Explanation:

This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large NUMBER of different molecules which COMBINE and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules.

Keywords: fluorescence microscopy, optical tweezers, spatiotemporal control.

For a century, fluorescence microscopy has come a long WAY since its inception by Oscar in 1911. Recent developments in novel fluorescent dyes and fluorescent proteins have further boosted the research in fluorescence microscopy. The key parameters that define the quality of a microscope are the resolution, magnification and contrast.1 The most important of these being the resolution, which is explained as the minimum distance between two bright points in the luminous object that can be DISTINGUISHED. For any form of far-field fluorescence microscope lateral and axial resolution is limited by diffraction and given by the (modified) Abbe relation2 [4-6]:

rlateral ≈ 0.61λ ∕ NA and raxial ≈ 2ηλ ∕ NA2,

where λ is the wavelength of emitted light, NA the numerical aperture of the collecting objective3 and η the index of refraction. Taking NA ≈ 1.4 and η ≈ 1.45, the lateral resolution turns out to be nearly half of the wavelength, and axial resolution, nearly on the order of the wavelength. One of the most sought after challenges in fluorescence microscopy has been on the advancement of better depth-resolution (also called axial- or z-resolution) in an optically thick specimen (where background fluorescence results in blurring of the image) as evidenced by the development of confocal [7] and multiphoton [8,9] fluorescence laser-scanning microscopic techniques [5,6]. In confocal microscopy, out-of-focus fluorescence is effectively rejected by making use of a pin-hole placed at the conjugate focal-plane of the image-plane (thus the name confocal) while in multiphoton microscopy confocality is inherent due to confined nonlinear fluorescence generation within the focus. A schematic explaining fluorescence generation and DETECTION for these two types of microscopy is given in Figure 1. As shown in the figure, an optically thick specimen can be considered to be composed of several fluorescing layers. The total fluorescence from the specimen is a sum total of fluorescence of each layer. The confocal pin-hole effectively collects fluorescence from the middle layer only (Figure 1(a)) while the multiphoton fluorescence arises only from focal volume, thus, giving rise to a background-free fluorescence detection