Confocal microscopy has a unique optical sectioning property which allows three-dimensional images at different depths. Use of a microlens array is a potential alternative to the Nipkow disk for parallel imaging with high throughput in real-time confocal microscopy. The use of variable-focal-length microlenses can provide a way to axially scan the foci electronically avoiding the inflexible mechanical movement of the lens or the sample. Here we demonstrate a combination of a variable-focal-length microlens array and a fiber optic bundle as a way to create a high throughput aperture array that would be potentially applied as confocal imaging in vivo biological specimens. Variable focal length microlenses that we use consist of a liquid crystal film sandwiched between a pair of conductive substrates with patterned electrodes. The incident side of the microlens array was determined by examining the focus distribution in the axial direction. The variation of the focal length obtained by changing the voltage and corresponding focus intensity were measured through a conventional microscope. Meanwhile, the fiber bundle was characterized by coupling with either coherent or incoherent light source. We use the fiber bundle as both a multiple aperture and an image-carrying element and combine it with a microlens array to built up a confocal system. Axial responses are measured in two optical arrangements as a route to investigate endoscope potential.
We present a description of our work in recent years on imaging in
confocal microscopy in the context of biological applications. The
first system presented considers a Nipkow disc type arrangement where a detailed investigation of optimal aperture arrangements and spacings is performed. The effect of varying these parameters on the optical sectioning characteristics and on the light throughput is evaluated. Novel routes to achieving alternative multi-aperture configurations are presented. A programmable array microscope demonstrator is described using a ferroelectric liquid crystal SLM. A novel system is also proposed which uses variable focus microlenses in a confocal imaging system. We also discuss current trends in confocal microscopy in biology.
Images of the microspheres are studied in three-dimensions by using the confocal and conventional scanning polarization microscopes. It is found that the polarization of the detected signals is mainly parallel to the initial polarization which is due to the high extinction coefficient of the confocal system. Arc pairs are observed at the edge of the microspheres with the conventional polarization microscope with a crossed analyzer. Theoretical analysis are given by using the vector field theory and the image formations of the two systems.
The direct-view microscope is a confocal microscope which allows faster image acquisition rates than typical confocal scanning optical microscopes through the use of a pinhole array rather than the usual single pinhole. We present a theoretical investigation of the effects of source coherence on optical sectioning in direct-view microscopy. As a first step we present an equation which describes the optical sectioning strength of a coherent source brightfield DVM employing an infinite pinhole array. By simulation of both this and the finite array equation which show the existence of certain `principal' sidelobes which are likely to represent the most problematic artifact of coherent source imaging. By further analysis of the infinite array equation, we arrive at an expression which describes the defocus positions where the principal sidelobes occur. Finally, we move on to show how rectangular arrays are predicted, by the infinite array equation, to outperform square arrays and we show examples of this.
We combine the differential confocal microscopy and annular pupil filter together. The theory of differential confocal microscopy and the property of the annular pupil filter are discussed in detail. The results of computer simulation and experiment prove that this method can extend the axial dynamic range and improve transversal resolution of 3D profilometry at sacrificing axial resolution.
In confocal scanning imaging system, using apodization masks can improve the system resolution. A mask based on circle grating function with different spacing is presented and made, which is simple and easy to practically make and apply. The experiments of using a mask are performed in a reflection-mode fiber confocal scanning microscope. The intensity response curves in transverse and axial direction of this system are given. A sharp straight edge of a flat object is measured. And the 2D image of a biological organism is also given. The results demonstrate that adding this apodization mask does not reduce the axial resolution while improve the transverse resolution.
Two optical sectioning imaging technologies with visible laser and near infrared laser illumination to apply industrial and biologic detection are presented respectively in this paper. One of them is based on fiber optical confocal scanning microscopy, the other is imaging-relay arrangement with small visible field. The systems have resolution of submicron at lateral direction and nanometer resolution in axial direction and wider work range about submillimeter magnitude.
In fiber optical confocal scanning imaging system of reflection mode, interference of the reflection light beams from terminal face of the fiber coupling tip and from the surface of the sample will always destroy the image of low reflection index samples, such as CD-R pregroove basal disc and biotic samples. A quantitative analysis was given to find out the influence of fiber terminal face reflection on the system axial response. In order to avoid the influence of the fiber reflected light, the reflectivity ratio of the fiber tip to the sample should be low. Interference noise was effectively reduced by immersing the unused fiber tip into glycerol and cleaving the fiber tip end face at an angle. The proof experimental results of axial response have been shown. Finally, good quality confocal images of the recordable CD pregroove and the chromosome were presented.
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