However, the interpretation of dark field images must be done with great care, as common dark features of bright field microscopy images may be invisible, and vice versa. Phase contrast is a widely used technique that shows differences in refractive index as difference in contrast. The basic principle to make phase changes visible in phase contrast microscopy is to separate the illuminating background light from the specimen scattered light, which make up the foreground details, and to manipulate these differently. The ring shaped illuminating light that passes the condenser annulus is focused on the specimen by the condenser.
Some of the illuminating light is scattered by the specimen.
The remaining light is unaffected by the specimen and forms the background light. This entails that the foreground and the background nearly have the same intensity, resulting in a low image contrast a. In a phase contrast microscope, the image contrast is improved in two steps. This eliminates the phase difference between the background and the scattered light, leading to an increased intensity difference between foreground and background b.
To further increase contrast, the background is dimmed by a gray filter ring c. Some of the scattered light will be phase shifted and dimmed by the rings. However, the background light is affected to a much greater extent, which creates the phase contrast effect. The above describes negative phase contrast. This means that the scattered light will be subtracted from the background light in b to form an image where the foreground is darker than the background.
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Differential interference contrast DIC microscopy is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples. A relatively complex lighting scheme produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo. The system consists of a special prism Nomarski prism, Wollaston prism in the condenser that splits light in an ordinary and an extraordinary beam.
The spatial difference between the two beams is minimal less than the maximum resolution of the objective. After passage through the specimen, the beams are reunited by a similar prism in the objective. In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated.
However, near a refractive boundary say a nucleus within the cytoplasm , the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a polarized light source to function; two polarizing filters have to be fitted in the light path, one below the condenser the polarizer , and the other above the objective the analyzer.
The image has the appearance of a three-dimensional object under very oblique illumination, causing strong light and dark shadows on the corresponding faces. The direction of apparent illumination is defined by the orientation of the Wollaston prisms. As explained above, the image is generated from two identical bright field images being overlaid slightly offset from each other typically around 0.
This interference may be either constructive or destructive, giving rise to the characteristic appearance of three dimensions. This is due to the similarity of refractive index of most samples and the media they are in: for example, a cell in water only has a refractive index difference of around 0. DIC has strong advantages in uses involving live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms. Its resolution and clarity in conditions such as this are unrivaled among standard optical microscopy techniques.
The main limitation of DIC is its requirement for a transparent sample of fairly similar refractive index to its surroundings. DIC is unsuitable in biology for thick samples, such as tissue slices, and highly pigmented cells. DIC is also unsuitable for most non biological uses because of its dependence on polarisation, which many physical samples would affect.
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. The specimen is illuminated with light of a specific wavelength or wavelengths which is absorbed by the fluorophores, causing them to emit light of longer wavelengths. The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter.
Typical components of a fluorescence microscope are a light source xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers , the excitation filter , the dichroic mirror or dichroic beamsplitter , and the emission filter. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. In this manner, the distribution of a single fluorophore is imaged at a time.
Multi-color images of several types of fluorophores must be composed by combining several single-color images. Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path i.
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These microscopes are widely used in biology and are the basis for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope TIRF. Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complex environment and to provide three-dimensional information of biological structures. However, this information is blurred by the fact that, upon illumination, all fluorescently labeled structures emit light, irrespective of whether they are in focus or not.
So an image of a certain structure is always blurred by the contribution of light from structures that are out of focus. This phenomenon results in a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture. However, blurring is not caused by random processes, such as light scattering, but can be well defined by the optical properties of the image formation in the microscope imaging system.
If one considers a small fluorescent light source essentially a bright spot , light coming from this spot spreads out further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "hourglass" shape of this point source in the third axial dimension. This shape is called the point spread function PSF of the microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be "convolved by the point spread function". Knowing this point spread function means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as deconvolution microscopy.
There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only the restorative methods can actually reassign light to its proper place of origin.
Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from confocal microscopy, because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken from different focal planes called a Z-stack plus the knowledge of the PSF, which can be derived either experimentally or theoretically from knowing all contributing parameters of the microscope.
Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. It enables the reconstruction of three-dimensional structures from the obtained images. In a conventional i. All parts of the specimen in the optical path are excited at the same time and the resulting fluorescence is detected by the microscope's photodetector or camera including a large unfocused background part.
In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal - the name "confocal" stems from this configuration.
As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity, so long exposures are often required.
As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster i.
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The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.
Spinning-disk confocal microscopes use a series of moving pinholes on a disc to scan spot of light. Since a series of pinholes scans an area in parallel each pinhole is allowed to hover over a specific area for a longer amount of time thereby reducing the excitation energy needed to illuminate a sample when compared to laser scanning microscopes.
Decreased excitation energy reduces photo-toxicity and photo-bleaching of a sample often making it the preferred system for imaging live cells or organisms. The typical FCS setup consists of a laser line, which is reflected into a microscope objective by a dichroic mirror. The laser beam is focused in the sample, which contains fluorescent molecules in such high dilution, that only a few are within the focal spot usually 1— molecules in one fL. When the particles cross the focal volume, they fluoresce. This light is collected by the same objective and, because it is red-shifted with respect to the excitation light, it passes the dichroic mirror reaching a detector.
The resulting electronic signal can be stored either directly as an intensity versus time trace to be analyzed at a later point, or computed to generate the autocorrelation directly. The FCS curve by itself only represents a time-spectrum. Conclusions on physical phenomena have to be extracted from there with appropriate models. The parameters of interest are found after fitting the autocorrelation curve to modeled functional forms.
When an appropriate model is known, FCS can be used to obtain quantitative information such as diffusion coefficients, hydrodynamic radii, average concentrations and kinetic chemical reaction rates. Fluorescence-lifetime imaging microscopy FLIM is an imaging technique for producing an image based on the differences in the decay rate of the fluorescence from a fluorescent sample.
This has the advantage of minimizing the effect of photon scattering in thick layers of sample. Fluorescence-lifetime imaging yields images with the intensity of each pixel determined by the fluorescence lifetime, which allows one to view contrast between materials with different fluorescence decay rates even if those materials fluoresce at exactly the same wavelength , and also produces images which show changes in other decay pathways, such as in FRET imaging. The main focus of these hybrid techniques is on the combination with optical techniques as there has been much progress in the past few years and we can contribute our expertise in this field.
For each combination, the working principle is explained briefly and applications of such combinations are pointed out. Finally, a short comprehension is given with basic information on resolution and field of application of all major combinations. We envision that this review is helpful to those confronted with combined measurements for the first time, for experienced researchers, needing quick access to recent literature as it may convey new ideas for problems in analysis, as well as for researchers, who develop novel combined methods.
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