Programmable diffractive optics for laser scanning confocal microscopy

Abstract

Coherent light beams find applications in several exciting research areas where it is important to have absolute control over the phase and amplitude properties of the light beams.To name a few, in applications such as microscopy, optical trapping, interferometry etc. it is imperative to have dynamic control over the properties of the beam. One way of achieving this is with the help of a computer controlled diffractive optical element such as a liquid crystal spatial light modulator (LCSLM). Based on the principle of holography, such a technique can provide dynamic control over amplitude, phase and polarisation state across a coherent beam.This work describes about a ferro-electric liquid crystal spatial light modulator (FLC-SLM) system acting as a programmable diffractive optical element. The efficiency of the system to detect and correct aberrations in laser beams were demonstrated. The ultra sensitivity of singular light beams to the presence of azimuthal aberrations were observed and a novel aberration sensor based on these findings were implemented. The synchronisation of the display holograms on the FLCSLM panel with the acquisition of a CCD camera is demonstrated in a programmable reference beam phase stepping interferometry experiment.Laser scanning confocal microscopy is a widely used technique for the electronic visualization of optically sectioned microscopic structures. The information available from such a system is limited by the intensity and polarisation properties of the point spread function (PSF) at the sample plane, which are in turn dependent on the optical system and the sample being imaged. Aberrations due to incorrect optics or the sample itself may significantly degrade the PSF there by reducing the resolution and the signal available for image formation. Also the information obtained from a laser scanning confocal system working in the epi-fluorescence mode will also depend on the polarisation properties of the PSF, as molecular absorption and emission cross-sections are polarisation dependant. These polarisation properties can be particularly important for high resolution microscopy such as STED and molecular alignment studies when high numerical aperture lenses are used that can result in complex polarisation structure within the PSF. The thesis develops the theory necessary to understand the vectorial behaviour of the 3D PSF for an arbitrarily polarised beam. The design and implementation of a beam scanning optical microscope system, that uses an FLCSLM to control the optical field in the illumination pupil of the microscope objective, are described. The performance of the system in engineering and reconfiguring the 3D PSF is demonstrated. Use of programmable diffractive optics to generate different depletion beams for STED microscopy is also demonstrated. 2To My Parents 3

A method for generating a three-dimensional dark spot using a radially polarized beam

“…The − → E z component of a radially polarized beam plays an important role so that a significant improvement in axial super-resolution is achieved. This method has this advantage over the former experiments in [2] and [13,15]. When the incoherent superposition result is used in a superresolution fluorescent microscope system as a STED beam, the effective FWHM of the super-resolution focal spot in the X Z plane is similar to the former results, and a nearly spherical 3D super-resolution spot is generated.…”

“…As indicated in figure 3, in the XY plane, the radially polarized beam with a quadrant 0/π phase plate (beam (2)) generates a dark spot uniformly and completely surrounded by light. As for the X Z plane, this beam gives a high intensity tube-shaped spot around the optical axis, and no confinement at all along the optical axis; notice that [15]), the dark spot is surrounded more uniformly and completely by high intensity light in all three dimensions, and the circular π phase plate and quadrant 0/π phase plate are easily made with high accuracy, compared with vortex phase plates [15]. At the same time, in this simulation, the proposed method just requires one type of polarized beam-a radially polarized beam, which brings superiority over the former methods that use two different types of polarized beams.…”

“…As we can see from figures 4(a) and (b), beam (3) has advantages over beam (4). Beam (4) is the coherent superposition of a right-handed circular polarized beam with a vortex phase plate and a left-handed circular polarized beam with circular π phase plate [13,15]. However, the circular polarized beam may excite redundant fluorescent on the edge of the excitation spot, which will add an unnecessary burden to the erase beam.…”

“…In many former experiments for creating a three-dimensional dark spot, two different and appropriately designed polarized beams are indispensable. For example, a radially polarized beam with a circular π phase plate and an azimuthally polarized beam [2] or a left-handed circularly polarized beam with circular π phase plate and a right-handed circularly polarized beam with vortex phase plate [13,15]. Although these methods can yield a threedimensional dark spot in the focal plane, they still have some shortcomings, which will be discussed in detail in this paper.…”

Confocal laser scanning microscopy measurement of the morphology of vanadium pentoxide nanorods grown by electron beam irradiation or thermal oxidation

“…8,9 A CLS microscope can provide optically sectioned images with a high lateral resolution, 10 and optical three-dimensional images without physical sectioning. 11 Although CLSM has a poor resolution of about 300 nm because of diffraction limits, 12 it has several advantages compared with nonoptical methods such as SEM, TEM, and AFM.…”