Sub-100nm Axial Resolution in 3D Widefield Optical Microscopy Using Two Opposing Objective Lenses

1997 ◽  
Vol 3 (S2) ◽  
pp. 801-802
Author(s):  
Mats G.L. Gustafsson ◽  
David A. Agard ◽  
John W. Sedat
Keyword(s):  
Optical Microscopy ◽  
Numerical Aperture ◽  
Optical Microscope ◽  
The Other ◽  
Objective Lens ◽  
Back Side ◽  
Angular Range ◽  
Axial Resolution ◽  
Lens Development ◽  
Microscope Slide

It has been understood since the days of Ernst Abbe that an optical microscope can reach higher resolution the wider the set of angles from which it can observe the specimen — this angular range, of course, is what is indicated by the Numerical Aperture (NA) of the objective lens. Over more than a century of objective lens development, this light collecting angle has been increased closer and closer to the full 180° angle available above a microscope slide. There is, however, a second, equally large and easily accessible set of observation angles available on the other side of the specimen slide, and a standard microscope makes no use whatsoever of light emitted in these directions. One might then ask whether there is not a substantial resolution advantage to be had by devising a microscope that does make use of this "back side" light. The answer, in fact, is yes.

scholarly journals Morphology of Endothelial Cells from Different Regions of the Swine Cornea

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Lídia Luzia Clerot ◽  
Paula Stieven Hünning ◽  
Marcelle Bettio ◽  
Marcela Torikachvili ◽  
Michelle Beck Petersen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Optical Microscopy ◽  
Repeated Measures ◽  
Corneal Endothelium ◽  
Anterior Segment ◽  
Optical Microscope ◽  
Specular Microscopy ◽  
Microscope Slide ◽  
Large White ◽  
Alizarin Red

Background: The corneal endothelium is a monolayer of polygonal cells which constitute the last layer of the cornea. The integrity of this layer is critical to cornea transparency. The characterization of normal corneal endothelial morphology is important not only to clinical evaluation but also to selection of areas of the cornea with better quality to be employed as donor tissue. The aim of the present study was to evaluate the morphology of endothelial cells from different regions of the swine cornea after alizarin red staining using optical microscopy.Materials, Methods & Results: Twenty-four healthy eyes from 12 swine Large White breed, with 14-month-old, males or females obtained from a licensed Brazilian commercial slaughterhouse were studied. Immediately after humane slaughter, the eyes were enucleated and submitted to ophthalmic examination. Eyes with signs of diseases of the anterior segment were excluded. The cornea, with 3 mm of the sclera, was removed and placed on a glass microscope slide with the endothelial side up. Four radial incisions were made in the periphery of the cornea to better accommodate the cornea on the microscope slide. Alizarin red was diluted in isotonic solution (0.2 g/100 mL) and the pH was adjusted to 4.2 with hydrochloric acid. Three drops of alizarin red were placed on the corneal endothelium. After 90 s, the dye was removed from the cornea with balanced saline solution. The corneal endothelium was examined and photographed using an optical microscope. All evaluations were performed by the same investigator. Photomicrographs were taken of central, superior, inferior, nasal and temporal corneal areas. Parameter studied included endothelial cell morphology. For the statistical analysis, was employed the ANOVA variance test (repeated measures). Differences were considered statistically significant at P < 0.05. Normal endothelium cells were mainly hexagonal (83.7%), pentagonal (7.45%) and heptagonal (8.8%), with a minimal number of cells of other shapes present. There were no significant statistical differences in the proportion of the morphology and the different regions of the cornea (P = 0.31).Discussion: Different techniques are available for the analysis of corneal endothelium, including mainly scanning electron microscopy, specular microscopy and optical microscopy. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and to obtain images of the swine endothelium of all regions of the cornea.  The analysis of the cellular morphology or the percentage of hexagonal cells are among the main parameters used to evaluate the health of the corneal endothelium. In this study, the endothelium had the predominance of the hexagonal shape in all regions studied. In swine, there are no studies evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea.

Laser-feedback microscopy: Fundamental principles and application to high-resolution biological imaging

1993 ◽  
Vol 51 ◽  
pp. 84-85
Author(s):  
Alan Bearden ◽  
Terrence L. Wong ◽  
Morgan W. Mitchell ◽  
Leslie C. Osborne ◽  
Michael P. O’Neill
Keyword(s):  
Confocal Microscopy ◽  
Near Field ◽  
Numerical Aperture ◽  
Optical Microscope ◽  
Lateral Resolution ◽  
Objective Lens ◽  
Scanning Confocal Microscopy ◽  
Gaussian Profile ◽  
Phase Interference

As discovered by Abbé, a fundamental limit to the lateral resolution of a conventional optical microscope is produced by “far-field” aperture diffraction at the objective lens. This limitation can be expressed in terms of the Airy disc which relates the obtainable resolution to the wavelength of light used and the objective’s numerical aperture (NA). In scanning confocal microscopy with a laser-produced Gaussian profile illumination beam, the lateral resolution in given by 1.22λ/NA. The axial resolution in this design is improved by the use of a pinhole aperture to pass light only from the objective’s focal plane. Typical best values for lateral (x,y) and axial (z) resolution for visible wavelength scanning confocal microscopy are ~200nm and ~300nm with a NA=1.4 oil immersion objective.Although optical microscopy displays limited resolution when compared to some forms of electron and scanning-probe microscopies (STM, AFM), the convenience of sample preparation and its ease in performing in vivo studies has given rise to new methods of increased resolution (e.g., “near field” techniques, photon-tunneling, phase interference contrast).

Practical Correction of Three Fold Astigmatism in the Philips CM TEM

1996 ◽  
Vol 54 ◽  
pp. 418-419
Author(s):  
Arno J. Bleeker ◽  
Mark H.F. Overwijk ◽  
Max T. Otten
Keyword(s):  
Optical Properties ◽  
Phase Contrast ◽  
The Other ◽  
Objective Lens ◽  
Symmetric Part ◽  
A Posteriori ◽  
Contrast Transfer Function ◽  
High Brightness ◽  
Rotationally Symmetric ◽  
Brightness Field

With the improvement of the optical properties of the modern TEM objective lenses the point resolution is pushed beyond 0.2 nm. The objective lens of the CM300 UltraTwin combines a Cs of 0. 65 mm with a Cc of 1.4 mm. At 300 kV this results in a point resolution of 0.17 nm. Together with a high-brightness field-emission gun with an energy spread of 0.8 eV the information limit is pushed down to 0.1 nm. The rotationally symmetric part of the phase contrast transfer function (pctf), whose first zero at Scherzer focus determines the point resolution, is mainly determined by the Cs and defocus. Apart from the rotationally symmetric part there is also the non-rotationally symmetric part of the pctf. Here the main contributors are not only two-fold astigmatism and beam tilt but also three-fold astigmatism. The two-fold astigmatism together with the beam tilt can be corrected in a straight-forward way using the coma-free alignment and the objective stigmator. However, this only works well when the coefficient of three-fold astigmatism is negligible compared to the other aberration coefficients. Unfortunately this is not generally the case with the modern high-resolution objective lenses. Measurements done at a CM300 SuperTwin FEG showed a three fold-astigmatism of 1100 nm which is consistent with measurements done by others. A three-fold astigmatism of 1000 nm already sinificantly influences the image at a spatial frequency corresponding to 0.2 nm which is even above the point resolution of the objective lens. In principle it is possible to correct for the three-fold astigmatism a posteriori when through-focus series are taken or when off-axis holography is employed. This is, however not possible for single images. The only possibility is then to correct for the three-fold astigmatism in the microscope by the addition of a hexapole corrector near the objective lens.

On resolving fine periodic microstructures in the optical microscope

1989 ◽  
Vol 47 ◽  
pp. 364-365
Author(s):  
W.S. Putnam ◽  
C. Viney
Keyword(s):  
Liquid Crystalline ◽  
Numerical Aperture ◽  
Polarized Light ◽  
Normal Incidence ◽  
Optical Microscope ◽  
Angle Of Incidence ◽  
Resolution Limit ◽  
Crystalline Materials ◽  
Periodic Microstructures ◽  
Liquid Crystalline Materials

Many sheared liquid crystalline materials (fibers, films and moldings) exhibit a fine banded microstructure when observed in the polarized light microscope. In some cases, for example Kevlar® fiber, the periodicity is close to the resolution limit of even the highest numerical aperture objectives. The periodic microstructure reflects a non-uniform alignment of the constituent molecules, and consequently is an indication that the mechanical properties will be less than optimal. Thus it is necessary to obtain quality micrographs for characterization, which in turn requires that fine detail should contribute significantly to image formation.It is textbook knowledge that the resolution achievable with a given microscope objective (numerical aperture NA) and a given wavelength of light (λ) increases as the angle of incidence of light at the specimen surface is increased. Stated in terms of the Abbe resolution criterion, resolution improves from λ/NA to λ/2NA with increasing departure from normal incidence.

Microspectroscopy Using a Solid Immersion Lens

2001 ◽  
Vol 7 (S2) ◽  
pp. 148-149
Author(s):  
C.D. Poweleit ◽  
J Menéndez
Keyword(s):  
Spatial Resolution ◽  
Focal Plane ◽  
Numerical Aperture ◽  
Normal Incidence ◽  
Spot Size ◽  
Index Of Refraction ◽  
Objective Lens ◽  
Improvement Factor ◽  
Oil Immersion ◽  
Long Time

Oil immersion lenses have been used in optical microscopy for a long time. The light’s wavelength is decreased by the oil’s index of refraction n and this reduces the minimum spot size. Additionally, the oil medium allows a larger collection angle, thereby increasing the numerical aperture. The SIL is based on the same principle, but offers more flexibility because the higher index material is solid. in particular, SILs can be deployed in cryogenic environments. Using a hemispherical glass the spatial resolution is improved by a factor n with respect to the resolution obtained with the microscope’s objective lens alone. The improvement factor is equal to n2 for truncated spheres.As shown in Fig. 1, the hemisphere SIL is in contact with the sample and does not affect the position of the focal plane. The focused rays from the objective strike the lens at normal incidence, so that no refraction takes place.

scholarly journals Focal Surface Detection of High Numerical Aperture Objective Lens Based on Differential Astigmatic Method

2021 ◽  
Vol 13 (4) ◽  
pp. 1-8
Author(s):  
Jia-Lin Du ◽  
Wei Yan ◽  
Li-Wei Liu ◽  
Fan-Xing Li ◽  
Fu-Ping Peng ◽  
...  
Keyword(s):  
Numerical Aperture ◽  
Objective Lens ◽  
High Numerical Aperture ◽  
Surface Detection ◽  
Focal Surface

The Structure of Sycamore Callus Cells During Division in a Partially Synchronized Suspension Culture

1970 ◽  
Vol 6 (2) ◽  
pp. 299-321
Author(s):  
K. ROBERTS ◽  
D. H. NORTHCOTE
Keyword(s):  
Cell Wall ◽  
Electron Microscope ◽  
Cell Plate ◽  
Optical Microscope ◽  
The Other ◽  
Active Movement ◽  
Developmental Sequence ◽  
Golgi Bodies ◽  
Dividing Cells ◽  
Peripheral Cytoplasm

Sycamore suspension callus cells have been partially synchronized to give a culture with a mitotic index of 15%. Living dividing cells of the culture have been examined with Nomarski differential interference optics and a comparable study made on fixed cells with the electron microscope. An organized band of reticulate cytoplasm partially encircles the nucleus at mitosis. The cell divides by the formation of a phragmosome which grows across the large vacuole; this allows the organization of the cytoplasm which forms the cell plate to be examined separately from the more general cytoplasm of the cell. The cell plate grows from one side of the cell to the other and down its length a complete developmental sequence can be seen. The Golgi bodies and the endoplasmic reticulum are probably involved in the formation of material for the construction of the cell plate and young cell wall. Microfibrils are formed within the plate in the more mature regions, while material contained within vesicles is incorporated at the young growing edge. At the edge of the plate microtubules are found and these correspond to the fibrillar appearance of the phragmoplast seen with the optical microscope. In the living cell an active movement of organelles along the peripheral cytoplasm can be seen and with fixed cells viewed with the electron microscope microtubules are often found adjacent to the plasmalemma and lying close to mitochondria, crystal-containing bodies and plastids. The appearance of crystal-containing bodies and plastids containing phytoferritin is described.

Sign in / Sign up

Export Citation Format

Share Document