- clinical microscopy : employment of the microscope in making clinical diagnoses.
- operating microscope : a specially designed magnifying instrument employed in the performance of delicate microsurgical procedures, as in operations on the middle ear, on small blood vessels, or on a vocal cord
is placed for viewing of the specimen.- mechanical stage : a platform of a microscope by which the specimen being viewed can be moved in either of 2 mutually perpendicular directions.
- filters
- anisotropic diffusion filter : an image processing method for reduction of shot noise without degradation of an image
- acousto-optic tunable filters (AOTFs) are birefringent crystals bonded to piezoelectric transducer. Application of a radiofrequency signal to the transducer generates acoustic waves in the crystal that alter the index of diffraction of the crystal and result in diffraction of the crystal and result in diffraction of certain wavelengths of light. A change in the frequency or amplitude of the applied radio signals leads to a change in the wavelength and intensity of the diffracted light. As the radio signal can be altered rapidly, the intensity and wavelength selection are rapidly altered accordingly.
- zoom : the optical zoom lens system assists the objective in image magnification. It determines the size of the scan region and the apparent magnification of the image. Pixels in a zoom-1 image have areas twice the width and length of those in a zoom-2 image. Increasing the zoom magnification leads to increased irradiation time per unit area and therefore to increased photobleaching
- capillary microscope : an instrument for giving an enlarged image of capillaries, often used for viewing the capillaries of the nail bed.
- hypodermic microscope : a combination of a fiberoptic probe (housed in a hypodermic needle) and a microscope for examining cell structure in tissue and muscle without a cutaneous incision
- epiluminescent microscopy : a technique for the examination of pigmented skin lesions in which the lesion is covered with immersion oil and a glass slide to make the epidermis translucent; then it is examined with a binocular surface microscope.
- corneal microscope : a specially designed instrument with lenses of high magnifying power, for observing minute changes in the cornea and iris
- examination of the cornea or the lens by a combination of Gullstrand's slit lamp biomicroscope (one embodying a diaphragm containing a slitlike opening, by means of which a narrow flat beam of intense light may be projected into the eye. It gives intense illumination so that microscopic study may be made of the conjunctivae, cornea, iris, lens, and vitreous, the special feature being that it illuminates a section through the substance of these structures) and corneal microscope.
- fundus microscopy : examination of the fundus of the eye with an instrument which combines a corneal microscope with an ophthalmoscope
- ultrasonic microscope : one which utilizes the reflection of ultrasonic or mechanical vibration to reveal the detail of the specimen.
- laser scanning microscopy (LSM) : focuses laser light on each point in the field of view to generate the image by scanning the laser beam across the field of view
- confocal microscopy : a term that is mainly applied to specific light microscopy techniques that are designed to minimize out-of-focus contributions from the vertical axis to an image. Typical single-photon microscopy makes use of a pinhole aperture to eliminate out-of-focus contributions., whereas multiphoton (also termed 2 photon) microscopy makes use of the non-linear dependece of excitation on photon density to only excite molecules in a single plane of focus For researchers who make live-action movies of cellular processes, Jena, Germany-based Carl Zeiss' new LSM 5 LIVE confocal microscope system enables you to collect images much faster and still maintain a reasonable sensitivity for biological work. 2 improvements give the 5 LIVE those advantages. First, the system captures full 512 × 512-pixel images at 120 frames per second, about 25 times faster than previous models. (In contrast, big-screen movies display 30 frames per second.) Typical confocals illuminate and capture 1 pixel at a time, scanning a sample point-by-point through a pattern of pinholes, but the 5 LIVE projects light through a confocal slit the full width of the image. Second, the 5 LIVE boasts greater sensitivity than previous Zeiss microscopes, processing > 90% of the light returning from the sample. The improvement is due in part to Zeiss' new "Achrogate" beam splitter. Unlike conventional splitters, the Achrogate can reflect excitation energy of any given wavelength over the entire spectrum of light. The splitter works in tandem with a detector specially designed for line-based imaging, which alone, Zeiss claims, raises the 5 LIVE's quantum efficiency 3-fold over previous Zeiss systems. SNRs with the 5 LIVE are well beyond those with point-by-point scanners under similar conditions. The new speed and sensitivity rob the resulting images of a few percentage points of resolution compared to other Zeiss confocals. But that loss is so slight that the advantages outweigh it. Samples showing greater structural contrast are affected less than the more homogeneous ones. A one-channel model of the 5 LIVE costs about $250,000 (US), nearly 25% more than a standard, real-time confocal system. The higher-contrast 2-channel system is priced at about $350,000.
- differential interference contrast light microscopy
- electronic and computer image enhancement in light microscopy
- fluorescence microscopy : microscopy of natural fluorescent materials or of specimens stained with fluorochromes, which emit light when exposed to blue or ultraviolet light, by means of fluorescence microscope (one used for the examination of specimens stained with fluorochromes or fluorochrome complexes, e.g., a fluorescein-labeled antibody, which fluoresces in ultraviolet light)
- deconvolution fluorescence light microscopy
- multiphoton fluorescence microscopy : a microscopy technique that uses a laser beam to focus a high density of photons of twice the excitation wavelength of a fluorophore on a single point : simultaneous absorption of 2 or more photons of a long wavelength excite fluorophores that are normally excited by a single photon of shorter wavelength. It allows 3D time-lapse imaging of fluorescent signals deep below the surface of living tissues, allowing cell migration and cell-cell interactions to be tracked. The fluorescent images are of slightly lower resolution compared with those obtained by traditional confocal microscopy. However, multi-photon microscopy in intravital imaging has several notable technical advantages compared with other fluorescence-based techniques, includingref :
- bulk fluorophor bleaching and generation of by-products toxic to living cells is reduced because of the lack of fluorophore excitation in regions away from the focal plane
- multi-photon images are less prone to degradation by light scattering for 2 reasons. First, the longer wavelengths that are used for excitation suffer less scattering from microscopic refractive-index differences within the sample, allowing penetration further into tissues and excitation of fluorophores located deep within thick samples (the maximal imaging depth in mouse lymph nodes is 100 mm for confocal microscopy vs. 400 mm for multi-photon excitation)ref. Second, as all the resolution is defined by the geometry of the excitation beam, the fluorescence emission is unaffected by light scattering. Live tissues contain several refractive index interfaces, which cause significant light scattering.
- multiphoton imaging is more efficient. As excitation in multiphoton imaging is confined to the optical section being observed, a pinhole aperture is not required for confocality. So, for identical detector configurations, 100% of the collected light is measured at the external detector as compared with 1% of the collected light in a conventional pinhole confocal microscopy
- true registration of the Z-axis is achieved. Multiphoton microscopy excites at one discrete diffraction-limited point and collects the emitted light without deconvolution to different focal planes. In standard multiprobe confocal microscopy, because wavelengths of different colours of light that originate from one point source focus at different focal planes, registration of many probes along the Z-axis is problematic
- the ability to excite several different fluorophores with the same excitation beam
- the phenomenon of second harmonic generation of UV photons allows non-invasive imaging of ECM components that contains a-helical proteins such as collagen and laminin in a confocal plane without the need for fluorescent labelingref
- two-photon fluorescence-correlation microscopy (TPFCM) is a 3D microscopy technique that allows in vivo measurement of the concentration and diffusion of fluorescently labelled molecules within heterogenous samples, such as tumours, or even within cells.
- immunofluorescence microscopy : fluorescence microscopy using immunofluorescence staining methods
- interference reflection microscopy
- total internal reflection microscopy
- brightfield and darkfield illumination
- direct illumination : the throwing of light upon a microscopical object from above or from the direction of observation
- darkfield microscope : one with a central stop in the condenser, permitting diversion of the light rays and illumination of the object from the side, so that the details appear light against a dark background
- darkfield or dark-ground illumination : the throwing of peripheral rays of light upon a microscopical object from the side, the center rays being blocked out: the object appears bright upon a dark background
- ultramicroscopy : the employment of the ultramicroscope (a special darkfield microscope for the examination of particles of colloidal size)
- Rheinberg color-contrast microscope : a darkfield microscope in which the condenser is modified by having a colored instead of an opaque stop, with the annulus in a complementary color
- opaque microscope : one with vertical illumination or with the condenser built around the objective (epimicroscope) for viewing opaque specimens
- Köhler illumination : an improved method of illumination by adjustment of the substage Abbe condenser, for obtaining the best image detail in microscopical work.
- phase-contrast microscopy : a microscope that converts variations of the refracting index in the object into variations of intensity in the image. Altering the phase relationship of light passing through and that passing around the object allows details of living cells to be seen without the fixation and staining that is normally necessary
- polarizing light microscopy : a microscope equipped with a polarizer, analyzer, and means for measurement of the alteration of the polarized light by the specimen
- rectified polarizing microscope : a polarizing microscope corrected for depolarization from curved lens surfaces so that full apertures can be used
- single-molecule light microscopy
- schlieren microscope : one in which light is deviated by the insertion of 1-2 diaphragms in the optical system, to reveal differences in refractive index in a specimen
- ultraviolet microscope : a microscope which utilizes reflecting optics or quartz and other ultraviolet-transmitting lenses, with radiation of less than 400 mm wavelength as the image-forming energy.
- x-ray microscope : one in which a beam of x-rays is used instead of light, the image usually being reproduced on film.
- projection x-ray microscope : a microscope using soft x-radiation for high resolution; the images may be photographed or observed directly on a fluorescent viewing screen.
-
selective
plane illumination microscopy (SPIM) : when mapping relatively large
millimeter-sized samples in vivo, optical
projection tomography can image embryos at high resolution, but of
fixed specimens only. Magnetic resonance imaging and optical
coherence tomography feature noninvasive imaging, but do not easily
provide specific contrasts. Laser
scanning microscopy (LSM) can use green fluorescent protein (GFP) or
similar labels as contrasting agents for high resolution imaging of protein
localization patterns in living organisms, but suffers from limited penetration
depth in heterogeneous samples. SPIM shines a sheet of laser light 2 to
8 mm thin through a slice of the sample and
then systematically moves the specimen to capture images from each layer.
Samples are embedded in a low-concentration agarose cylinder. Since any
fluorescence imaging system suffers from scattering and absorption in tissue
that can reduce image quality in large samples, the microscope can employ
multi-view reconstruction, in which multiple 3D data sets of the same object
are collected from different directions and combined in post processing
for an optimal representation of the specimen. In experiments in which
the researchers imaged embryos of the teleost fish Medaka
(Oryzias latipes)
of the transgenic Arnie line that expresses GFP in somatic and smooth muscles
as well as in the heart, SPIM was capable of resolving the internal structures
of an entire 4-day-old fixed specimen with better than 6-mm
resolution as deep as 500 mm inside the fish.
Confocal imaging can only image 100 to 200 mm
deep into tissue : SPIM achieved greater penetration depth because it employs
lower numerical aperture illumination than confocal imaging, receiving
more light. SPIM should improve in resolution—confocal can go down to 0.2
mm
in the planar direction and about 1 mm in the
axial direction. This microscope fills a niche, a middle ground between
techniques : with preparations a bit thinner than this, we're able to use
2-photon microscopy to look at events over time at much higher resolution,
and using MRI microscopy, we can look at preparations slightly larger than
this at lower resolution. The investigators imaged all the muscles in vivo
in Medaka as well as the inner surface of the Medaka embryo heart, a structure
barely accessible by confocal LSM. Fast frame recording also allowed SPIM
to image the heartbeat. Previously, similar imaging was only demonstrated
when the heart was exposed and the embryo was cooled to reduce the heart
rate. Recording speed is excellent, at up to 6 frames per second at 1300
by 1000 at 10 bits, but can still be improved. Shrinking the frame size
can increase speed up to 17 frames per second. By using thin slices of
light instead of illuminating the entire sample at once, SPIM greatly reduces
photodamage to samples. The research team reported they routinely imaged
live Medaka and Drosophila embryos over periods of up to 3 days,
apparently without detrimental effects on embryogenesis and development.
SPIM can be automated for large-scale studies of developing organisms and
the systematic collection of expression data (spatial temporal expression).
You could imagine the goal of imaging the gene expression patterns from
the moment of taking a fertilized egg, dropping it in the microscope, and
actually following every single cell division and movement to reconstruct
a digital embryo. What's exciting about this and other techniques that
collect 4-D images—3D images over time—is that instead of guessing at what
might have happened between 2 time points, you can follow a specimen, which
makes it much easier to come up with proper interpretations. It's surprising
how often the ability to follow something over time reveals what we thought
was happening wasn't what was really going on. Although SPIM is far simpler
to build than a confocal microscope, it would probably prove difficult
for the average biologist to implement. The team is currently negotiating
with companies to commercialize the instrument within 2 yearsref.
The efficiency of excitation depends on the square of photon density in two-photon fluorescence light microscopy. Higher number of photons can also be used: for example, simultaneous absorption of 3 photons at one-third the excitation energy will excite the fluorophore, with a cubic dependence on photon density
Companies offering tools for fluorescence imaging : Active Motif, AnaSpec, Andor Technology, Applied Precision, BD Pharmingen, Biocat, BioPAL, BioVision, Cambridge Research & Instrumentation (CRi), Carl Zeiss, Chroma Technology, Clontech, Covalys, Evident Technologies, Evrogen, GE Healthcare, Hamamatsu, HORIBA Jobin Yvon, Intracellular Imaging, Invitrogen/Molecular Probes, ISS, Kodak, Leica Microsystems, LI-COR Biosciences, Lightools, LUX biotechnology, MAG Biosystems, MBL International, MCID Scientific Imaging Software, Media Cybernetics, Molecular Devices, Nanoco Technologies, Nikon, Olympus America, Omega Optical, PerkinElmer, Pierce Biotechnology, Prior Scientific, Promega, Roche Applied Sciences, Semrock, Stratagene, UVP, VisEn Medical, Visiopharm
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|
|
|
| lateral (x, y) resolution | 250 nm | 250 nm |
| axial (z) resolution | 700 nm | 700 nm |
| acquisition speed | dependent on fluorescence intensity; up to 50 frames/s | dependent on fluorescence intensity and pixel number; up to 20 frames/s with low-resolution images |
| photon efficiency | higher than confocal microscope | lower than deconvolution microscope |
| evaluation of images during experiment | difficult; full-resolution images only available after off-line computational image restoration | immediate access to full-reslution images |
| image-processing artefacts | can appear with inappropriate deconvolution algorithm parameters or too low signal-to-noise ratio or raw data | unprocessed images serve as raw data |
| maximum thickness of specimen | 40 mm | 150 mm |
| multi-colour imaging | sequential acquisition of different channels by framewise filter switching | simultaneous and pseudosimultaneous recording in multiple channels by using linewise switching of laser wavelength |
| flexibility in excitation wavelength | full flexibility | limited by available laser wavelengths |
| selective photobleaching | not possible except in specialized systems, in which lasers have been incorporated into the light path for this purpose | possible in interactively defined regions |
| recent applications | Cajal body movements, centromere dynamics, biogenesis of nucleoli, interphase chromosome dynamics | nuclear envelope breakdown and assembly, mitotic chromosome dynamics |
- atomic force microscopy (AFM) : a type of scanning-probe microscope, in which a fine needle attached to the tip of a soft cantilever scans the surface of a specimen. The shape and physical properties of the surface can be detected from the bending of the cantilever. This type of microscope can be used to manipulate single molecules or transfect single cellsref
- tapping mode : a form of AFM in which the tip is vibrated perpendicular to the specimen plane to avoid gouging the specimen as the tip is scanned laterally
- scanning electron microscopy (SEM) : an EM in which a beam of electrons scans over a specimen point by point, causing the emission of a secondary beam that makes an image on the fluorescent screen of a cathode ray tube; differences in depth over the surface may be imaged in 3 dimensions. QuantomiX wet SEM technology enables EM of wet samples, simply by using the affordable single-use QX capsules, which fit standard SEMs easily and quickly. Using the QX capsules, fully hydrated cells, tissue biopsies, food, ink and more can now be imaged and analyzed in their native environment.
- serial block-face SEM offers an effective means for the generation of serial microscopic images to reconstruct high-resolution 3D tissue nanostructureref
- transmission electron microscopy (TEM)
- cryoEM / electron cryomicroscopy : a technique by which, using a special cryoholder, cryofixed biological smaples are directly imaged in the transmission electron microscope under low-dose conditions and at low temperature (at least 170°C). The sample can be either a frozen layer of suspension that contains many isolated macromolecules for single-particle reconstruction techniques, or a thin (100-500 nm) section.
- electron cryomicroscopy and 3D computer reconstruction of biological molecules
- immuno-EM : the detection of identified proteins by electron microscopy, which makes use of specific antibodies that are tagged with a marker, usually colloidal gold, for visualization in the electron microscope
- ion microscope : an electron microscope modified to use ions (e.g., of lithium), instead of electrons.
Alicona Imaging GmbH, Able Software Corp., Axon Instruments, BAL-TECH, BOC Edwards, Campden Instruments, Ltd., CamScan USA,
Charles River Laboratories, CELLVIS, Cressington Scientific Instruments Inc., Diatome U.S., Digital Biomedical Imaging Systems AG, Delong Instruments,
Denton Vacuum, D'Outils Dumont SA, Electron Microscopy Sciences, EBSciences, EMITECH, Ernest F. Fullam Inc., FEI Company, Fischione Instruments, Focus Electronics GmbH, 4pi, Gatan Inc., Hitachi High-Technologies America Inc., JEOL, K.E. Developments, Leica Microsystems, Marivac Canada Inc., M. E. Taylor Engineering Inc., Mercury Computer Systems Inc., Micro Star Technologies, Millbrook Scientific Instruments PLC, National A.T.E., Numerical Algorithms Group, Omicron NanoTechnology GmbH, Optronics, Oxford Instruments, PARAGON Bioservices, Photometrics (Roper Scientific Inc.), Polysciences Inc., Princeton Gamma-Tech, Proscan, ProSciTech, Quorum Technologies, RMC Products, SemTech Solutions Inc.,
Sidec Technologies, Soft Imaging System, South Bay Technology Inc., Structure Probe Inc., Ted Pella Inc., Tousimis, Tietz Video and Image Processing Systems, Vibratome, Visitec, Zeiss
Because the 2 image paths in a CMO scope are parallel rather than diverging, additional components, such as extra lenses, fluorescence capabilities, and imaging equipment can be added in a modular fashion. But you pay for that modularity. While a basic Greenough system (without illumination) such as the Olympus SZ 51 or Leica S6 might cost $1,500, CMOs start at around $3,500 and can run into the tens of thousands of dollars at the high end. That's pretty expensive when you consider a regular tissue-culture or embryology-type inverted microscope would sell for $3,000-5,000. As a rule, high-quality optics, such as apochromatically corrected lenses (which align the whole of the visible spectrum in one plane), are the preserve of high-end CMO scopes. The exception is Leica's S8 APO. What this Greenough instrument lacks in modularity, it makes up for in optical quality, achieving higher resolution and double the magnification (80×) of other Greenough models. For some jobs, such as fluorescence, the CMO scope's modularity is essential. Adding fluorescence capabilities to a CMO microscope is a snap, given the design's modular architecture. Researchers are just continuing to push the boundaries with a broader range of fluorochromes, labeling fewer and fewer cells, maybe deeper and deeper within tissues. And fluorescence work is increasingly carried out on living specimens. Illumination intensity means energy, and energy destroys living animals. Low-intensity excitation is another driver for increased sensitivity, and has also led to the development of highly automated models such as Leica's MZ 16 FA, which illuminates in short bursts, enabling time-lapse image capture. All of which pushes the total system cost higher. A full imaging system including a monitor and a sensitive digital camera might add an extra $15,000 to the instrument's cost. Meanwhile, different fluorochromes need different excitation and emission filters, which might cost $1,500 per set. Most people use 3 of those. Ergonomics is another advantage of CMO modularity. In the standard biological lab, you have people who are 4'10" up to 6'5" working on the microscope. Having the ability to tilt the binocular head is a big advantage. In this respect, St. Johnston has a gripe with the high-end Leica models. The eyepieces are just too high for any normal human being to use, and you have to spend another £1,000 on an adjustable head. St. Johnston has long used Leica scopes (he has about 10 in his lab) but is currently testing other makes and models. In terms of optical qualit, Leica is definitely the Cadillac, but Zeiss' are catching up and will maybe even beat them. Zeiss recently launched 2 new stereoscopes. Both its new flagship CMO-style Discovery microscope and its fluorescence-capable partner, the Lumar, are characterized by unusually large, high-numerical aperture objective lenses to achieve the highest possible resolution while maintaining depth-of-field. This has trimmed the working distance from 60 mm in previous models to 35 mm, but it is a price many biologists will be willing to pay. If you've got a weak signal, you need a bright machine. But it doesn't come cheap: between £20,000 and £30,000. The new Leica does not have the ergonomic drawbacks of its predecessors. The final decision on which instrument to buy might come down to which manufacturer offers the best deal. M2 Bio Quad, a rotating objective turret built by Kramer Scientific to fit a Zeiss SV11, enables switching between stereo and high-power compound lenses. Adapting an SV11 (a discontinued model) in this way costs between $17,000 and $20,000 : the company is developing a similar unit for Zeiss' Discovery. Leica provides an equivalent product in its Fluo Combi. The trend for increasing motorization of such features as focus and zoom, which, via foot-pedal control, can leave both hands free for manipulations.
- optical tweezers : a laser light that is focused by the objective lens of a microscope can be used to trap a plastic bead with a diameter of 0.1-10 mm. The trapped bead can be used as a "handle" to allow the manipulation of molecules. They offer high resolution for trapping single particles, but have a limited manipulation area owing to tight focusing requirements
- electrokinetic forces and other mechanisms provide high throughput, but lack the flexibility or the spatial resolution necessary for controlling individual cells
- electrophoresis
- dielectrophoresis
- travelling-wave dielectrophoresis
- optical image-driven dielectrophoresis technique that permits high-resolution patterning of electric fields on a photoconductive surface for manipulating single particles. It requires 100,000 times less optical intensity than optical tweezers. Using an incoherent light source (a light-emitting diode or a halogen lamp) and a digital micromirror spatial light modulator, we have demonstrated parallel manipulation of 15,000 particle traps on a 1.3 x 1.0 mm2 area. With direct optical imaging control, multiple manipulation functions are combined to achieve complex, multi-step manipulation protocolsref.
- magnetic tweezers
- acoustic traps
- hydrodynamic flows cannot achieve high resolution and high throughput at the same time.
