Aperture Correlation Microscopy (ACM) is a fluorescence microscopy technique capable of depth resolved imaging
and enhanced lateral resolution at real-time acquisition rates. It relies on the subtraction of 2 separate images
from different cameras which must be registered to the sub-pixel level. In order to achieve real-time registration
and subtraction, the graphics processing unit (GPU) is used to apply a transformation from one frame to the
other, resulting in a system capable of processing over 200 frames per second on modest hardware (GeForce
330M). Currently, this rate is limited by camera acquision to 16fps.
Additionally, a novel reflection mode correlation microscope is introduced which functions on similar principles
as the fluorescent system but can be used to examine reflective samples. Images and z-stacks taken with this
system are presented here.
In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic
scan rate. This means that the focal spot can be scanned at high speed along any direction in the
specimen, without introducing systematic aberrations. We present experimental point spread function measurements
for this system using an Olympus 0.8 NA 40X water dipping objective lens that demonstrates an axial
range of operation greater than 200 μm. We give details of a novel actuator device used to displace the focusing
element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present a bioscience application of this
system to image dendritic processes that follow non-linear paths in three-dimensional space. The focal spot was
scanned along one such process at 400 Hz with an axial range of more than 90 μm.
In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic
scan rate. This means that the focal spot can be scanned at high speed along any direction in the
specimen, without introducing systematic aberrations. We present experimental point spread function measurements
for this system using an Olympus 1.4 NA 60X oil immersion lens that demonstrates an axial range of
operation greater than 70 μm. We give details of a novel actuator device used to displace the focusing element
and demonstrate axial scan responses up to 3.5 kHz. Finally, we present an application of this system in liquid
crystal research to image the dynamic response of a nematic device during switching. Information about the
director field at different levels in the device can be inferred from images acquired with a temporal resolution of
2.5 ms.
In this paper, we present a novel technique that permits sectioning microscopes to refocus and acquire images
from a large range of specimen depths without introducing movements near the specimen. In contrast to other
such remote focusing methods, this technique avoids systematic aberrations that degrade image quality when
imaging planes away from the optimal focal plane. Furthermore, the specific geometry that is employed in this
work enables refocusing to be carried out at high speed and hence permits, for the first time, a number of
dynamical biological processes to be observed. Although this technique can be applied to any optical imaging
system, it is particularly suited to the case of high numerical aperture microscope systems. To demonstrate
this we present results from two prototype systems built in our laboratory based on a slit scanning confocal
fluorescence microscope and a two photon fluorescence microscope.
In this paper we describe a simple method of optical refocusing for high numerical aperture imaging systems.
As the first stage of this new method satisfies both the sine and Herschel conditions of geometric optics, images
obtained are free from spherical aberration over a large scan range.
A standard method in confocal microscopy to form an extended focus image is to merely add together (integrate) a number of optical sections taken throughout the specimen volume of interest. If we use this method in a conventional microscope the image that results is of rather poor quality. However since the image has been degraded in a known fashion and it is straightforward, by using simple inverse filtering techniques, to restore a high quality extended depth of focus image. Examples will be shown obtained in both the fluorescence and brightfield imaging modes. The method is also suited to high resolution stereo imaging.
We demonstrate a simple and light-efficient way of generating non-diffracting Bessel beams for use in confocal microscopy. A number of imaging modalities using such beams is discussed. Preliminary experimental results including brightfield, fluorescence and two-photon images are presented.
The use of radially polarized light is known to produce, when focused by a high numerical aperture objective lens, a spot of light whose polarization is predominantly axial. We have generated radially polarized beams accurately and have confirmed this behavior experimentally and have also shown that the axial polarization leads to a ring type image of sub-resolution gold beads.
We describe a simple modification to a rigid endoscope so as to provide both high quality conventional endoscopic as well as and confocal endoscopic images of reasonably accessible regions of the body in real time. The systems are based around either host lenslet-array tandem scanning microscope together with laser illumination or a structured illumination approach together with a conventional incoherent illumination source. Images taken in fluorescence are presented using this combined conventional and confocal endoscope.
We report a whole-field fluorescence imaging microscope that combines 3-D spatial resolution by optical sectioning, using structured illumination, with fluorescence lifetime imaging and spectrally-resolved imaging. We show the potential of this technique in the elimination of common artefacts in fluorescence lifetime imaging and apply it to study the dependence of the lifetime on the emission wavelength in biological tissue.
We describe a simple method to obtain an arbitrary complex optical field. The system employs a ferroelectric liquid crystal spatial light modulator acting as a binary optical element in a 4-f optical system. The ability to produce an arbitrary wavefront permits us to modify the pupil function of an objective lens in any desired manner. Examples of pupil plane filters which enhance certain aspects of the point spread function are described.
We present a novel light efficient technique for obtaining optically sectioned images in wide-field microscopy. The technique is a further development of correlation microscopy and is based on using complementary structured light patterns to illuminate the specimen together with uncomplicated post- processing of the captured images.
We present a novel technique for testing of the high quality microscope objective lenses. The characterization of the lens is achieved by using a point light source approximated by a 40 nm colloidal gold bead scatterer and simultaneously measuring the field distribution in the pupil plane (pupil function) and light intensity in the image plane (point spread function). Aberrations introduced by the lens are then expanded into Zernike polynomials. The proposed technique is particularly suited for measuring apodization and vignetting effects and allows for easy measurements of the off-axis aberrations.
An analysis is presented of the image formation in widefield fluorescence microscopy using standard light. The region of support of the resulting optical transfer function is discussed.
We describe a method of obtaining optically scanned fluorescence images in a widefield conventional microscope by interfering two beams on an object so as to illuminate it with a single spatial frequency fringe pattern. Images taken at three spatial positions of the fringe pattern are processed in real time to produce optically sectioned images which are substantially similar to those obtained with confocal microscopes.
We discuss a number for configurations for confocal microscopes and measure the extinction coefficient as a function of pinhole size. The introduction of a Babinet- Soleil compensator/polarizer combination is found to result in an extremely high extinction coefficient.
We describe a new form of light-efficient real time confocal microscope which requires neither a laser nor scanning in order to obtain a real time confocal image. The approach is based on aperture correlation techniques.
We describe a real time white light reflection confocal microscope incorporating an optical fiber bundle. We investigate the optical sectioning properties of the fiber bundle in detail.
We describe a semiconductor laser confocal interference microscope that is capable of producing both confocal images and high resolution surface profiles. The system is based on an optical fiber interferometer together with injection current modulation. We also address the issue of resolution in phase imaging.
We discuss the differences between polarization contrast imaging in conventional and confocal systems and show that because of the fundamentally different imaging properties of the two systems, the extinction coefficient is non-zero in a conventional system even with perfect polars. We then discuss the axial response of high numerical aperture systems and show that a polarization effect leads to an asymmetric response when the objective lens numerical aperture is greater than the refractive index of the specimen.
A new spatial multiplexing technique which uses a confocal scanning microscope to address fibre—optic
sensors is proposed. Multiplexing ofmore than 1000 sensors has been achieved both in amplitude and
phase sensor regimes.
We will discuss a variety of design simplifications which lead to the realization of an extremely simple confocal microscope based on signal feedback into a single transverse mode laser. Semiconductor and helium-neon gas lasers are considered.
In recent years, the use of endoscopic instruments for examining and operating on internal regions of human body has become commonplace. Apart from improving the resolution and reducing the diameter of the probe current research is aimed at providing a 3-D imaging capability for these devices. Diverse approaches have been demonstrated ranging from binocular-view endoscope I to holographic imaging2. Because of its optical sectioning property the confocal scanning microscope is a tool especially suited for imaging of volume objects3. This property means that only thin region adjacent to the focal plane of the microscope is imaged while volume scattering is rejected by confocal filtering (in conventional imaging systems scattering causes blurring of the focused image). The object's appearance is then reconstructed by combining the recorded optical slices. Fibre-optic modification of the confocal microscope4 is most promising candidate for endoscopy applications due to it's compactness and flexibility. The fibre scanner is however the main obstacle to the system integration under given geometrical constraints (typically less than 2 mm outer diameter). We suggest here replacing the bulk focusing lens by gradient-index (GRIN) rod that is long enough to move the scanner off the zone of strict dimensional constraints. We demonstrate in this work a practical compromise between the dimensions of the probe and the endoscope 3-D imaging capacity.
Optical sectioning capability of fiber-optic scanning microscope using 9 cm long GRIN-rod lens probe has been demonstrated. Transverse resolution 1.3 micrometers and longitudinal resolution 30 micrometers were achieved. Dimensions of the probe allow the use of the device as an endoscope.
We describe a simple method of obtaining confocal, differential phase, and differential amplitude contrast in a scanning microscope. Our system permits these imaging modes to be obtained simultaneously. We extend the versatility of the system by describing its operation as a non-contacting surface profilometer.
A phase sensitive scanning fiber-optic microscope is described. Confocal operation with single mode fiber acting as a spatial filter is demonstrated. Application of two mode optical fiber for the differential imaging is proposed.
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