Confocal laser scanning microscopy. Confocal microscopes Confocal microscope

Melitek LLC supplies laser confocal microscopes. This is modern research equipment, which differs significantly in its capabilities from conventional light microscopes. With its help, you can obtain not only two-dimensional, but also three-dimensional images, obtain information about the surface profile, measure the thickness of translucent coatings, etc.

A laser confocal microscope is used as an instrumental microscope for measuring linear dimensions, angles, radii, etc. Due to the measurement accuracy, such microscopes are suitable for quality control of measuring, metalworking and medical instruments, microelectronic components and other parts, the control of which involves measurements in planes XY, XZ and YZ, One of the features of the LEXT OLS5000 microscope is the ability to build models and take measurements of surfaces with an inclination angle of up to 87.5 0

A laser microscope makes it possible to study the structure and topography of a surface with the ability to determine height differences of up to 6 nm in transparent and opaque samples, which makes it widely used in materials science, forensics and microelectronics. Thanks to a laser light source with a wavelength of 405 nm, illumination, confocal optical design, as well as application. PMTs, instead of conventional CMOS or CCD detectors, LEXT OLS5000 microscopes have a very shallow depth of field, which allows you to determine the height of the focal plane and build high-precision 3D models of the surface. Special software allows you to measure the profile, area and volume of the resulting models. In the cut plane of the model, you can measure linear dimensions, radii of circles and angles. Images and models are saved in a special format that allows additional measurements to be taken if necessary.

Characteristic features of the new LEXT OLS5000 confocal microscope:

detection of height difference 6 nm
lateral resolution 120 nm
guaranteed accuracy of measuring linear dimensions on images obtained by panoramic stitching;
high scanning speed thanks to special algorithms;
increased contrast due to dual confocal design
color images with 4K resolution;
ability to work with samples various sizes and shapes
innovative scanning noise reduction algorithms.

A special frame model allows measurements of samples up to 210 mm in height, and new lenses, adjusted to work with a 405 nm laser with an increased working distance, allow you to examine surfaces in recesses up to 25 mm Descriptions and specifications microscopes for measuring linear dimensions presented in the Melytek LLC catalog can be found on our website. If you have questions, our staff is ready to answer them.

Margolin 389p.

Optical microscopy used all the achievements of both technology and technology, as well as information and computer technology. This led to significant improvements in existing equipment and methods for its use, which, in turn, led to the emergence of new methods, in particular, confocal microscopy. A confocal microscope differs from a classical optical microscope in that at each moment in time an image of one point of an object is recorded, and a full image is constructed by scanning (moving the sample or rearranging the optical system). Thus, the principle of scanning electron microscopy is implemented in a unique form, which makes it possible to record and process the signal from each individual point for as long as desired.

In a classical microscope, light from various points of the sample enters the photodetector. In a confocal microscope, in order to record light from only one point, a small diaphragm is placed after the objective lens in such a way that the light emitted by the analyzed point passes through the diaphragm and will be recorded, and the light from the remaining points is mainly blocked by the diaphragm, like this shown in Fig. 7.28.

Rice. 7.28. Scheme of beam transmission in a confocal optical microscope

Another feature is that the illuminator does not create uniform illumination of the field of view, but focuses the light onto the analyzed point. This can be achieved by placing a second focusing system behind the sample, but this requires that the sample be transparent. In addition, objective lenses are usually expensive, so using a second focusing system for illumination is of little preference. An alternative is to use a beam splitter so that both incident and reflected light are focused by a single lens. This scheme also makes adjustment easier.

Let us now consider how and how quantitatively the contrast changes when using confocal microscopy. Since light passes through the lens twice in a confocal microscope, the point blur function (hereinafter denoted PSF) will be the product of the independent probabilities that a photon will hit a point with its coordinates or a photon will be detected from this point.

If we use the Rayleigh criterion for resolution, it turns out that the resolution in a confocal microscope increases, but not significantly. For a confocal microscope we have an expression for resolution r:

While for a conventional microscope:

However, the main advantage of a confocal microscope is not an increase in resolution in the sense of the Rayleigh criterion, but a significant increase in contrast. In particular, for a conventional PSF in the focal plane, the ratio of the amplitude at the first side maximum to the amplitude at the center is 2%, and for a confocal microscope this ratio will be 0.04%. It follows from this that a dim object with an intensity, for example, 200 times less than that of a bright object, cannot be detected in a conventional microscope, although the distance between objects may be significantly greater than the distance prescribed by the Rayleigh criterion. At the same time, such an object should be well recorded in a confocal microscope.

An important parameter is the size of the apertures in the focal plane of the irradiating and collecting lenses. The image of the aperture in the object plane determines from which areas the light is detected by the photodetector. Obviously, reducing the aperture size leads to a decrease in the amount of light transmitted, increases the noise level and ultimately can negate any achieved contrast benefits. Thus, the question arises about the optimal choice of aperture size and a reasonable compromise.

An aperture with a hole size smaller than the Airy spot simply results in a loss of intensity and has no effect on resolution. A single-spot Airy aperture allows maximum use of the resolving power of the objective lens. However, a diaphragm with an opening size approximately 3 to 5 times larger than the Airy spot appears to be the most suitable compromise. It should be understood that the size discussed here refers to the size of the image in the object plane, and therefore the actual size of the aperture hole depends on the magnification of the lens. Specifically, when using a 100x lens, an aperture with a 1 mm aperture will project into the object plane into a circle of 10 µm radius.

The development of the idea of confocal microscopy was the development of a confocal laser scanning microscope (KJICM), which was caused by the need for more sensitive and metrologically rigorous methods for analyzing the shape and spatial structure of observed objects. A schematic diagram of the CLSM with the main functional connections is shown in Fig. 7.29.

The main feature of CLSM is the possibility of layer-by-layer imaging of the object under study with high resolution and low noise level. This is achieved by step-by-step scanning of the object with a focused beam of light from a coherent source or by moving the stage using special fluorescent probes and special methods for limiting light fluxes.

Rice. 7.29. Structural scheme KJICM:

1 - scanning table; 2 - test sample; 3, 6 - lenses; 4 - scanning device; 5 - beam splitter plate; 7, 9 - needle diaphragms; 8 - radiation receiver; 10 - laser; 11 - Control block; 12 - computer; 13 - axis scanning drive z.

The use of a pinhole diaphragm in CLSM, the dimensions of which are coordinated with the microscope magnification and wavelength, makes it possible to increase the resolution by more than 10%. It is obvious that the resolution of CLSM and, accordingly, the capabilities of analyzing fine structures can exceed the similar capabilities of a conventional microscope by no more than 40% under conditions of scanning a sample with a thin beam. The resolution of KLCM depends on the microscopy method and lighting. KLCM resolution is determined both optical system and electronic path information processing. Therefore, in the design of the KLCM, its circuits, parameters such as the resolution of the optical system, scanning step, detector characteristics must be coordinated, and optimal processing algorithms and appropriate software must be selected.

IN general case The depth of field of KLCM depends on the aperture, wavelength, coherence of light sources and the size of the needle diaphragm. The needle diaphragm is the main design element that distinguishes KLCM from other types of microscopes. Needle diaphragms are designed to create conditions for maximum or complete filtration of light entering the image formation plane from points that do not coincide with the focal plane or are located next to the analyzed element of the object in the focal plane.

Selecting the optimal needle diaphragm diameter is important to obtain the required device characteristics. Relationships for estimating the lateral resolution and depth of field of KJICM are obtained under the assumption that the needle diaphragm has a small aperture, being a luminous point. In reality, the size of the needle diaphragm is finite and the transverse resolution of the device and the brightness of the illuminated elements of the sample, shifted relative to the focal plane along the axis, depend on it z, and depth of field. With small needle diaphragm diameters, the luminous flux becomes low, which reduces the signal-to-noise ratio and reduces contrast. At larger diameters, the effectiveness of the needle diaphragm is reduced by reducing the aperture.

Optical microscopy is also intensively developing using the latest advances in technology, information and computer technologies. This leads to the improvement of existing equipment and methods of its use, which leads to the emergence of new methods, in particular confocal microscopy.

Confocal microscope differs from a “classical” optical device in that at each moment of time an image of one point of the object is recorded, and a full picture is built by scanning (moving the sample or rearranging the optical system). Thus, the principle of scanning electron microscopy is implemented in a unique form, which makes it possible to record and process the signal from each individual point for as long as desired.

In a conventional microscope, light enters the photodetector simultaneously from different points of the sample. In a confocal microscope, in order to record light from only one point, a small diaphragm is located after the objective lens in such a way that the light emitted by the analyzed point passes through the diaphragm and is recorded, and the light from other points is delayed by the diaphragm, as shown in Fig. . 15.31.

Rice. 15.31

Another feature is that the illuminator does not create uniform illumination of the field of view, but focuses the light in the vicinity of the analyzed point. This can be achieved by placing a second focusing system behind the sample, but this requires that the sample be transparent. In addition, objective lenses are usually expensive, so using a second focusing system for illumination is not always justified. An alternative is to use a beam splitter so that both incident and reflected light are focused by a single lens. This scheme also makes adjustment easier.

Let us now consider a mathematical model for quantitatively assessing the change in contrast when using confocal microscopy. Since light passes through the lens twice in a confocal microscope, the point blur function is the product of the independent probabilities that a photon will hit a point with its coordinates and that a photon leaving that point will be detected.

In accordance with the Rayleigh criterion for resolution, it turns out that the resolution in a confocal microscope increases, but not significantly. For a confocal microscope we have an expression for resolution G

While for a conventional microscope

where A." = H/n; P- refractive index; 0 - aperture angle; D- aperture diameter; F- focal length.

The main advantage of a confocal microscope is not an increase in resolution (in the sense of the Rayleigh criterion), but a significant increase in contrast. A dim object with an intensity, for example, 200 times less than that of a bright one, is not visible in a conventional microscope, although the distance between objects can be much greater than that prescribed by the Rayleigh criterion. On the contrary, a confocal microscope should register such an object.

An important parameter is the size of the apertures in the focal plane of the irradiating and collecting lenses. The image of the diaphragm in the object plane determines the light from which areas is recorded by the photodetector. It is clear, however, that reducing the aperture size reduces the amount of light transmitted, reduces the signal-to-noise ratio and, ultimately, can negate any contrast benefits achieved. Thus, the question is about optimal choice aperture size and reasonable compromise.

An aperture with an opening smaller than the Airy spot simply results in a loss of intensity and has no effect on resolution. The aperture size of one Airy spot allows for maximum use of the resolving power of the objective lens. But an aperture size approximately 3-5 times larger than the Airy spot seems most suitable. It should be understood that the size discussed here refers to the size of the image in the object plane, and therefore actual size The opening in the aperture depends on the magnification of the lens. Specifically, when using a 100x lens, an aperture with a 1 mm aperture will project into the object plane into a circle of 10 µm radius.

The development of the idea of confocal microscopy was the development confocal laser scanning microscope(CLSM), which was caused by the need for more sensitive and metrologically rigorous means of analyzing the shape and spatial structure of observed objects. The CLSM diagram with the main functional connections is shown in Fig. 15.32.

Rice. 15.32.1 - coordinate table; 2- test sample;

3,6 - lenses; 4 - scanning device; 5 - beam splitter; 7, 9- needle diaphragms; 8- radiation receiver; 10 - laser; 11 - Control block; 12 - computer; 13 - drive for scanning along the axis Z

The peculiarity of CLSM is the possibility of layer-by-layer imaging of the object under study with high resolution and low noise level. This is achieved by step-by-step scanning of an object with a focused beam of light from a coherent source or using a stage with special fluorescent probes, as well as special methods for limiting light fluxes.

The resolution of CLSM is determined by both the optical system and the electronic information processing path. Therefore, in the design of the CLSM and its circuits, such parameters as the resolution of the optical system, the scanning step, the characteristics of the detector must be coordinated, and in addition, optimal processing algorithms and appropriate software must be selected.

In general, the depth of field of CLSM depends on the aperture, wavelength, coherence of light sources and the size of the needle diaphragm. Needle diaphragm(ID) is the main design element that distinguishes CLSM from other types of microscopes. Needle diaphragms are designed to create conditions for maximum or complete filtration of light entering the image formation plane from points that do not coincide with the focal plane or are located next to the analyzed element of the object in the focal plane.

Selecting the optimal ID diameter is important to obtain the required device characteristics. Relations for estimating the lateral resolution and depth of field of CLSM are obtained under the assumption that the ID has a small aperture and is a luminous point. In reality, the size of the ID is finite, and the lateral resolution of the device, the brightness of the illuminated elements of the preparation, shifted relative to the focal plane along the Z axis, and the depth of field depend on it.

With a small ID diameter, the luminous flux becomes low, which reduces the signal-to-noise ratio and reduces the contrast. With a large diameter, the efficiency of the diaphragm is reduced by reducing the aperture.

Story

In the 1950s, biologists needed to increase the contrast of observing fluorochrome-labeled objects in thick tissue sections. To solve this problem, Marvin Minsky, a professor in the USA, proposed using a confocal scheme for fluorescence microscopes. In 1961, Minsky received a patent for this scheme.

Principle of operation

A confocal microscope has the same resolution as a conventional microscope and is limited by the diffraction limit.

where is the radiation wavelength, is the numerical aperture of the lens, is the refractive index of the medium between the sample and the lens, is half the angle that the lens “captures.” In the visible range, the resolution is ~ 250 nm (NA=1.45, n=1.51). However, in recent years, microscope designs that use the nonlinear properties of fluorescence of samples have been successfully developed. In this case, a resolution is achieved that is significantly lower than the diffraction limit and is ~ 3-10 nm.

A confocal microscope produces a clear image of a sample that would appear blurry with a conventional microscope. This is achieved by cutting off the background light coming from deep within the sample with the aperture, that is, the light that does not fall on the focal plane of the microscope lens. The result is an image with better contrast than a conventional optical microscope.

The image is a two-dimensional (2D) picture.

Advantages in biology over other microscopes

The refractive index of biological objects is almost the same as that of glass, so observing these objects on the surface of a glass slide is very difficult in a conventional microscope. A confocal microscope, which has high contrast, provides two invaluable capabilities: it allows you to study tissue at the cellular level in a state of physiological activity, and also evaluate the results of the study (that is, cellular activity) in four dimensions - height, width, depth and time.

Notes

Links

Molecular Expressions: Laser Scanning Confocal Microscopy
Nikon's MicroscopyU. Comprehensive introduction to confocal microscopy.
Emory's Physics Department. Introduction to confocal microscopy and fluorescence.
The Science Creative Quarterly’s overview of confocal microscopy - high res images also available.
Programmable Array Microscope - Confocal Microscope Capabilities.

Wikimedia Foundation.

2010.

See what a “Confocal microscope” is in other dictionaries:

This term has other meanings, see Microscope (meanings). Microscope, 1876 ... Wikipedia

Atomic force microscope Atomic force microscope (AFM atomic force microscope) is a high-resolution scanning probe microscope. Used to determine surface topography with a resolution from des... Wikipedia The general name for methods of observing objects indistinguishable to the human eye through a microscope. For more details, see Art. (see MICROSCOPE). Physical encyclopedic dictionary. M.: Soviet Encyclopedia. Editor-in-chief A. M. Prokhorov. 1983 ...

Physical encyclopedia

- (English: nitrogen vacancy center) or nitrogen-substituted vacancy in diamond is one of the many point defects in diamond. A defect is a disturbance in the structure of the diamond crystal lattice that occurs when an atom is removed... ... Wikipedia

Wikipedia has articles about other people with this surname, see Minsky. Wikipedia has articles about other people with this surname, see Minsky. Marvin Lee Minsky English Marvin Lee Minsky ... Wikipedia Minsky, Marvin Lee American scientist in the field artificial intelligence

Date of birth: August 9, 1927 (19270809) ... Wikipedia

Minsky, Marvin Lee American scientist in the field of artificial intelligence Date of birth: August 9, 1927 (19270809) ... Wikipedia

Books

Confocal microscopy and ultramicroscopy of a living cell, Georgy Mikhailovich Svishchev. A confocal microscope is a type of scanning light microscope. When examining thick objects, it produces background-free images that conventional microscopes produce...

Confocal laser scanning microscopy (CLSM) is a high-resolution optical three-dimensional (3D) surface profiling technique.

The high numerical aperture of the lens objectives (up to 0.95) and the short wavelength of laser radiation provide high-resolution images along the optical and transverse directions.

Confocal microscopes also have significant contrast compared to classical optical microscopes due to the use of a special diaphragm (pinhole) that cuts off the flow of background scattered light - this helps improve image quality.

In a confocal microscope, an image of one point of an object is recorded at each moment of time, and a full image is constructed by scanning (moving the sample or rearranging the optical system). In order to register light from only one point, a pinhole is located after the lens in such a way that the light emitted by the analyzed point passes through it and will be recorded, and light from other points is cut off by this pinhole.

An increase in image contrast is also achieved due to the fact that the illuminator does not create uniform illumination of the field of view, but focuses the light onto the analyzed point. This can be achieved by using a beam splitter so that both the incident and reflected light are focused by a single lens. This scheme also makes adjustment easier.

Real-time image acquisition is achieved through a fast scanning module and a signal processing algorithm. It takes less than 1 second to obtain a 3D profile of the sample surface. Confocal laser scanning microscopy is an optical non-destructive testing technique for profiling the surfaces of microstructures at high resolution. It is an ideal solution for measuring and inspecting semiconductor wafers, FPD products, MEMS devices, glass surfaces and other materials.

Height measurement capability is achieved through a confocal arrangement of source, sample and detector. When the sample is in the focal plane of the objective, the light reflected from the sample surface is focused onto the confocal pinhole, and the photodetector collects the signal from the sample. However, the sample is placed in an out-of-focus position and the light signal is deflected by the confocal diaphragm. Thus, only the signal in focus reaches the photodetector. This explains the optical selective ability of CLSM technology.

To obtain a 3D profile of the sample surface, optical images are collected along the Z axis. Thanks to the confocal pinhole, light intensity is maximum when the sample is positioned in the focal plane.

The maximum radiation intensity is recorded in the focal plane. The intensity decreases as the sample is moved away from the focal plane.

To accurately find the maximum intensity, multi-point positioning is used near the maximum position. This ensures maximum repeatability of height measurements. The height is calculated by fitting the curve at each pixel. Using this height map, a surface profile of the sample is constructed.

As mentioned above, important parameters This technology is high resolution and high contrast. Our confocal microscopes (NS-3500 series) improve on these parameters by using piezoelectric actuators to move the scanning head between 200 µm and 400 µm in 0.1 nm increments in precision scanning mode, allowing for even higher resolution imaging of sample surfaces and contrast than other confocal microscopes. This feature allows you to analyze and obtain three-dimensional images of the smallest structures, examine up to several layers of transparent coatings on various microcircuits, analyze micro-deep structures (for example, analysis of micro- and nanocracks in oil and gas pipes, car engine pistons, airplane flaps, etc.).

Another important aspect for confocal microscopy is the need to have a tool to analyze the information obtained. Our simple and intuitive software makes it easy to analyze acquired images with digital resolution down to 0.001 µm. It also gives you the ability to analyze large samples by scanning small areas and then stitching them together, analyze roughness and individual pyramidal and cone-shaped microstructures (which is especially important when testing solar cells), etc. An additional autofocus system and the presence of a CCD camera further simplify the measurement procedure and allow you to fully concentrate on the study, without being distracted by unimportant actions.

CLSM has many applications in industrial fields as it is fast, non-destructive and reliable way 3D surface profiling. Laser confocal microscope can measure 3D shape, step height, volume of microstructures, LCD panels, semiconductor wafers, MEMS devices, material surfaces, transparent glass surfaces. In addition, confocal microscopy is widely used for biological research.