained by using confocal microscopy. Its high resolution and deep depth of focus enable detailed observation of cellular and viral structures. MIP images can be acquired by the following mechanisms.

• Acquisition of optical slice (cross-sectional) images in pixels (two-dimensional scanning)
• Capturing the maximum intensity value while moving in the optical axis direction (Z-direction)

Confocal microscopy allows the peak brightness value of each pixel to be combined into a single image. As a result, MIP images can be obtained by combining images focused on different depth positions.

Non-invasive Observation

Confocal microscopy enables observation without touching the sample, so even soft samples can be measured without damaging them. In addition, it can be used without any pre-treatment such as addition of chemicals or processing of cells, which is necessary when using electron microscopes.

Unconstrained by Sample Structure

Confocal microscopy allows observation without being constrained by the sample structure as long as it can receive fluorescence. For example, scanning probe microscopes cannot measure narrow grooves or deep holes that cannot be penetrated by the probe tip, but this is not a problem with confocal microscopes.

Three-dimensional Images

Three-dimensional (3D) imaging can be also performed using confocal microscopy. In 3D imaging, two-dimensional (2D) confocal images (cross-sectional images) are captured one at a time while the objective lens is moved in the depth direction of the sample. This is repeated to acquire multiple cross-sectional images of the sample at all depths. These cross-sectional images can be connected in the depth direction by image processing to generate a 3D image of the sample, which can then be utilized to measure 3D shapes such as step-height, width, surface roughness, and film thickness.

 

Drawbacks of Confocal Microscopy

Although confocal microscopy is very sophisticated, it has the following drawbacks.

Scanning Time

One of the drawbacks of confocal microscopy is that scanning is time consuming. This is because confocal microscopes scan only a single point. However, the spinning disk confocal technique, described later, can shorten the time to some extent. 

Sample Photobleaching and Phototoxicity

As mentioned earlier, confocal microscopy performs single-point scanning, which shortens the irradiation time per unit area. This requires exposing the sample to a higher intensity of light, making the sample more susceptible to photobleaching and phototoxicity. However, as with scanning time, this can also be minimized with the spinning disk confocal technique.

 

Principles of Spinning Disk Confocal Microscopy

The principle of spinning disk confocal microscopy is a technique that overcomes the above-mentioned drawbacks of confocal microscopy. Since confocal microscopy performs single-point laser scanning, image formation takes longer, and the sample is more susceptible to photobleaching and phototoxicity due to the high intensity of light that is applied to the sample for the shorter illumination time per unit area. Spinning disk confocal microscopy, on the other hand, can reduce these problems by scanning multiple points through disk rotation.

Spinning disk confocal microscopy uses a disk with multiple pinholes arranged in a spiral pattern as a point light source and pinholes for confocal microscopy, and scanning is performed by rotating this pinhole array disk. The disk was originally invented for video transmission in 1884 and is also called the Nipkow disk after its inventor. However, this disk alone has not been widely used as a confocal microscope for biological use with dark specimens because most of the illumination light is blocked by small pinholes, resulting in a low signal-to-noise ratio (S/N). In contrast, Yokogawa’s confocal scanner unit, the “CSU” series, dramatically increases S/N by placing a microlens array disk with microlenses arranged in the same spiral pattern over the pinhole array disk and using the microlenses to focus the illumination light through the pinholes.

The laser beam is spread out in parallel to cover the field of view, forming a multi-beam with individual microlens and pinhole pairs through the two disks. The multi-beam illuminates the sample at multiple points through the objective lens. The fluorescence generated there passes through the same pinholes, is split from the laser beam by a dichroic mirror, and is reflected and imaged by the camera. As the disk rotates, the multi-beams of fluorescence fill the imaging surface of camera, forming an image in exactly one cycle of the disk pattern. In this type of scanning, increasing the number of points to be illuminated speeds up image formation, but darkens the image. To make it brighter, the intensity of the original laser beam must be increased, or multiple scans must be performed. Multiple scanning is achieved by setting the camera exposure time to an integer multiple of the scan cycle time.
 

 

Features of Yokogawa’s Confocal Scanner Unit CSU

Yokogawa’ s confocal scanner unit “CSU” scans the field of view with about 1000 beams. A single beam is divided into 1000 beams for raster scanning, which allows scanning in 1/1000 of the time, but since the power per beam is reduced to 1/1000, it is necessary to make the beam 1000 times stronger or perform 1000 multiple scans to obtain the same brightness. Although multiple scanning ends up taking the same amount of time, there is another great benefit. Photobleaching and phototoxicity are non-linear, and are significantly reduced with multiple scans with weak light than a single scan with strong light.

The actual CSU also increases the beam power and finds conditions that balance brightness, speed and photobleaching/phototoxicity to suit the sample, and this flexibility is a great benefit. Galvanometer confocal scanning system has been improved by using a resonant scanner to increase speed and a high sensitivity photomultiplier tube (PMT) to reduce laser power for lower damage. However, since it is not inherently parallel scanning, it cannot perform multiple scans and is not as flexible as a CSU.

As an example, if a galvanometer confocal scanning system can capture the motion of a sample with just barely enough brightness under the condition of laser 1mW30fps (33ms), but fading prevents observation for a long time, the CSU-X1 can find conditions such as laser 3mW11ms exposure (11 multiple scans at 1000fps) to obtain a fast, bright image with less fading.
 

CSU Features: The de facto standard for live cell imaging

• High-speed, full-field confocal imaging, ideal for observing moving objects
• Low photobleaching and phototoxicity, ideal for long-term observation of living cells and organisms

 
Comparison of Confocal Scanner Unit CSU and Galvanometer Scanner

	CSU-X1	CSU-W1	High-speed galvanometer scanner
Features	High speed	Wide field of view/High resolution	High speed
Scan method	Spinning disk 1,500 - 10,000rpm Spirals:12	Spinning disk 1,500 - 4,000rpm Spirals: 3	Resonant scanner 7.8kHz
Image formation	2,000fps (0.5ms) max Depends on camera	200fps (5ms) max Depends on camera	30fps (512 x 512 pixels) 420fps (512 x 32 pixels: limited field of view)
Field of view	10 x 7mm	17 x 16mm	30fps (512 x 512 pixels) 420fps (512 x 32 pixels: limited field of view) 12.7 x 12.7mm
Number of beams in field of view	Approx. 1,000	Approx. 1,000	1

 

Principles and Features of CSU-W1 SoRa Super-Resolution

Even with an ideal lens, light can only be focused to a finite size (airy disk) due to diffraction, which results in micro-blur, the limit of resolution. This optical principle is universal and inevitable, but various innovations in imaging have realized super-resolution that exceeds the limits of diffraction.

Confocal microscopy consists of a reciprocal focusing process of excitation and emission light, which in principle allows for greater resolution than a single process. The excitation light from a point light source is focused on the object, and the light generated there is focused back to the pinhole. Looking at this microscopically, light corresponding to the illumination distribution of the micro-blur that cannot be narrowed down is generated at the object, and that individual light also returns and becomes a micro-blur that cannot be narrowed down, passing through the pinhole according to the light distribution. Here, if the pinhole is minimized with respect to the micro-blur, the detection distribution equals the light distribution of the micro-blur.
The light generated is weak at the edge of micro-blur of the illumination, and the light that returns and is detected by the pinhole is also weaker at the edge of the micro-blur. Thus, the light distribution of the micro-blur works double, sharpening the distribution, narrowing its width, thereby increasing resolution. In actual confocal microscopy, the resolution cannot be that high because the pinhole is expanded to a reasonable size (usually the size of an airy disk) to obtain light intensity. 

Considering the case where there is a misalignment between the position of a point light source and a minimal pinhole, the light distribution of illumination and detection is misaligned, resulting in areas where light hits but cannot be detected and areas where light can be detected but does not come, so the position of the midpoint where the light distribution of the two overlaps is viewed as the center. However, it is detected as the position of a minimal pinhole, and this is where the position error occurs. Considering a pinhole as a collection of tiny pinholes, tiny pinholes at the edges are more likely to deviate from the center point light source, resulting in a larger position error. Therefore, the larger the pinhole, the lower the resolution. If this positional error can be corrected, resolution can be restored while maintaining light intensity.
To achieve this, the light distribution in the pinhole should be reduced by half, and this is called Optical Photon Reassignment, which can be achieved by using a pixel in the pinhole for arithmetic conversion or by rescanning the light that has passed through the pinhole for optical conversion.

The CSU-W1 SoRa super-resolution achieves this Optical Photon Reassignment with the spinning disk confocal system. By placing a microlens behind the pinhole on the pinhole array disk and doubling the focusing angle of the light returning to the pinhole with this microlens, the focused spot diameter, which is determined by the diffraction limit, is halved to achieve conversion. Stable and real-time conversion is possible without a large conversion mechanism.

 

Usage Scenarios of Confocal Microscopy

The properties of confocal microscopy are used in research. In this section, we introduce the usage scenarios of confocal microscopy.

Intracellular Molecular Analysis

Confocal microscopy enables us to observe cells at the molecular level. Cells have a variety of organelles, including the nucleus, mitochondria, and Golgi apparatus. The non-invasiveness characteristic of confocal microscopy allows detailed analysis of these microscopic movements and formation processes. For example, protein localization, distribution, and intracellular trafficking can be observed on a molecular basis by utilizing fluorescent dyes in cells.

Study of Cell Formation Processes

Confocal microscopy is very useful for studying the process of cell formation because it allows observation of 3D images. By continuously acquiring multiple images with different focal positions, you can observe the three-dimensional positioning of cells during their formation process. For example, observation of fine structural changes and chromosome movement with confocal microscopy is considered to lead to an understanding of the mechanisms of diseases and physical disorders.

Observation of Plant Mitochondria

Confocal microscopy can also be used to observe the detailed structure of mitochondria, an organelle of the plant cell structure.

• Mitochondria moving linearly on actin filaments
• Carbon nanotubes migrating into mitochondria

Even specimens that are difficult to focus with a confocal microscope can be observed by selecting the optimum wavelength and detection range.

Observation of Cell Structure in Fungi

Confocal microscopy enables high-resolution analysis of not only the human body but also the cell structure of fungi. For example, you can observe the following fungi.

• Arbuscular mycorrhizal fungi (AM fungi), a type of soil fungi: morphology of hyphae and spores, number of nuclei, etc.
• Fungi: structure of cell wall and cytoplasm, etc.

Confocal microscopy is expected to provide a foothold for treatment by enabling detailed observation of fungi.

Corneal Examination

Using confocal microscopy to observe the cornea enables quantitative evaluation of subbasal nerve fibers and dendritic cells in the cornea. This allows observation of the number and even length of corneal nerves. These two factors are also useful parameters that can determine the severity of neuropathy that diabetic patients develop. Derived from this, confocal microscopy has also been used to evaluate neurological disorders in patients with sequelae after infection with the novel coronavirus (COVID-19).

 

Applications of Confocal Microscopy

Here are specific examples of confocal microscopy applications using Yokogawa’s products.

Interface of Plants and Atmospheric Environment Captured by Live Imaging

The opening/closing of the stomata is strictly regulated in response to changes in various environmental factors, such as light and humidity. Stomatal movement originates from changes in turgor pressure of the guard cells, which is under control of the mechanics of the cell wall of guard cells. That is, when the guard cell volume increases, the stoma opens, when the guard cell volume decreases, the stoma closes. Understanding of the mechanism of the stomatal movement is not only the main focus of plant cell biology but also is regarded as basic research toward the improvement of the atmospheric environment through the carbon-dioxide assimilation of plants. To investigate interactive dynamics of the intracellular structures and organelles in the stomatal movement through live imaging technique, a CSU system was used to capture 3-dimensional images (XYZN) and time-laps images (XYT) of guard cells.
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New Era in Mammalian Genetics Research; Utilize the Same Embryo After Observation

In the fertilization and early embryonic development process, various events are spatiotemporally controlled, and many events are connected in the cause-effect relations toward the final goal of ontogenesis. To understand the mechanism of this process, conventional experimental techniques by fixing and destruction of the cells have limitations. If this process can be observed over time and the development process can be continued after the observation, it will open a new era in the Genetics research. A mammalian developmental biology researcher, Dr. Kazuo Yamagata, established such technique by using the CSU system.
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fMCI - The Functional Multineuronal Calcium Imaging Technique

The neuronal network is a computing system that transforms input to output. This computation involves complex nonlinear processes through polysynaptic feedforward and feedback microcircuitry, and thus cannot be addressed either with isolated neuron responses or averaged multineuronal responses. Functional multineuron calcium imaging (fMCI) is promising to solve this problem. This technique is also available with confocal microscopy.
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Unraveling Molecular Mechanism of Pathological Conditions of Metabolic Syndrome by in vivo Molecular Imaging

To elucidate the molecular mechanisms of pathological conditions consisted by the complicated and multi-cellular abnormal interactions in remodeling tissues, an “in vivo molecular imaging” based on the CSU system was developed. By using this technique, it becomes possible to precisely evaluate the three-dimensional changes in the structures in living tissue, and the multi-cellular dynamics in vivo with high time and spatial resolutions.
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Three-Dimensional Real-Time Observation of the Dehydration and Contraction Process of Cells

The damage cells suffer after freeze-thawing is influenced by the electrolyte concentration associated with ice growth. As extracellular electrolyte concentration increases, osmotic pressure rises, resulting in dehydration and contraction of the cells. The rate of this process depends on the water permeability of the cell membrane, and its rate is believed to have a significant impact on the mechanism of cell freezing damage. Conventional microscopic observations of the osmotic behavior of cells provide only two-dimensional information. The only way to determine the rate of dehydration and contraction was to estimate the volume from the projected area and calculate the rate from the change in volume using a sample of isolated cells suspended in solution. Thus, a confocal scanner unit capable of high-speed scanning was introduced into a perfusion microscope, which can observe the response of cells to changes in surrounding solution concentration, and the dehydration and contraction behavior of cells was observed in three-dimensions in real time. In addition to the isolated cells, cultured cells in an adherent state were observed to compare the results.

Long-time 4-color 3D in vivo Imaging in Mouse Intraperitoneal Cancer Tissue

Leukocytes are attracted to cancerous or infected sites, and certain types of leukocytes help tumors grow and metastasize instead of attacking them. Although understanding the various behaviors of leukocytes in response to cancer should lead to a better understanding of cancer, the details of leukocyte behavior in vivo in real time have not yet been elucidated. In this study, real-time 4D analysis of the behavior of various leukocytes in mouse intraperitoneal cancer tissue revealed that their behavior differs significantly depending on the cell type and microenvironment within the tumor, demonstrating the utility of in vivo imaging technology at the cellular level.
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Prospects on Confocal Microscopy

Confocal microscopes are emphasized not only as observation devices but also as measurement instruments, and are expected to:

• Enhance integration with applications such as image processing and image analysis
• Improve the performance of optics technology and optical components for a variety of samples

Confocal microscopy is expected to meet the above two requirements and build enough trust and achievements to become an international standard as a technology using non-contact measuring instruments.