A new kid on the microscopy block, Vutara Salt Lake City, UT was on hand to discuss its SR, designed for acquiring and analyzing super-resolution images with "speed, simplicity, and stability. Among other new products on display was the Simoa single-molecule measurement system, a fluorescence-based diagnostic platform promising sensitivity X greater than any other technology on the market.
Maker Quanterix Lexington, MA delivered a poster describing a pilot study done with the Swedish Hockey League using Simoa to analyze a single drop of blood for biomarkers indicating brain injury—an alternative to spinal tap. Prior Scientific Rockland, MA previewed its ultra-quiet HLD, "the most repeatable microscope stage available today" with 50 nm encoders. And Intelligent Imaging Innovations 3i; Denver, CO offered "the only" commercially available spatial light modulator SLM designed for patterned and 3D point photomanipulation in optical microscopy.
Finally, Inscopix Palo Alto, CA discussed its strategy of selling only to selected customers; the company's Neuroscience Early Access Program NEAP invites labs to apply for the opportunity to work with nVista HD, "the first end-to-end solution for visualizing large-scale neural circuit dynamics in freely behaving animals. Lin and A. Hoppe, Microsc. The work could lead to a way to noninvasively control transplanted neural progenitor cells with light to restore lost functions in stroke recovery.
Using advanced imaging techniques and novel fluorescence biosensors, the research team has revealed new insights into CaMKII's activity at the single synapse level.
An optogenetics technique showed that a group of hormone neurons increase REM sleep when the need for body temperature defense is minimized. The brain imaging method is based on a recent bioluminescence-based indicator of membrane voltage to record brain activity simultaneously in multiple, freely moving mice. The confocal microscopy system could better observe how large networks of neurons interact during various behaviors. The operation of modern microscopes, although conceptually similar, often requires training to understand not only the physics of the optics but also the associated systems that integrate the hardware with the imaging software.
The spatial resolution of traditional microscopy is limited by the wavelength-dependent diffraction of the illuminating light; in theory, visible light resolution is about 0. In the optical microscope, image formation occurs at the intermediate image plane, through interference between direct light that has passed through the specimen and light diffracted by structures present in the specimen. The image produced by the objective i.
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Biological tissues in general, and living cells in particular, are transparent to visible light, thus making traditional bright field and reflection-based techniques e. However, these techniques do slightly alter the phase of the light diffracted by the specimen. This phenomenon was first studied in the s by Frits Zernike, who devised a method that uses destructive or constructive light interference to enhance the contrast between the imaged specimen and the background.
Phase contrast microscopy continues to be a widely used and important tool, particularly for the study of cells and tissues in culture. However, it has some disadvantages, such as halo artifacts halos around the boundaries of images and image distortion in thick specimens, because of phase shifts occurring in planes other than the one in focus. Also, the system components introduce limitations in the working numerical aperture a measure of the light-gathering ability of the microscope , causing a reduction in spatial resolution.
The basic differential interference contrast DIC system, first devised in , is a modified polarized light microscope. Because the optical components required for DIC microscopy do not obstruct the objective and condenser apertures, the instrument can be employed at full numerical aperture.
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The results are the capacity to use thick specimens, the elimination of halo artifacts, and a dramatic improvement in resolution. To enhance their quality, images can be manipulated using digital and video imaging techniques that further modify contrast. Typical DIC applications include visualization of living cell cultures, blood cells, subcellular organelles, chromosomes, embryos, and either relatively thick or ultrathin microtome sections Friedl Both phase contrast microscopy and DIC table 1 rely on specimen phase differences between sampling and reference beams to produce an image.
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The two techniques are complementary and are capable of producing high-contrast images of transparent biological specimens that do not affect the amplitude of visible light waves passing though them. A primary advantage of DIC over phase contrast is that its system architecture makes it possible to utilize the instrument at full numerical aperture, resulting in improved axial resolution.
Also, the halo artifact present in phase contrast microscopy is largely absent in DIC images. Another significant advantage of DIC is its capacity for creating images of very small features and of much larger ones at the same time. In phase contrast microscopy, halo shadows center around very fine structures, a problem not presented by DIC. Halo artifacts and image contrast are also affected by the optical thickness of the specimen to a much larger degree in phase contrast than in DIC microscopy.
An advantage of phase contrast over DIC, on the other hand, is its ability to create images of birefringent specimens. DIC systems rely on polarized light, resulting in image distortion when applied to birefringent specimens or materials, whereas phase contrast uses nonpolarized light and is free of optical disturbances. Tissue cultures are often prepared with polymers that present birefringence, thus making phase contrast the favored imaging system.
In general, phase contrast and DIC microscopy should be considered complementary techniques. Only by taking full advantage of both techniques is it possible to completely investigate specimen properties, dynamics, and morphology. The basis for fluorescence microscopy is the property of some atoms and molecules to absorb light at a particular wavelength and subsequently to emit light at longer wavelength. In practice, this is achieved by illuminating the object with monochromatic light at the wavelength that excites fluorescence emission in the fluorophores fluorescent molecules of the sample figure 1.
The object is viewed using filters that exclude the illuminating wavelength and select the wavelength of the light emitted by the fluorophores. Each fluorophore behaves as a separate light source, thus avoiding imaging limitations caused by diffraction. Using several different fluorophores with different optical properties, it is possible to create images of living cells in which different organelles appear in different colors.
Characteristic problems of fluorescence microscopy include photobleaching permanent loss of fluorescence due to photon-induced chemical damage and covalent modification and photodamage tissue damage induced by excessive absorbed energy , which are critical factors when imaging living cells. A conventional fluorescence microscope bathes the entire specimen in high-intensity light from a mercury or xenon source figure 2. This results in a significant amount of signal caused by autofluorescence and by emitted background light originating from areas above and below the focal plane.
Secondary fluorescence emitted by the specimen often occurs through the excited volume of tissue and obscures the resolution of features that lie in the objective focal plane. Confocal microscopy, by contrast, exploits a different method of illumination to form an image, scanning one or more focused beams of light across the specimen.
The point of illumination in the specimen is brought into focus by the objective lens and laterally scanned with a device under computer control. The light from the specimen is detected by a photomultiplier tube through a pinhole, which eliminates out-of-focus light by acting as a spatial filter, and the output is displayed by the computer.
Confocal spot size ranges from approximately 0. A major feature of confocal microscopy is its ability to optically section a specimen, allowing the collection of thin 0.
Thanks to spatial filtering, background noise is significantly reduced and the resolution along the z- axis greatly increased in comparison with wide-field fluorescence. Data can be collected from stained specimens using single-, double-, triple-, or multiple-wavelength modes of illumination, and the images collected with the various illumination and labeling strategies will be in register with each other. Live cell imaging and time-lapse sequences are possible, and digital image processing methods applied to sequences of images allow not only three-dimensional representation of specimens but four-dimensional imaging through the time-sequence presentation of three-dimensional data; Emptage The effects of photobleaching and photodamage can be further reduced by combining this technique with nondestructive ones, such as DIC and phase contrast, which can be used to locate the specific area of interest in a specimen before proceeding with the fluorescence analysis.
Multiphoton fluorescence microscopy MFM combines laser scanning microscopy with long-wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of stained specimens. The technique is based on the capability of fluorophores to be excited not only by photons at a wavelength corresponding to the absorption band for fluorescence but also by those at twice the wavelength. Fluorescence excitation will occur only following two-photon excitation in a small volume at the focal point of the microscope, resulting in reduced photobleaching and photodamage Piston , Kawano et al.
The lack of absorption from out-of-focus fluorophores allows light to penetrate deeper into the specimen, permitting the imaging of samples up to three times thicker than those used in confocal microscopy. MFM permits scanning in the xy plane and through the depth of the specimen, allowing the three-dimensional imaging of living cells and tissues Tauer However, confocal microscopy has a slightly higher spatial resolution, because it uses shorter wavelengths, resulting in lower diffraction.
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In MFM, the low background noise and the absence of pinholes make the efficiency of the process much higher in comparison with both wide-field and confocal microscopy. Most of the current microscopy techniques can be used in real time, creating high-resolution, time-lapse images of living cells. Through the use of these techniques, a variety of molecular mechanisms are now being uncovered. For example, with regard to time-lapse imaging of dendritic spines, mechanisms involving changes in intracellular calcium concentration are being unraveled that may control both the formation or elongation and the pruning or retraction of spines Segal In another example, time-lapse imaging has helped researchers view the nuclear envelope, a highly dynamic structure that reversibly disassembles and re-forms at mitosis and breaks down during apoptosis Buendia et al.
Time-lapse imaging has also been used in conjunction with green fluorescence protein, which can be introduced into intracellular structures and proteins, making them fluorescent and permitting visualization of them in living cells. X rays are short-wavelength electromagnetic waves with high energy 5 to kiloelectron volts that differentially penetrate solid objects and tissues according to their absorbance, which is proportional to their density.
To produce an X-ray image, radiation passes through the subject, resulting in an image on photographic film that displays the variation in the subject's density box 1.
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Radiation intensity is defined as dose rate intensity per unit of time and the X-ray quanta per exposure as dose. High-quality images depend on an adequate dose rate. If the intensity of the X rays is too low, the target will not be penetrated adequately, and the image will be underexposed; if the dose is too high, the image will be overexposed. Contrast is very good between hard and soft tissue, and it may be further increased by introducing strong X-ray absorbers, making it possible to view otherwise invisible structures.
Some X-ray systems are more sophisticated than others at measuring the dose, which ultimately has an effect on the sensitivity of dose rate adjustments. Spatial resolution is high in the X-ray system, on the order of less than 0. However, one major pitfall is the obvious loss of depth information. Computed tomography CT also uses X rays to visualize body structures box 1.
Computed tomographic images are reconstructed from a large number of X-ray transmission measurements throughout the target. Beams are passed through the region of interest from several different angles to create cross-sectional absorbance maps, which can be assembled by a computer into a three-dimensional picture. Different designs exist for the CT machinery, with progressively shorter acquisition times and better resolution. The latest generations of CT scanners can reconstruct acquired images in less than 1 second, and some designs can acquire tomographic sections in less than 50 milliseconds, making them usable in real-time imaging.
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