1 Ray Optics , Wave Optics and Imaging SystemDesign

Fluorescence imaging has its roots in optics and molecular physics. The light-matter interaction is the key for understanding molecular phenomenon, be it Raman or fluorescence microscopy. For the simplistic reason of strong light-matter interaction, fluorescence imaging has gained prominence over other imaging techniques and has found wide applications in fields ranging from biophysics to optical engineering. The key advantage of this interaction is the ability to follow biological processes in their native state, thereby opening-up a large number of unanswered questions that have been asked for centuries. For example, protein migration and binding mechanisms were known to occur inside the cell, but were never observed directly. Moreover, many internal functioning of the cells is still a mystery. Only now, we are able to unravel these mysteries with the development of powerful super-resolution microscopes that allow visualization of molecule/protein functioning with near molecular-scale resolution. Molecules of interest are often tagged with fluorescent markers and followed while cell performs key molecular level tasks. The advantage of fluorescence microscopy is its ability to perform functional studies that are rare in other forms of imaging. Visible light is a minute part of what we generally refer to as a electromagnetic (EM) spectrum. So, visible light is governed by the same principles that are applicable to other members (radio waves, X-rays, γ -rays etc.) of EM spectrum All the members of EM spectrum (that include visible light) propagate in the form of two mutually coupled vector fields i.e., electric field and magnetic field. This stems from the fact that, electric field effects gives rise to magnetic field and vice-versa. The very fact that, light has been manipulated for centuries before the existence of its vectorial description of light infers that light can be treated in a simplified manner as well for explaining day-to-day events. An approximate way of treating light (which is indeed a vectorial quantity) is referred as scalar theory (i.e., ray optics and wave optics). When light propagating through and around the objects whose dimensions are much greater than the dimension of wave (i.e., wavelength), the wave nature is not evident. In such a situation, one can describe light as a bunch of rays emanating from a source and propagating towards the object. During this, the light gets slowed down and diverted obeying a set of geometrical rules and postulates. It will be shown in the later part of the chapter that, ray optics is the limit of a more general theory called, wave optics which assumes that the dimensions of objects through which the light is passing are comparable to the wavelength of light. Specifically specialization such as, beam and Fourier optics has caved an important space within wave optics. Overall, the following picture (Fig. 1.1) emerges for describing the classical theory of light. Light is also known to produce unusual effects that are classically elusive and unexplainable. So, the theory of light can be broadly divided into two streams: classical optics and quantum optics. Classical optics encompasses, ray optics, wave optics and electromagnetic optics, while quantum optics deals with the non-classical effects (such as, squeezing, entanglement, bunching etc.). It is important to realize that, within wave optics, specializations such as, Fourier optics and beam optics have gained prominence due to its wide applications in applied sciences. The optimal choice of the theory for an observed optical effect is the simplest theory that can satisfactorily explain all the aspects of the phenomena. The choice of a particular theory is often based on experience, so a-priori it is difficult to judge an appropriate theory. In this book, we will follow the order of simplicity and progress towards more complex theory as and when the need arise. In this chapter, we begin with the most simplistic theory of light i.e., ray optics or geometrical optics.