Confocal microscopy

Confocal microscopy (confocal laser scanning microscopy, CLSM) is a type of light optical microscopy with significant contrast and spatial resolution compared to classical light microscopy, which is achieved using a point aperture (pinhole, pinhole) located in image plane and restricting the flow of background scattered light emitted not from the focal plane of the lens ^[1] . This allows one to obtain a series of images at various depths of the focal plane inside the sample (the so-called optical sectioning of the sample by depth), and then reconstruct a three-dimensional image of the sample from these series. Confocal microscopy is widely used in the fields of biology, medicine, materials science, and semiconductor physics.

History

First Prototypes

Scheme of a confocal microscope from a patent by Marvin Minsky

In 1940, Hans Goldmann, an ophthalmologist from Bern, Switzerland, developed a slit lamp system for documenting eye examinations ^[2] . This system is considered by some later authors as the first confocal optical system. ^[3] ^[4]

In 1943, Zün Koana published a confocal system. ^[3] In 1951, Hiroto Naora, a colleague of Koana, described a confocal microscope in the journal Science for spectrophotometry ^[5] .

In the 1950s, biologists needed to increase the contrast of images of fluorochrome-labeled objects in thick sections of tissues ^[6] . To solve this problem, Marvin Minsky , a professor at the Massachusetts Institute of Technology in the United States, suggested using a confocal scheme for fluorescence microscopes. In 1957, Minsky received a patent for this scheme ^[7] .

Scheme of the tandem scanning microscope

Tandem Scanning Microscope

In the 1960s, the Czechoslovak scientist Mojmir Petraš, a medical officer at Charles University in Pilsen, developed the Tandem Scanning Microscope, the first commercial confocal microscope to use a rotating disk - the Nipkow disk - to create and localize multiple point sources of excitation and radiation. ^[8] ^[9]

The Czechoslovak patent was filed in 1966 by Petrasz and his colleague Milan Hadravsky. The first scientific publication with data and images obtained using this microscope, sponsored by David Egger from Yale University and directly by Moymir Petrash, was published in the journal Science in 1967 ^[10] . The second publication of 1968 describes the theory and technical details of the device ^[11] . In 1970, a patent was obtained in the USA. ^[12]

Combining a confocal microscope with a laser illuminator

In 1969 and 1971 scientists David Egger and Paul Davidovich of Yale University published pioneering works describing the first confocal laser scanning microscope ^[13] ^[14] It was a point scanner, that is, only one spot of illumination was generated. They used epi backlight in reflected light to observe nerve tissue. A 5 mW helium-neon laser with a wavelength of 633 nm was used as a source of coherent radiation. The laser beam was reflected by a translucent mirror in the direction of the lens . The lens was a simple lens with a focal length of 8.5 mm. Unlike all previous (and subsequently later designs of confocal systems), the sample was scanned by the movement of this lens (scanning by the lens), which led to the displacement of the focal point. The reflected light returned to the translucent mirror, focused by another lens on the diaphragm ( pinhole ), behind which the photomultiplier was placed. The signal was visualized using a CRT oscilloscope , the cathode ray moved simultaneously with the lens. Using a special adapter, it was possible to take photos on a Polaroid camera. Three of the photographs thus obtained were published in 1971 ^[14] .

The schemes of the confocal scanning microscope of Marvin Minsky using laser radiation were also developed ^[15] . Subsequently, the main attention of the researchers was directed to the analysis of the use of fluorescent dyes for in vivo studies and to improving the quality of confocal images by increasing the intensity of fluorescent radiation.

Scanning System Development

In 1977, Colin J. R. Sheppard and Amargioti Chowdhury published a theoretical analysis of confocal and laser scanning microscopes. ^[16] This was probably the first scientific publication to use the term “confocal microscope”. ^[17] In 1978, Christoph Kremer and Thomas Kremer published a project for a confocal laser scanning microscope using fluorescence excitation with electronic autofocus. ^[18] In this CLSM model, the laser scanning method was first combined with volumetric detection of biological objects labeled with fluorescent markers. In 1978 and 1980, the Oxford group of Colin Sheppard and Tony Wilson described a confocal system with epi-laser illumination, a scanning object table and photomultipliers as detectors. The table could move along the optical axis, which allowed for three-dimensional optical layer-by-layer partitioning. ^[17] In 1979, Fred Brackenhoff and his colleagues demonstrated that the theoretical advantages of optical sectioning and improved optical resolution are indeed achievable in practice. ^[19] In 1983, I. Cox and S. Sheppard published the first work in which a confocal microscope was controlled by a personal computer. ^[20]

Laser beam spot scan

Laboratory model of a confocal laser scanning microscope

In the mid-1980s, William Bradshaw Amos and John Graham White and colleagues at the Cambridge Molecular Biology Laboratory built the first confocal laser scanning microscope. ^[21] ^[22] In its optical design, scanning was carried out by sequentially moving the light beam through the sample, and not by moving the table with the sample. This scheme allowed to significantly increase the scanning speed due to the rejection of inertial mechanical scanning systems and to achieve a speed of up to four frames per second (512 lines each). ^[21]

In parallel, the UK Medical Research Council (MRC) sponsored the development of a prototype of a modern commercial confocal microscope, which was later acquired by Bio-Rad, complemented by computer control and commercialized as the “MRC 500”. The successor to the MRC 600 later became the basis for the development of the first two-photon fluorescence microscope, developed in 1990 at Cornell University. ^[nineteen]

Research at Stockholm University around the same time also transformed into the commercial KLSM Sarastro. ^[23]

The company was acquired in 1990 by Molecular Dynamics ^[24] , but further development of the system was eventually discontinued.

In 1989, Fritz Karl and Eckhard Priikshat invented a scanning laser-diode microscope for particle size analysis. ^[25] ^[26]

In Germany, Heidelberg Instruments, founded in 1984, developed the CLSM technology, which was originally intended for industrial use, not biology. In the early 1990s, this technology was actively developed by Leica Lasertechnik and Carl Zeiss , which at that time had already successfully produced light microscopes with an implemented laser beam scanning scheme, which were later upgraded to confocal systems ^[27] .

Principle of Operation

Play media file

The principle of operation of a fluorescence and confocal microscope

Scheme of a confocal microscope. The scattered light coming from the depth of the sample (dashed lines in blue) is cut off by apertures, which ensures high image contrast

The optical scheme of a conventional light microscope forms an image of the entire part of the sample located in the depth of field of the used micro lens, and the confocal microscope forms an image of a very thin section of the object at the same depth level. In essence, the CLSM method is achieved through a controlled limitation of the focus depth of the optical system.

The principle of confocal imaging was patented in 1957 by Marvin Minsky ^[28] ^[29] and is aimed at overcoming some of the limitations of traditional fluorescence microscopes. In a conventional (wide-field) fluorescence microscope, the entire sample is uniformly illuminated by the radiation source of the microscope. In this case, the entire sample is simultaneously irradiated and excited, and the resulting fluorescence is detected using a photodetector or a microscope camera, including a large background part of the object. In contrast, a confocal microscope uses spot illumination (see Point Spread Function ) and a pinhole in an optically conjugated plane in front of the detector to exclude an out-of-focus signal. Thus, fluorescence radiation is detected only from the focal plane; therefore, the optical resolution of the image, especially along the Z axis (along the depth of the sample), is much higher than that of conventional light microscopes. However, since most of the fluorescence from the sample is diaphragmed, an increase in resolution is accompanied by a decrease in the intensity of the useful signal. To compensate for this side effect, a longer exposure time of the detector and highly sensitive photodetectors, usually PMTs or an avalanche photodiode , which convert the optical signal to an electric one, followed by registration on a personal computer, are used ^[30] .

Since only one fluorescent point is recorded on the sample, raster scanning of the sample is required to form a two-dimensional or three-dimensional image. The laser beam moves along the sample in a horizontal plane using one or more mirrors with a controlled angle of inclination. This scanning method usually has a low scanning speed, which, however, can vary. Thus, a slower scan provides a better signal to noise ratio, which leads to better contrast and higher resolution.

As is known, the KLSM depth of field is directly proportional to the wavelength of the radiation used and inversely proportional to the numerical aperture of the micro-lens, and also depends on the optical properties of the sample. Due to this, in KLSM using various algorithms, a software reconstruction of the point of 3D objects occurs. The most common is the maximum intensity search algorithm. ^[31]

Confocal microscope has a resolution $\delta$ ${\ displaystyle \ delta}$ the same as a conventional microscope and it is limited by the diffraction limit .

\delta ={\frac {\lambda }{\pi (NA)}}

{\ displaystyle \ delta = {\ frac {\ lambda} {\ pi (NA)}}}

Where $\lambda$ ${\ displaystyle \ lambda}$ Is the radiation wavelength, $(NA)=n\sin \alpha$ ${\ displaystyle (NA) = n \ sin \ alpha}$ - numerical aperture of the lens, $n$ ${\ displaystyle n}$ - the refractive index of the medium between the sample and the lens, $\alpha$ ${\ displaystyle \ alpha}$ - half the angle that “captures” the lens. In the visible range, the resolution is ~ 250 nm (NA = 1.45, n = 1.51). However, in recent years, microscope designs have been successfully developed that use the nonlinear fluorescence properties of the samples. In this case, a resolution much lower than the diffraction limit is achieved and amounts to ~ 3–10 nm ^[32] ^[33] ^[34] ^[35] .

Let us now consider the issue of increasing contrast when using a confocal optical scheme. Firstly, since in a confocal microscope light passes through the lens twice, the point blur function ^[36] (hereinafter referred to as PSF ) has the following form:

$Pconf(\xi ,\rho )=p(\xi ,\rho )\times p(\xi ,\rho )$ ${\ displaystyle Pconf (\ xi, \ rho) = p (\ xi, \ rho) \ times p (\ xi, \ rho)}$ ,

where Pconf is the confocal point blur function, and p is the regular point blur function.

Visualization of GFP-tagged virus proteins in tobacco leaf using confocal microscopy

Thus, the achievable focal plane thickness is determined mainly by the wavelength of the radiation used, divided by the numerical aperture of the lens, and also depends on the optical properties of the sample. Thanks to the thin optical cut, these types of microscopes are especially good for 3D visualization and surface profiling of samples.

Sequential slices form a “z-stack”, which can be processed with certain software to create a reconstructed 3D image or presented in a two-dimensional stack for publication, thanks to the common algorithm for finding the maximum intensity. ^[31]

Confocal microscopy provides the possibility of direct, non-invasive sequential optical sectioning of intact thick live samples with minimal requirements for their preparation, as well as higher lateral resolution compared to conventional light microscopy ^[37] ^[38] . Typically, biological samples are contrasted with fluorescent dyes to visualize their specific regions or organelles. However, the actual concentration of the dye can be very low in order to minimize the impact on biological systems. So, some confocal systems can track individual fluorescent molecules ^[39] . In addition, transgenic technologies can create organisms that produce their own fluorescent chimeric molecules (labeled GFP, green fluorescent protein) ^[40] .

A high-contrast confocal laser scanning microscope provides two invaluable opportunities: it allows you to examine tissues at the cellular level in a state of physiological activity, as well as evaluate the results (dynamics) of cellular activity in four dimensions - height, width, depth and time. ^[41]

Spatial resolution in confocal microscopy

Point Blur Function (PSF) in a confocal microscope

Multichannel spectral confocal microscopy. Organelles in Marchantia polymorpha thallus cells

Applying the Rayleigh criterion for resolution (a dip of 26% of the distribution maximum), we find that the resolution in the confocal microscope increases, but not significantly. For a confocal microscope, the resolution (r _c ) is determined as follows ^[8] ^[42] [1] :

$r_{c}=0.44{\lambda \over n\cdot sin\Theta }=0.88{\lambda ' \over D}\cdot F$ ${\ displaystyle r_ {c} = 0.44 {\ lambda \ over n \ cdot sin \ Theta} = 0.88 {\ lambda '\ over D} \ cdot F}$ ,

where n is the relative refractive index, D is the diameter of the entrance pupil of the optical system, λ is the wavelength, F is the focal length of the micro lens, θ is the aperture angle of the micro lens, λ '= λ / n. For a conventional light microscope, the resolution (r _r ):

$r_{r}=0.61{\lambda \over n\cdot sin\Theta }=1.22{\lambda ' \over D}\cdot F$ ${\ displaystyle r_ {r} = 0.61 {\ lambda \ over n \ cdot sin \ Theta} = 1.22 {\ lambda '\ over D} \ cdot F}$

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 in the first lateral maximum to the amplitude of the main maximum is 2%, for a confocal microscope this ratio will be 0.04%. The figure shows the intensity distribution of the fluorescent signal. On the upper part of the figure, we see that a dim object (intensity 200 times less than that of a bright one) cannot be detected using a conventional light microscope, although the distance between objects is much larger than the Rayleigh criterion determined for their resolution. At the same time, by the method of confocal microscopy, this object is well recorded.

Confocal microscopy provides an increase in image contrast due to the use of focused illumination (excitation) in the field of analysis and aperture of fluorescence radiation in the image plane. Such an increase in contrast makes it possible to resolve objects having a difference in intensity up to 200: 1, and also provides an increase in resolution both in the plane of the object and along the optical axis. Along with increasing contrast, fluorescence confocal microscopy allows for step-by-step three-dimensional reconstruction of the studied object through the use of multi-point illumination. Among the most advanced methods of scanning confocal microscopy, the use of a scanning disk with microdiaphragms and the use of matrix photodetectors should be highlighted [2] .

Today, there are methods that can significantly increase the resolution of a confocal microscope. In a conventional confocal microscope, the exciting light is focused at a single point on the sample, followed by the detection of the emission fluorescence signal. Out-of-focus emission radiation is cut off by a pinhole (pinhole), the size of which determines how many maxima of the Airy disk reach the detector. An increase in resolution can be achieved by reducing the diameter of the diaphragm (pinhole), but the signal-to-noise ratio will significantly decrease due to a decrease in the intensity of the emission radiation passing through the diaphragm. An alternative to such scanning is the use of matrix detectors ^[43] ^[44] , which simultaneously record the intensity distribution along the lateral plane of the sample simultaneously with the entire area of the Airy disk, where each photosensitive element performs the function of a pinhole aperture. Thus, using the detection algorithm with a 32-channel matrix detector (Airyscan ^[45] ^[46] ), the possibility was shown to exceed the classical resolution limit (diffraction limit) by more than 1.7 times in all three dimensions: up to 140 nm laterally and 400 nm axially at a wavelength of 488 nm ^[47] ^[48] ^[49] ^[50] ^[51] ^[52] ^[53] .

Application

KLSM is widely used in almost all branches of biology, from cell biology and genetics to microbiology and developmental biology. It is also used in quantum optics and nanocrystalline imaging and spectroscopy.

Biology and medicine

In clinical terms, KLSM is used to study various eye diseases, and especially for visualization, qualitative analysis and quantification of corneal endothelial cells ^[54] . It is used to localize and identify the presence of filamentous fungal filaments in the stroma of the cornea in cases of keratomycosis, which provides quick diagnosis and early introduction of correct therapy. It also seems promising to carry out endoscopic procedures ( endomicroscopy ) using the KLSM method ^[55] . In the pharmaceutical industry, it was recommended using this approach to monitor the production process of thin-film pharmaceutical forms, to control the quality and uniformity of the distribution of drug substances.

Optics and crystallography

CLSM is used as a data recovery mechanism in some 3D optical data storage systems or mapping of chemical compounds, as well as in semiconductor physics and spintronics (in particular, in studying the properties of NV centers ). ^[56] ^[57] .

Example of images obtained by the KLSM method

A series of confocal images (z-stack) showing the distribution of actin filaments in the cell line of osteosarcoma U2OS
3D reconstruction of a series of confocal images of the core of fixed cells of the HeLa line, transgenic chimeric histone H2B-GFP protein
β-tubulin in Tetrahymena infusoria
Pteridium aquilinum leaf slice
Confocal image of a cross section of the stem of Lycopodium annotinum
Cross section of Dryopleris filix-mas leaf
Confocal image of a fragment of a 1 Euro coin
Two-channel confocal image of mitotic microtubules

Notes

↑ Handbook of Biological Confocal Microscopy / JB Pawley. - 3rd ed. - Berlin: Springer, 2006 .-- 985 p. - ISBN 0-387-25921-X . - DOI : 10.1007 / 978-0-387-45524-2 .
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↑ Barry R. Masters. Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging . - SPIE Press, 2006-01-01. - 234 p. - ISBN 9780819461186 .
↑ Hiroto Naora. Microspectrophotometry in Visible Light Range . - 1958-01-01. - book p.
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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 (link not available) - Confocal Microscope Capabilities.
OPTELICS HYBRID - multifunctional light confocal and laser confocal microscope.

[Pawley_2006-1] Handbook of Biological Confocal Microscopy / JB Pawley. - 3rd ed. - Berlin: Springer, 2006 .-- 985 p. - ISBN 0-387-25921-X . - DOI : 10.1007 / 978-0-387-45524-2 .

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