" Using a 36-megapixel camera that has a 10x microscope objective attached to it, "I photograph the insect in approximately 30 different sections, depending on the size of the specimen.
|
|
Biss, who began photographing insects this way using specimens from his back yard, uses a 36 megapixel camera with a microscope lens ("10x microscope objective attached to it via a 200mm prime lens," he says) to shoot the insects, lighting each section of the insect separately and tailoring the lighting to suit the specific segment.
|
|
Commonly, a microscope objective is used to collect the object wave front. However, as the microscope objective is only used to collect light and not to form an image, it may be replaced by a simple lens. If a slightly lower optical resolution is acceptable, the microscope objective may be entirely removed. Digital holography comes in different flavors, such as off-axis Fresnel, Fourier, image plane, in-line, Gabor and phase-shifting digital holography, depending on the optical setup.
|
|
In this detection scheme a conventional scanning sample or laser-scanning transmission microscope is employed. Both, the heating and the probing laser beam are coaxially aligned and superimposed using a dichroic mirror. Both beams are focused onto a sample, typically via a high-NA illumination microscope objective, and recollected using a detection microscope objective. The thereby collimated transmitted beam is then imaged onto a photodiode after filtering out the heating beam.
|
|
Upconverting nanoparticles can also be used for barcoding. These micro- barcodes can be embedded onto various objects. The barcodes are seen under NIR illumination and can be imaged using an iPhone camera and a microscope objective.
|
|
Micrograph of amyloid in a section of liver that has been stained with the dye Congo red and viewed with crossed polarizing filters, yielding a typical orange-greenish birefringence. 20X microscope objective; the scale bar is 100 microns (0.1mm).
|
|
In practice, the most commonly used configuration is to use a microscope objective lens for focusing the beam, and an aperture made by punching a small, precise, hole in a piece of thick metal foil. Such assemblies are available commercially.
|
|
Focus variation requires an optics with very little depth of field. This can be realized if a microscopy like optics and a microscope objective is used. These objectives have a high numerical aperture which gives a small depth of field.
|
|
31—44 (1978) Since 1992, 4Pi microscopy has been developed by Stefan Hell (Max-Planck Institute for Biophysical Chemistry, Göttingen) into a highly efficient, high-resolution imaging process, using two microscope objective lenses of high numeric aperture opposing each other.
|
|
After mastering the problem of producing objectives based on theoretical calculation one problem remained, namely the production of suitable optical glass. At the time optical glass was obtained from England, France or Switzerland and left much to be desired in quality, reliable availability, selection of optical properties and prompt delivery. The optical properties were not consistent from batch to batch and, as important, those glasses which could be obtained were not ideal for the properties calculated to give the best correction in a microscope objective. Abbe and Zeiss were convinced that the optical qualities of the microscope objective could be improved further if glasses with certain properties could be obtained.
|
|
Optical aberrations are "corrected" by design of the reconstruction algorithm. A reconstruction algorithm that truly models the optical setup will not suffer from optical aberrations. Low cost In optical microscopy systems, optical aberrations are traditionally corrected by combining lenses into a complex and costly image forming microscope objective. Furthermore, the narrow focal depth at high magnifications requires precision mechanics.
|
|
In this detection scheme a conventional scanning sample or laser-scanning transmission microscope is employed. Both, the heating and the probing laser beam are coaxially aligned and superimposed using a dichroic mirror. Both beams are focused onto a sample, typically via a high-NA illumination microscope objective. Alternatively, the probe-beam may be laterally displaced with respect to the heating beam.
|
|
Pictured using transmission microscopy with collimated illumination. One of the methods for constructing such waveguides utilizes photorefractive effect in transparent materials. An increase in the refractive index of a material may be induced by nonlinear absorption of pulsed laser light. In order to maximize the increase of the refractive index, a very short (typically femtosecond) laser pulses are used, and focused with a high NA microscope objective.
|
|
Two Leica oil immersion microscope objective lenses; left 100×, right 40×. The objective lens of a microscope is the one at the bottom near the sample. At its simplest, it is a very high-powered magnifying glass, with very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus inside the microscope tube.
|
|
Xylene is used in the laboratory to make baths with dry ice to cool reaction vessels, and as a solvent to remove synthetic immersion oil from the microscope objective in light microscopy. In histology, xylene is the most widely used clearing agent. Xylene is used to remove paraffin from dried microscope slides prior to staining. After staining, microscope slides are put in xylene prior to mounting with a coverslip.
|
|
Optical tweezers are capable of manipulating nanometer and micron-sized dielectric particles by exerting extremely small forces via a highly focused laser beam. The beam is typically focused by sending it through a microscope objective. The narrowest point of the focused beam, known as the beam waist, contains a very strong electric field gradient. Dielectric particles are attracted along the gradient to the region of strongest electric field, which is the center of the beam.
|
|
The abnormal proteins in some proteopathies have been shown to fold into multiple 3-dimensional shapes; these variant, proteinaceous structures are defined by their different pathogenic, biochemical, and conformational properties. They have been most thoroughly studied with regard to prion disease, and are referred to as protein strains. α-synuclein (brown) in Lewy bodies (large clumps) and Lewy neurites (thread-like structures) in the cerebral cortex of a patient with Lewy body disease, a synucleinopathy. 40X microscope objective.
|
|
To image samples at low temperatures, two main approaches have been used, both based on the laser scanning confocal microscopy architecture. One approach is to use a continuous flow cryostat: only the sample is at low temperature and it is optically addressed through a transparent window. Another possible approach is to have part of the optics (especially the microscope objective) in a cryogenic storage dewar. This second approach, although more cumbersome, guarantees better mechanical stability and avoids the losses due to the window.
|
|
The refractive index (1.7) and optical dispersion properties of germanium dioxide makes it useful as an optical material for wide-angle lenses, in optical microscope objective lenses, and for the core of fiber-optic lines. See Optical fiber for specifics on the manufacturing process. Both Germanium and its glass oxide, GeO2 are transparent to the infrared spectrum. The glass can be manufactured into IR windows and lenses, used for night-vision technology in the military, luxury vehicles,"The Elements" C. R. Hammond, David R. Lide, ed.
|
|
As can be seen in Figure 2, the laser light is split into an object beam and a reference beam. The expanded object beam illuminates the sample to create the object wave front. After the object wave front is collected by a microscope objective, the object and reference wave fronts are joined by a beam splitter to interfere and create the hologram. Using the digitally recorded hologram, a computer acts as a digital lens and calculates a viewable image of the object wave front by using a numerical reconstruction algorithm.
|
|
By translating the focal spot through a bulk transparent material the waveguides can be directly written. A variation of this method uses a low NA microscope objective and translates the focal spot along the beam axis. This improves the overlap between the focused laser beam and the photorefractive material, thus reducing power needed from the laser. When transparent material is exposed to an unfocused laser beam of sufficient brightness to initiate photorefractive effect, the waveguides may start forming on their own as a result of an accumulated self-focusing.
|
|
Optical transfection is the process of introducing nucleic acids into cells using light. Typically, a laser is focussed to a diffraction limited spot (~1 µm diameter) using a high numerical aperture microscope objective. The plasma membrane of a cell is then exposed to this highly focussed light for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane. The generation of a photopore allows exogenous plasmid DNA, RNA, organic fluorophores, or larger objects such as semiconductor quantum nanodots to enter the cell.
|
|
A generic optical tweezer diagram with only the most basic components. The most basic optical tweezer setup will likely include the following components: a laser (usually Nd:YAG), a beam expander, some optics used to steer the beam location in the sample plane, a microscope objective and condenser to create the trap in the sample plane, a position detector (e.g. quadrant photodiode) to measure beam displacements and a microscope illumination source coupled to a CCD camera. An Nd:YAG laser (1064 nm wavelength) is a common choice of laser for working with biological specimens.
|
|
Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique based on the principle of time-domain OCT with line illumination using a broadband laser and line detection using a line-scan camera. LC-OCT produces B-scans in real-time from multiple A-scans acquired in parallel. En face images can also be obtained by scanning the illumination line laterally. The focus is continuously adjusted during the scan of the sample depth, using a high numerical aperture (NA) microscope objective to image with high lateral resolution.
|
|
Illustration of different LSFM implementations. See text for details. Legend: CAM=camera, TL=tube lens, F=filter, DO=detection objective, S=sample, SC=sample chamber, PO=projection objective, CL=cylindrical lens, SM=scanning mirror In this type of microscopy, the illumination is done perpendicularly to the direction of observation (see schematic image at the top of the article). The expanded beam of a laser is focused in only one direction by a cylindrical lens, or by a combination of a cylindrical lens and a microscope objective as the latter is available in better optical quality and with higher numerical aperture than the first.
|
|
Fourier ptychography can be easily implemented on a conventional optical microscope by replacing the illumination source by an array of LED and improve the optical resolution by a factor 2 (with only bright-field illumination) or more (when including dark-field images to the reconstruction.) A major advantage of Fourier ptychography is the ability to use a microscope objective with a lower numerical aperture without sacrificing the resolution. The use of a lower numerical aperture allows for larger field of view, larger depth of focus, and larger working distance. Moreover, it enables effective numerical aperture larger than 1 without resorting to oil immersion.
|
|
These images acquired by a CCD camera are combined in post-treatment (or on-line) by the phase shift interferometry method, where usually 2 or 4 images per modulation period are acquired, depending on the algorithm used. The "en-face" tomographic images are thus produced by a wide- field illumination, ensured by the Linnik configuration of the Michelson interferometer where a microscope objective is used in both arms. Furthermore, while the temporal coherence of the source must remain low as in classical OCT (i.e. a broad spectrum), the spatial coherence must also be low to avoid parasitical interferences (i.e.
|
|
Next, the light source is focused onto a small patch of the viewable area either by switching to a higher magnification microscope objective or with laser light of the appropriate wavelength. The fluorophores in this region receive high intensity illumination which causes their fluorescence lifetime to quickly elapse (limited to roughly 105 photons before extinction). Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot. As Brownian motion proceeds, the still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region.
|
|
Two Leica oil immersion microscope objective lenses: 100× (left) and 40× (right) Some microscopes make use of oil-immersion objectives or water-immersion objectives for greater resolution at high magnification. These are used with index-matching material such as immersion oil or water and a matched cover slip between the objective lens and the sample. The refractive index of the index-matching material is higher than air allowing the objective lens to have a larger numerical aperture (greater than 1) so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. Numerical apertures as high as 1.6 can be achieved.
|
|
Defocusing is the most basic variant of the technique and it does not provide a complete separation between excitation and collection zones, rendering this variant less effective. Nonetheless, defocused measurements have the great advantage to be easily performed with a conventional micro-Raman without any hardware nor software modifications. Defocusing consists in the enlargement of the excitation and the collection zones that is achieved by moving the microscope objective out of focus (Δz movements) from the surface of the object or sample under analysis. The Δz movements range goes typically from few tens to two millimeters, depending on the numbers and thicknesses of the materials.
|
|
In 2009, McConnell began working with William Bradshaw Amos and built a new lens, Mesolens, that can allow 3D imaging with a depth resolution of a few microns for objects up to 6 mm wide and 3 mm thick. The Mesolens is a giant optical microscope objective supported by the Medical Research Council (MRC). It can be used to image large biomedical specimens, including embryos, tumours and areas in brain, as well as scanning large areas of samples in a short amount of time. The lens has 260 megapixal effective camera and a magic ratio of 8:1, which can even resolve individual bacteria.
|
|
As an example, the figure on the right shows the 3D point-spread function in object space of a wide-field microscope (a) alongside that of a confocal microscope (c). Although the same microscope objective with a numerical aperture of 1.49 is used, it is clear that the confocal point spread function is more compact both in the lateral dimensions (x,y) and the axial dimension (z). One could rightly conclude that the resolution of a confocal microscope is superior to that of a wide-field microscope in all three dimensions. A three-dimensional optical transfer function can be calculated as the three-dimensional Fourier transform of the 3D point-spread function.
|
|
By using a supercontinuum laser as a light source, a quasi-isotropic spatial resolution of ~ 1 µm is achieved at a central wavelength of ~ 800 nm. On the other hand, line illumination and detection, combined with the use of a high NA microscope objective, produce a confocal gate that prevents most scattered light that does not contribute to the signal from being detected by the camera. This confocal gate, which is absent in the full-field OCT technique, gives LC-OCT an advantage in terms of detection sensitivity and penetration in highly scattering media such as skin tissues.. So far this technique has been used mainly for skin imaging in the fields of dermatology and cosmetology.
|
|
This way a thin sheet of light or lightsheet is created in the focal region that can be used to excite fluorescence only in a thin slice (usually a few micrometers thin) of the sample. The fluorescence light emitted from the lightsheet is then collected perpendicularly with a standard microscope objective and projected onto an imaging sensor (usually a CCD, electron multiplying CCD or CMOS camera). In order to let enough space for the excitation optics/lightsheet an observation objective with high working distance is used. In most LSFMs the detection objective and sometimes also the excitation objective are fully immersed in the sample buffer, so usually the sample and excitation/detection optics are embedded into a buffer-filled sample chamber, which can also be used to control the environmental conditions (temperature, carbon dioxide level ...) during the measurement.
|
|
The image reconstruction algorithms are based on iterative phase retrieval, either related to the Gerchberg–Saxton algorithm or based on convex relaxation methods. Like real space ptychography, the solution of the phase problem relies on the same mathematical shift invariance constraint, except in Fourier ptychography it is the diffraction pattern in the back focal plane that is moving with respect to the back-focal plane aperture. (In traditional ptychography the illumination moves with respect to the specimen.) Many reconstruction algorithms used in real-space ptychography are therefore used in Fourier ptychography, most commonly PIE and variants such as ePIE and 3PIE. Variants of these algorithms allow for simultaneous reconstruction of the pupil function of an optical system, allowing for the correction of the aberrations of the microscope objective, and diffraction tomography which permits the 3D reconstruction of thin sample objects without requiring the angular sample scanning needed for CT scans.
|
|