SUMMARY
The effect of refractive-index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.

Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index

Since Gabor's first hologram, the meanings of that term have grown with increased use of the invention. Computers expanded applications further, and this paper surveys the methods and techniques of computer generated holograms.

Computer generated holograms: an historical review

Imaging systems with circular and annular pupils aberrated by primary aberrations are considered. Both classical and balanced (Zernike) aberrations are discussed. Closed-form solutions are derived for the Strehl ratio, except in the case of coma, for which the integral form is used. Numerical results are obtained and compared with Marechal’s formula for small aberrations. It is shown that, as long as the Strehl ratio is greater than 0.6, the Marechal formula gives its value with an error of less than 10%. A discussion of the Rayleigh quarter-wave rule is given, and it is shown that it provides only a qualitative measure of aberration tolerance. Nonoptimally balanced aberrations are also considered, and it is shown that, unless the Strehl ratio is quite high, an optimally balanced aberration does not necessarily give a maximum Strehl ratio.

Strehl ratio for primary aberrations: some analytical results for circular and annular pupils.

It was recently shown that, when a converging spherical wave is focused in a diffraction-limited system of sufficiently low Fresnel numbers, the point of maximum intensity does not coincide with the geometrical focus but is located closer to the exit pupil. In the present paper both qualitative and quantitative arguments are presented that elucidate the modifications that the whole three-dimensional structure of the diffracted field undergoes as the Fresnel number is gradually decreased. Contours of equal intensity in the focal region are presented for systems of selected Fresnel numbers, which focus uniform waves.

https://ir.nctu.edu.tw:443/bitstream/11536/4876/1/A1984TD14300001.pdf

Three-dimensional intensity distribution near the focus in systems of different Fresnel numbers

Three-dimensional intensity distribution near focus in systems of different Fresnel numbers (A)

On the assumption that a hologram is a superposition of zone plates, one from each point in the object, the authors have produced, by sequential printing of zone plates, holograms which reconstruct a pattern of several bright points on a dark field. The pattern may be prescribed in advance and may be three dimensional. The validity of the method is examined mathematically.

The experimental production of synthetic holograms.

CONTOUR VISUALIZATION USING COHERENT OPTICAL SYSTEMSAlan WertheimerLeeds g Northrup Co.North Wales, Pa. 19454andM. Parker GivensThe Institute of OpticsRochester, N.Y. 14627IntroductionThis paper discusses two optical process-ing systems for the rapid extraction of con-tours from a pair of stereo transparencies.For a given x- parallax, these systems pro-vide a display of all contours present at theelevation corresponding to the parallax set-ting.Two assumptions form the basis for theapproaches described here. The first is thatthe photography is taken vertically, or hasbeen rectified to a vertical orientation, sothe stereo images of all points at a givenelevation have the same value of x- parallax.Accordingly, when the stereo transparenciesare overlapped and registered properly, imagecoincidence occurs for all areas at one spe-cific elevation. The task, then, is to lo-cate these regions of image coincidence andisolate them from the rest of the scene.The Positive -Negative SystemOne technique for defining the areas ofmatching imagery is to make a positive trans-parency from one input photograph and a neg-ative transparency from the other. A simpli-fying assumption is that the copies are madewithin the straight line portion of the H & Dcurve, where the slope is defined as the con-stant, y. With T1 (x,y) and T2 (x,y) repre-senting the intensity transmittances of theinputs, the copies TA and TB, may be expressed

Contour Visualization Using Coherent Optical Systems

An expression derived for hologram exposures made along the straight-line portion of an H–D curve of a photographic plate gives the first-order transmittance of a hologram made of several object points exposed simultaneously (conventional holograms). This expression is compared with a similar expression derived previously for holograms made of several object points exposed sequentially (synthetic holograms). Theory and experiments show the effect of the nonlinearity of the photographic process on the contrast of the reconstruction of conventional holograms. Synthetic and conventional holograms are studied theoretically and experimentally to determine how the total amount of light in the reconstruction image depends upon the number of object points when the total amount of light in the object is constant. It is shown that the reconstructed image formed by a conventional hologram contains more light than the image formed by a synthetic hologram of the same number of object points. Both synthetic and conventional holograms are also studied to determine the ratio of reference-beam illuminance to object-beam illuminance that maximizes the amount of light in the reconstructed image.

Effects of Photographic Gamma on Hologram Reconstructions

Focal shifts in diffracted converging spherical waves

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Papers

The experimental production of synthetic holograms.

Contour Visualization Using Coherent Optical Systems

Effects of Photographic Gamma on Hologram Reconstructions

Focal shifts in diffracted converging spherical waves