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Super-Resolution Microscopy (SRM): Brief Introduction

Zhigang Yang, Soham Samanta, and Yingchao Liu

Center for Biomedical Photonics (CBMP) & Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China

1.1 Optical Microscopy

There is a well-known saying "Seeing is believing." Undoubtedly, visualization is one of the most trusted means of perceiving an object. Hence, bioimaging using various optical microscopy techniques constitutes the foundation of biological research.

To improve the performance of microscopes, scientists have realized significant technological advancements over the last few centuries to achieve the present epoch of biological research. The quest for improving the performance of microscopes resulted in the discovery of newer optical microscopic methods, since its first invention. In the sixteenth and seventeenth centuries, the earliest functional microscopes were introduced to overcome the limitation of the human eyes, which revolutionized the study of the natural world from a new perspective, even though a low magnification of the object was achieved. Therefore, to appreciate the current quest for developing more advanced optical systems, it is important to first briefly review the history of the evolution of the development of microscopes.

1.1.1 History and Background

Direct observation of objects using the unaided eyes was the only means of perceiving the outside world until the emergence of the first optical microscope. Initially, lenses were significant components of any microscopic system and performed a function similar to that of the lens of the eye. Since the invention of the first compound microscope in the late sixteenth century, which consisted of an eyepiece, objective lens, wooden tube covered in fish skin, and cardboard, [1] it has become increasingly popular. In 1663, Robert Hooke initially employed his self-made compound microscope to observe microscopic objects, and 2 years later, Hooke obtained microscopic images of many familiar objects using his microscope. The term "cell" was first coined by him, which was later recognized as the basic building block of every living organism [2, 3]. In the 1670s, Antonie van Leeuwenhoek pioneered hand-made stamp-sized microscopes to observe freshwater microorganisms at a magnification of approximately 300. In 1674, with the assistance of the simple microscope, Leeuwenhoek effectively launched a new area of research- microbiology. Nevertheless, single-lens microscopes remained popular until the 1850s [4]. Figure 1.1 depicts images of an antiquated (left) and a modern (right) optical microscope.

Figure 1.1 Images of antiquated (a) and modern (b) optical microscopes.

The resolution of a simple microscope is limited owing to the constraints of using a single lens, which is improved in a compound microscope with an objective lens and an eyepiece. The focal length, numerical aperture (NA), and field of view (FOV) are the common features of an objective lens. In a compound microscope, the objective lens possesses a short focal length and is therefore placed close to the specimen. It enables the formation of a real image in the front focal plane of the second lens, whereas the eyepiece can form a magnified virtual image at the observing end. The total magnification of a microscope is the product of the magnification associated with the objective and eyepiece lenses. In addition, a compound microscope can be constructed using a converging lens, a body tube, and illumination sources. Typically, the aim of the converging lens is to focus the image of the light source onto a sample. However, in an alternative setup, the source image is focused onto the condenser so that it could ultimately be focused onto the entrance pupil of the microscope objective lens. This is known as Köhler illumination, which typically offers the advantage of averaging the nonuniformities of the source in the imaging process (Figure 1.2) [5].

For modern microscopes, the objective lens enables a standard magnification ranging from 2 to 100 folds. In the imaging process, the objective lens collects rays from each target point, which are imaged at the front focal plane of the eyepiece. The common rules of ray tracing are used in image formation. Without consideration of aberration, geometric rays from each object point form a point image. However, in the presence of aberrations, each fine object point is replaced by a blur spot. The eyepiece is designed to observe the relayed image at a distance that is convenient for the viewer. In this system, the brightness of an image is determined by the size of the lens aperture and the pupil of the eye. By adjusting the focal length, the resultant magnification of the objective can be modulated according to the requirements for viewing the object through the eyepiece with a suitable resolution. The light source imaged in the focal plane suffers from diffraction and other effects in the imaging system, which complicates the image formation process in the microscope. The German physicist Ernst Abbe is regarded as the founder of the modern theory of image formation using a microscope. In 1873, Abbe investigated microscopes in which objects in the focal plane were illuminated by focusing light from a condenser. The convergent light can be thought of as a collection of many plane waves, propagating in specific directions, which form the incident illumination via superimposition. Each of these effective plane waves is diffracted by features in the object plane, which implies that the smaller the objective features, the larger the diffraction angle [6].

Figure 1.2 Schematic diagram of Köhler illumination.

Source: Adapted from Education in Microscopy and Digital Imaging; http://zeiss-campus.magnet.fsu.edu/articles/basics/kohler.html.

1.2 Specialized Optical Microscopes

Gradual improvements in optical microscopy with different adaptations have been espoused for specific purposes. In 1958, the most successful microscope was invented by the British doctor John McArthur, and since then, different "McArthur" microscopes with slight modifications have been manufactured by different manufacturers. To meet the needs of professional researchers, different specialized microscopes (e.g. inverted, phase-contrast, and confocal) have been subsequently developed for modern optical applications. In this chapter, the inverted and confocal microscopes will be primarily discussed, given that these two categories of microscopes form the basis of super-resolution microscopy (SRM).

1.2.1 Inverted Microscopes

In 1850, the first inverted microscope was invented by John Lawrence Smith at Tulane University. Inverted microscopes are specifically designed for various life science, material science, and industrial applications, wherein images are mounted upside down, unlike an upright microscope. In inverted microscopes, the light source and condenser are positioned at the topmost part and point downward toward the stage, the objective lens is set below the stage pointing upward, and the eyepieces are angled upward to facilitate the observation of specimens. Inverted microscopes are crucial for various types of biological and medical research, considering their vast application in cell biology and biomedicine [7, 8].

1.2.2 Confocal Microscopes

In the 1950s, Marvin Minsky [9, 10] first conceived the idea of eliminating out-of-focus light to improve the image quality of a microscope so that only light in the focal plane can reach the detector. Based on this principle, the confocal microscope was constructed, wherein a pinhole was inserted in the image plane to restrict out-of-focus light from reaching the detector (Figure 1.3). A focused spot of illumination is generated in confocal microscopy via spatial filtering, which must be raster scanned across the sample to generate an image. The confocal microscope is equipped with pinhole apparatuses at the illuminator and the detector sides in a conjugate image plane in front of the detector, which is called "confocal." The construction of a confocal microscope includes the positioning of a pinhole in front of a light source (zirconium arc lamp) to generate a point of light, which is focused on the sample by an objective lens. A second objective lens is used to focus the illuminated specimen onto the second pinhole put in front of the detector. In this arrangement, the out-of-focus rays from the illuminated sample are successfully removed using this "double focusing" system since they cannot reach the detector (photomultiplier, PMT, or avalanche photodiode, APD).

Figure 1.3 Schematic representation of a confocal microscope.

Therefore, a confocal microscope only detects the clear structures in the focal point, and the out-of-focus light is filtered. Since only the light from the focal point contributes to forming the final image in the confocal imaging system, this system can facilitate the three-dimensional...