confocal microscopy resolution

The aliased image results from the undersampling mismatch between the resolved data and the detector characteristics. Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310. In this work, we report a highly practical approach to improve confocal microscopy resolution in three dimensions. In order to quantify resolution, the concept of contrast is employed, which is defined for two objects of equal intensity as the difference between their maximum intensity and the minimum intensity occurring in the space between them. The confocal pinhole acts to reduce the effect of diffraction on image formation. The contrast transfer function (CTF) of an optical system is constructed by plotting the measured contrast in an image of test patterns consisting of periodic arrays having alternating dark and light bars at a range of frequencies, or spacing intervals. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes. The zoom control on the microscope provides the capability to match the pixel size to the optical characteristics, and to meet the Nyquist criterion of acquiring samples at a minimum of twice the highest spatial frequency of the specimen signal. I wrote a brief discussion here of … The concept of resolution is inseparable from contrast, and is defined as the minimum separation between two points that results in a certain level of contrast between them. The axial spreading of light from an illuminated point-source is called the point spread function (PSF). L'image confocale (ou coupe optique) est ensuite obtenue par le déplacement de ce point de focalisation de la lumière d'excitation dans les trois dimensions de l'échantillon XYZ (2). Sampling at greater than 6 cycles per micrometer would satisfy the Nyquist criterion, and accurately reproduce the specimen features, but if this is not possible, the most practical solution in most cases is to adjust the microscope zoom setting to provide a match between the sampling and resolution mechanisms. Such specimens are optically thick and inhomogeneous, resulting in a far-from-ideal imaging situation in the microscope. The intensity point spread function in the confocal case is, therefore, the product of the independent illumination intensity and detection intensity point spread functions. When actual features are missed by so-called "blind spots" produced by too-large pixel size, the aliasing may cause features to appear that are larger, smaller, or in different locations than they are in the specimen. Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT Nat Commun . The chief disadvantage of this approach is that it reduces light collection efficiency, and the consequent increase in specimen exposure time (for image acquisition) risks greater damage to sensitive specimens. Resolution is a somewhat arbitrary variable that is meaningful only when considered within the framework of other related factors. The optical sectioning properties result from the use of point-like illumination and detection configurations that restrict the relevant confocal observation volume to the overlap region of the illumination and detection volumes. When the two objects are widely separated, their images are easily distinguished on the basis of the variation in image intensity. In this case, however, resolution will decrease (slightly) at the expense of being able to image the sample at all. The spreading of light from a luminous object: The Point Spread Function. It is important, therefore, to scan a sample at a resolution that meets the criteria of your experiment and conforms to the anatomy of the sample. Confocal microscope resolution Resolution in the x, yplane As in wide-field microscopy, resolution at the focal plane is determined by the diameter of the Airy disc, and therefore is said to be diffraction limited. The fact that all digital confocal microscopy images are acquired, processed, and displayed in the realm of discrete partitions, or pixels, as opposed to being treated as a continuous representation of the specimen data is not a problem of fundamental significance, but rather is a practical matter of imaging technique. Super-resolution microscopes and nanoscopes overcome the diffraction limit of light and allow investigators to study subcellular structures in greater detail than achieved with a standard confocal microscope. A popular method of limiting optical resolution is to employ smaller numerical aperture optical components, or to underfill the rear focal plane of the objective to reduce its effective numerical aperture. Given that the available timescale may be dictated by these factors and by the necessity to record rapid dynamic events in living cells, it must be accepted that the quality of images will not be as high as those obtained from fixed and stained specimens. Reasonable contrast between point-like objects lying on the optical axis occurs when they are separated by the distance between the central maximum and the first minimum of the axial point spread function component. Simply adding a microscope to a Raman spectrometer does not give a controlled sampling volume—for this a spatial filter is required. The influence of noise on the image of two closely spaced small objects is further interconnected with the related factors mentioned above, and can readily affect the quality of resulting images. Even with higher numerical aperture objectives, the use of confocal pinholes restricts the illumination and detection light paths to a much smaller portion of the maximum solid angle of the optical system, resulting in elongation of the point spread function along the optical axis. The greater the depth of the gap between the peaks, the easier it is to distinguish, or resolve, the two objects. The challenge in digital microscopy is to manage the relationship among optical resolution, sampling interval in the spatial domain, and the pixel dimensions of the display device in order to maximize the capture and display of true specimen information, while minimizing visual artifacts that may be introduced by interactions among the various sampling (partitioning) stages. In the past decade, several pioneering approaches have been developed that bypass this limit. In effect, they appear under an "alias", or false identity and are often masqueraded as spatial frequencies that do not exist in the specimen. Consequently, the contrast cut-off distance is reduced in the confocal arrangement, and equivalent contrast can be achieved at a shorter distance compared to the widefield illumination configuration. Similarly, the measured maximum value of the intensity peaks is reduced by the area averaging, and the combined effect of increasing the minimum and decreasing the maximum is to reduce the contrast. The typical value of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy. All optical systems degrade contrast in the imaging process, and this effect is more severe for higher spatial frequencies (small spacing) than for lower frequencies (larger spacing). Furthermore, if pixel size relative to the resel size is too large, ambiguity is introduced in the positions of the minima and maxima of the intensity response in the image plane. By analogy to the resel, the size of which is determined by the optical characteristics of the system, the minimum detectable difference in intensity that can be resolved depends upon electronic properties of the detector, in particular its signal-to-noise ratio. The line scan of confocal laser scanning systems utilizing a single detection channel operate in this manner. Regardless of the instrument configuration, the lateral resolution displays a proportional relationship to wavelength, and is inversely proportional to the objective lens numerical aperture. An ideal solution for small labs with limited budget, but demanding tasks. Figure 5 illustrates a hypothetical CTF for an optical system, and includes curves for the system response to a test target having black and white bars (100 percent contrast), and to a target made up of gray and white bars yielding only 30 percent contrast. The conventional diffraction limit for visible light sets a resolution limit of ~200 nm when using a wide-field, confocal, deconvolution or two photon microscope. available in your country. As noted previously, lateral resolution is of primary interest in discussing resolution and contrast, although the axial extent of the microscope intensity point spread function is similarly reduced in the confocal arrangement as compared to the widefield fluorescence configuration. d is the size of the smallest object to be imaged. The resulting image is then composed of the sum of two Airy disks, the characteristics of which depend upon the separation distance between the two points. The properties of the intensity point spread function (PSF; see Figure 1) in the image plane as well as in the axial direction (Figure 3) are major factors in determining the resolution of a microscope. : Re-scan confocal microscopy scanning twice for better resolution. Because resolution, even under high illumination levels, requires a certain contrast level, and contrast is affected by the ratio of signal to noise, the contrast level attained in practice depends ultimately on the number of photons detected. Video monitors are able to vary the intensity of each displayed dot and achieve some tonal variation even while utilizing one dot per pixel. This situation is caused, as is the case for oversampling, by a mismatch in the partitioning mechanisms of the imaging sequence. In the confocal microscope, the optical resolution (the resel size) is primarily determined by the wavelength of the light and the numerical aperture of the objective. Low-pass filtration that is applied to the sampled data before the image is reconstructed is analogous to the "filtration" done by the eye and brain to smooth pixelated data such as half-tone images, or to moving farther away from a display such as a large screen television in order to eliminate visible scan lines. En microscopie optique à champ large, pour qu'une image soit nette, il faut que l'objet soit dans le plan focal du système optique. Recently, techniques have been created that combine super-resolution imaging with the simplicity and optical sectioning capability of spinning disk confocal, resulting in a spinning disk system capable of a twofold resolution improvement over the diffraction … As is apparent in Figure 2, when sufficiently separated, the intensity change in the area between the objects is the maximum possible, cycling from the peak intensity (at the first point) to zero and returning to the maximum value at the center of the second point. In three-dimensional confocal imaging, a volume resolution element is sometimes referred to as a voxel, although there is no reason to restrict the concept of the resel to two dimensions, and the term may be used to describe the minimum spatially-resolved element (in two or three dimensions) at the specimen, determined by the optics of the microscope system. Because of the cylindrical symmetry of the microscope lenses, the two lateral components (x and y) of the Airy pattern are equivalent, and the pattern represents the lateral intensity distribution as a function of distance from the optical axis. Confocal microscopy gives the highest spatial resolution, as well as allowing the acquisition of depth profiles in three-dimensional thin structures. Where r is resolution (the smallest resolvable distance between two objects), NA is a general term for the micro… The Airy pattern is generally assumed to be a smooth continuous function described by an infinite number of samples (or data points), as shown in the typical analog representation of the intensity variation across the pattern. Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657. A variety of equations are presented in the literature that pertain to different models for calculating axial resolution for various microscope configurations. It is obvious that when zero contrast exists (Figure 2(b)), the points are not resolved; the so-called Sparrow criterion defines the resolution of an optical system as being equivalent to the contrast cut-off distance. In practice, the best horizontal resolution of a confocal microscope is about 0.2 microns, and the best vertical resolution is about 0.5 microns. A useful approach to the concept of resolution is based on consideration of an image formed by two point-like objects (specimen features), under the assumption that the image-forming process is incoherent, and that the interaction of the separate object images can be described using intensity point spread functions. 244, Pt 2 2011, pp. After image subtraction, the result image features super-resolution together with a high SNR. Optics Express 4: 2644–56 (2013). The resolution predicted by diffraction limits is never achieved in fluorescence observations of biological material, and for performance to be maximized the number of photons detected must be optimized at every stage in the imaging process. Utilisation du microscope confocal. The zoom control on the microscope provides the capability to match the pixel size to the optical characteristics, and to meet the Nyquist criterion of acquiring samples at a minimum of twice the highest spatial frequency of the specimen … When taken at the same resolution as z, an optical section series (contiguous optical sections through z)  will contain all possible sample information. If divided into a finite number of measurement points or areas, by a scanner or digital imaging device, for example, the smooth curves are transformed into a series of intensity values, each of which can be stored in a computer memory location. The sample partitioning misses entire cycles of the specimen data, and outputs a 1-per-micrometer signal that does not exist in the specimen. It is important to recognize that pixelation at the image display stage is not unique to digital imaging. 2017 Dec 12;8(1):2090. doi: 10.1038/s41467-017-02028-8. The relationship of the pixel size to the diameter of the Airy disk determines the number of pixels that are required to sample two adjacent Airy disks to achieve a certain contrast. In a situation such as the one illustrated in Figure 7, since specimen features of this size cannot be accurately rendered at the chosen sampling rate, either the sampling rate must be increased, or some form of filtration should be employed to eliminate frequencies that are beyond those that can be dealt with by the system (those exceeding the Nyquist limit). As stated previously, the fact that digital confocal images must be not only recorded, but processed and displayed within discrete picture elements introduces imaging variables that may be unfamiliar to microscopists that are new to digital imaging. The relationship between contrast and separation distance for two point-like objects is referred to as the contrast/distance function or contrast transfer function. Experimental limitations and the properties of the specimen itself, which vary widely, dictate that imaging cannot be performed at the theoretical maximum resolution of the microscope. At decreased distance, as the two point spread functions begin to overlap, the dip in intensity between the two maxima (and the contrast) is increasingly reduced. Conventional Abbe theory describes the interaction of the parameters that can be modified to improve lateral resolution, including decreasing the wavelength of the incident light, increasing the refractive index of the specimen medium, and increasing the acceptance angle (numerical aperture) of the optical system. So a confocal microscope has slightly better resolution horizontally, as well as vertically. When stored by the computer, each pixel corresponding to a spatial location in the image has an associated intensity value ranging from 0 to 255 for 8-bit storage (256 gray levels). This field is commonly represented through the amplitude point spread function, and allows evaluation of the optical transfer properties of the combined system components. When considered as continuous functions, the Airy patterns exhibit their full intensity variations and produce the maximum theoretical contrast for a given separation distance. The operator must consider the size of the objects of interest and set pixel size and step size to appropriate values in order to collect enough information to faithfully reconstruct the object in question. It is common, however, to specify that greater contrast is necessary to adequately distinguish two closely spaced points visually, and the well-known Rayleigh criterion for resolution states that two points are resolved when the first minimum (zero crossing) of one Airy disk is aligned with the central maximum of the second Airy disk. A mirror is used to place behind the specimen, and two confocal images with large pinholes and small pinholes are recorded. Another mechanism of degrading resolution to match sampling properties is to utilize larger pinhole diameters (or other optical methods) to increase the effective size of the source disk beyond the diffraction-limited spot size. About Us, Terms Of Use | Figure 1 (airy disk and intensity function) illustrates diagrammatically the formation and characteristics of the Airy disk, the related three-dimensional point spread function, and Airy patterns in the fluorescence microscope. Confocal Raman microscopy refers to the ability to spatially filter the analysis volume of the sample, in the XY (lateral) and Z (depth) axes. There is only one optimal zoom setting for a particular combination of wavelength, numerical aperture, and objective magnification: the one that provides a pixel size that matches the Nyquist criterion when referenced to the specimen space. Chemical properties of solid and liquid components can be analyzed with diffraction-limited spatial resolution (λ/2 of the excitation wavelength, down to 200 nm). Actual specimens are not point sources, but can be regarded as a superposition of an infinite number of objects having dimensions below the resolution of the system. To learn more about how we use cookies on this website, and how you can restrict our use of cookies, please review our Cookie Policy. In all of these methods, as more fundamental dots or image elements are grouped to achieve greater tonal range, the appearance of the image becomes more "grainy", with the effect of reducing apparent resolution. It is important to be able to operate the microscope with spot sizes consistent with the specimen and fluorophore properties, and appropriate to the requirements of the experiment. The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities. 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