Principles and Technique of Fluorescence Microscopy

1961 ◽  
Vol s3-102 (60) ◽  
pp. 419-449
Author(s):  
M. R. YOUNG
Keyword(s):  
Fluorescence Microscopy ◽  
Focal Length ◽  
Dark Field Image ◽  
Wide Band ◽  
Dark Field ◽  
Microscopical Study ◽  
Light Sources ◽  
Bright Field ◽  
Alternative Systems ◽  
Light Filters

Interest in fluorescence microscopy has greatly increased in recent years. Technical considerations have to some extent prevented even wider application of the various fluorescence techniques now available for microscopical study of biological specimens. This paper outlines the basic requirements for optimal image quality, for the benefit of biologists and others who may not be conversant with the optical principles involved. The central problem of illumination is reviewed in some detail, and an assessment given of the two methods in current use, namely the bright-field and dark-field systems. Ratios of fluorescent to activating light received by the objective aperture, given by the two systems, have been compared, and measurements have been made of their relative light-concentrating power. Available light sources and their suitability for the excitation of fluorescence are discussed, with the problems of selecting appropriate light filters for use with the alternative systems of illumination. It is concluded that the dark-field system has decided advantages in practice and in theory for the following reasons: (1) The dark-field condenser serves as an efficient primary filter, contributing to a black background and hence good contrast. (2) The equivalent focal length is less than that of the bright-field condenser and it concentrates energy in a smaller area; this compensates in part for the loss of energy inevitably caused by the central stop. (3) It permits the use of wide-band primary filters of maximum transmission because contrast in the fluorescent image is affected only by a weak superimposed dark-field image produced in the object-plane by scattered residual activating light passed by the primary filter. With blue-light activation the visible dark-field image is effectively eliminated by means of a weak blue-absorbing secondary filter. (4) The loss of contrast due to veiling glare is minimized. A rational layout for fluorescence microscopy and methods for accurate alignment of the microscope in the vertical and horizontal positions are described. Factors influencing the choice of suitable objectives and eyepieces and some details of methods for mounting specimens are given.

Use of Astronomy Filters in Fluorescence Microscopy

2012 ◽  
Vol 18 (1) ◽  
pp. 196-211
Author(s):  
Jörg Piper
Keyword(s):  
Fluorescence Microscopy ◽  
Incident Light ◽  
Optical Design ◽  
Dark Field ◽  
Light Sources ◽  
Light Path ◽  
Bright Field ◽  
Energy Excitation ◽  
Halogen Light ◽  
Background Brightness

AbstractMonochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this “low energy excitation,” photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

Density-based discrimination of protein and RNA in the ribosome

1992 ◽  
Vol 50 (1) ◽  
pp. 464-465
Author(s):  
Joachim Frank
Keyword(s):  
Transfer Function ◽  
Spatial Frequency ◽  
Three Dimensional ◽  
Wiener Filter ◽  
Dark Field Image ◽  
Dark Field ◽  
Frequency Range ◽  
Bright Field ◽  
Contrast Transfer Function ◽  
Pass Filter

Cryo-electron microscopy combined with single-particle reconstruction techniques has allowed us to form a three-dimensional image of the Escherichia coli ribosome.In the interior, we observe strong density variations which may be attributed to the difference in scattering density between ribosomal RNA (rRNA) and protein. This identification can only be tentative, and lacks quantitation at this stage, because of the nature of image formation by bright field phase contrast. Apart from limiting the resolution, the contrast transfer function acts as a high-pass filter which produces edge enhancement effects that can explain at least part of the observed variations. As a step toward a more quantitative analysis, it is necessary to correct the transfer function in the low-spatial-frequency range. Unfortunately, it is in that range where Fourier components unrelated to elastic bright-field imaging are found, and a Wiener-filter type restoration would lead to incorrect results. Depending upon the thickness of the ice layer, a varying contribution to the Fourier components in the low-spatial-frequency range originates from an “inelastic dark field” image. The only prospect to obtain quantitatively interpretable images (i.e., which would allow discrimination between rRNA and protein by application of a density threshold set to the average RNA scattering density may therefore lie in the use of energy-filtering microscopes.

Structures and defects identified by dark-field HREM

1992 ◽  
Vol 50 (1) ◽  
pp. 124-125
Author(s):  
J.P. Zhang
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Image Interpretation ◽  
Dark Field Image ◽  
Dark Field ◽  
Bright Field ◽  
Field Mode ◽  
Transmitted Beam ◽  
Structure Information ◽  
The Difference

The tilted illumination dark field high resolution imaging technique was applied to structures and defects of semiconductors and superconductors. We used a Hitachi-H9000 top entry microscope with a high resolution pole-piece of Cs=0.9 mm, operated at 300 Kv. Proper apertures, tilting angle and imaging conditions were chosen to minimize the phase shift due to aberrations. Since the transmitted beam was moved outside the aperture, the noise ratio was greatly reduced, which resulted in a significant enhancement of image contrast and apparent resolution. Images are not difficult to interpret if they have a clear correspondence to structure - information from image simulations in bright field mode can be used to assist in dark field image interpretation.An example in a semiconductor, GaAs/Ga0.49In0.51P2 superlattice imaged along [110] direction is shown in Figure 1. In this dark field image the GaAs and GaInP layers can be easily distinguished by their different contrast, and the difference in quality between both sides of interfaces is clear. An enlarged image in Figure 1 shows the defective area on the rough side of interface. Since this image shows the same pattern as the [110] projection of an fee structure, the major structural information about {111}, {200}, {220} planes can be obtained from this zone. Note that in bright field mode, [110] is not a good zone for imaging such multilayers.

scholarly journals Dual-phone illumination-imaging system for high resolution and large field of view multi-modal microscopy

Lab on a Chip ◽  
2019 ◽  
Vol 19 (5) ◽  
pp. 825-836 ◽  
Author(s):  
Sara Kheireddine ◽  
Ayyappasamy Sudalaiyadum Perumal ◽  
Zachary J. Smith ◽  
Dan V. Nicolau ◽  
Sebastian Wachsmann-Hogiu
Keyword(s):  
High Resolution ◽  
Fluorescence Microscopy ◽  
Mobile Phone ◽  
Imaging System ◽  
Dark Field ◽  
Field Of View ◽  
Large Field ◽  
Bright Field

Bright-field, dark-field, Rheinberg, fluorescence microscopy on a mobile phone with phone screen illumination.

Displaced Aperture Dark Field Images by an Aberration Correction

1972 ◽  
Vol 30 ◽  
pp. 564-565
Author(s):  
K. Shirota ◽  
T. Yamamoto ◽  
T. Yanaka ◽  
O. Vingsbo
Keyword(s):  
Optical Axis ◽  
Dark Field Image ◽  
Image Plane ◽  
Dark Field ◽  
Objective Lens ◽  
Aberration Correction ◽  
Bright Field ◽  
Incident Electron Beam ◽  
Dark Field Microscopy ◽  
Electron Orbit

In displaced aperture dark field microscopy, the direction of the incident electron beam with respect to the specimen is maintained unaltered between bright field and dark field images, unlike the case of usual high resolution dark field microscopy by tilted illumination. The displaced aperture technique, however, gives a strong dark field image deterioration due to field aberrations. These are prounouncedly large since the deviation of the orbits of the imaging electrons from the optical axis is very large in this case. In the present paper, the improvement of the image quality by aberration correction is discussed.1. The chromatic field aberration is composed of components mainly from the objective and intermediate lenses. It can be corrected by changing the electron orbit by means of a beam deflector, introduced in the image plane of the objective lens.

scholarly journals THE LOCALIZATION OF CATECHOLAMINE FLUORESCENCE TO DOG HYPOTHALAMIC NEUROMELANIN-BEARING NEURONS

1973 ◽  
Vol 21 (2) ◽  
pp. 175-183 ◽  
Author(s):  
HERBERT BARDEN ◽  
ROBERT BARRETT
Keyword(s):  
Optical Properties ◽  
Fluorescence Microscopy ◽  
Ultraviolet Light ◽  
Dark Field ◽  
Fluorescence Method ◽  
Green Fluorescence ◽  
Bright Field ◽  
Neuronal Perikaryon ◽  
Histochemical Methods ◽  
Catecholamine Fluorescence

By use of histochemical methods, a survey has been made of the localization of catecholamine to neuronal perikarya and processes in relation to neuromelanin accumulation in neuronal perikarya in the hypothalamus of dogs of various ages. Catecholamine was visualized in sections as a green fluorescence by means of the formaldehyde gasinduced fluorescence method of Falck and Hillarp. Neuromelanin, if also present in these sections, was identified on the basis of its characteristic optical properties as visualized through bright field, dark field and fluorescence microscopy. The study demonstrated a sequential replacement of temporally diminishing catecholamine fluorescence by accumulating neuromelanin within the same neuronal perikaryon wherein they were found to overlap during part of the 1st year of life. Catecholamine in processes in the perifornical region of the hypothalamus, where the neuromelanin-accumulating neurons were situated, was also diminished with age. If neuromelanin was extensively exposed to ultraviolet light, as required for viewing and for photography, the initially nonfluorescent pigment was converted to one which appeared fluorescent yellow. The larger and older accumulations of neuromelanin were more resistant to this induced alteration than the smaller and younger accumulations. These results support the concept that catecholamine contributes to the formation of neuromelanin although they do not exclude the possibility that the presence of both these substances in the same perikaryon is entirely coincidental.

Two Investigations into Grain Boundary Structure

1977 ◽  
Vol 35 ◽  
pp. 688-691
Author(s):  
P. Humble
Keyword(s):  
Grain Boundary ◽  
Dark Field ◽  
Boundary Structure ◽  
Boundary Dislocation ◽  
Bright Field ◽  
Intrinsic Structure ◽  
Weak Beam ◽  
Grain Boundary Structure ◽  
Independent Information ◽  
Diffraction Patterns

There has been sustained interest over the last few years into both the intrinsic (primary and secondary) structure of grain boundaries and the extrinsic structure e.g. the interaction of matrix dislocations with the boundary. Most of the investigations carried out by electron microscopy have involved only the use of information contained in the transmitted image (bright field, dark field, weak beam etc.). Whilst these imaging modes are appropriate to the cases of relatively coarse intrinsic or extrinsic grain boundary dislocation structures, it is apparent that in principle (and indeed in practice, e.g. (1)-(3)) the diffraction patterns from the boundary can give extra independent information about the fine scale periodic intrinsic structure of the boundary.In this paper I shall describe one investigation into each type of structure using the appropriate method of obtaining the necessary information which has been carried out recently at Tribophysics.

Observation of Atom Rows in Thorium Pyromellitate Using a Field Emission STEM

1977 ◽  
Vol 35 ◽  
pp. 80-81
Author(s):  
H. Todokoro ◽  
S. Nomura ◽  
T. Komoda
Keyword(s):  
Field Emission ◽  
Amorphous Carbon ◽  
Carbon Film ◽  
Dark Field Image ◽  
Dark Field ◽  
Amorphous Carbon Film ◽  
Atomic Size ◽  
Wide Range ◽  
A Chain

It is interesting to observe polymers at atomic size resolution. Some works have been reported for thorium pyromellitate by using a STEM (1), or a CTEM (2,3). The results showed that this polymer forms a chain in which thorium atoms are arranged. However, the distance between adjacent thorium atoms varies over a wide range (0.4-1.3nm) according to the different authors.The present authors have also observed thorium pyromellitate specimens by means of a field emission STEM, described in reference 4. The specimen was prepared by placing a drop of thorium pyromellitate in 10-3 CH3OH solution onto an amorphous carbon film about 2nm thick. The dark field image is shown in Fig. 1A. Thorium atoms are clearly observed as regular atom rows having a spacing of 0.85nm. This lattice gradually deteriorated by successive observations. The image changed to granular structures, as shown in Fig. 1B, which was taken after four scanning frames.

Imaging of platinum-shadowed macromolecular complexes in dark field

1984 ◽  
Vol 42 ◽  
pp. 146-147
Author(s):  
D.W. Andrews ◽  
F.P. Ottensmeyer
Keyword(s):  
Thin Film ◽  
Three Dimensional ◽  
Average Diameter ◽  
Dark Field ◽  
Bright Field ◽  
Field Mode ◽  
Macromolecular Complexes ◽  
Average Mass ◽  
Topological Features ◽  
Projection Images

Shadowing with heavy metals has been used for many years to enhance the topological features of biological macromolecular complexes. The three dimensional features present in directionaly shadowed specimens often simplifies interpretation of projection images provided by other techniques. One difficulty with the method is the relatively large amount of metal used to achieve sufficient contrast in bright field images. Thick shadow films are undesirable because they decrease resolution due to an increased tendency for microcrystalline aggregates to form, because decoration artefacts become more severe and increased cap thickness makes estimation of dimensions more uncertain.The large increase in contrast provided by the dark field mode of imaging allows the use of shadow replicas with a much lower average mass thickness. To form the images in Fig. 1, latex spheres of 0.087 μ average diameter were unidirectionally shadowed with platinum carbon (Pt-C) and a thin film of carbon was indirectly evaporated on the specimen as a support.

A Many-Beam Technique for Enhanced Resolution of Partial Dislocations

1972 ◽  
Vol 30 ◽  
pp. 646-647
Author(s):  
J. M. Oblak ◽  
B. H. Kear
Keyword(s):  
Phase Shift ◽  
Field Method ◽  
Dark Field ◽  
Bright Field ◽  
Field Technique ◽  
Weak Beam ◽  
Partial Dislocations ◽  
Enhanced Resolution ◽  
Nickel Base ◽  
Beam Technique

The “weak-beam” and systematic many-beam techniques are the currently available methods for resolution of closely spaced dislocations or other inhomogeneities imaged through strain contrast. The former is a dark field technique and image intensities are usually very weak. The latter is a bright field technique, but generally use of a high voltage instrument is required. In what follows a bright field method for obtaining enhanced resolution of partial dislocations at 100 KV accelerating potential will be described.A brief discussion of an application will first be given. A study of intermediate temperature creep processes in commercial nickel-base alloys strengthened by the Ll2 Ni3 Al γ precipitate has suggested that partial dislocations such as those labelled 1 and 2 in Fig. 1(a) are in reality composed of two closely spaced a/6 <112> Shockley partials. Stacking fault contrast, when present, tends to obscure resolution of the partials; thus, conditions for resolution must be chosen such that the phase shift at the fault is 0 or a multiple of 2π.

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