1
Advanced Wide‐Field Fluorescent Microscopy for Biomedicine

Chong Chen and Hui Li

Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science, Suzhou, 215163, China

1.1 Introduction

Life was assembled from molecules, subcellular organelles, cells, tissues, organs, to the whole organism in totally different ways. The assembly at each level has its own structure, dynamics, and functions, making for such a complex and beautiful living world. To study such complex structures, as nobel prize winner Feynmann said, “It is very easy to understand many of these fundamental biology questions: you just look at the things.” However, different imaging tools need to be developed for different purposes to look at the various biological objects, with a scale from nanometers to centimeters.

Optical microscopy plays the most important role in inspecting the microscale biological world among all the imaging tools. Although optical microscopy has been invented for more than 300 years, we have witnessed significant improvement in the optical microscope technique in the last 30 years. These improvements mostly fall into two aspects: sample labeling technique and different imaging modalities.

Image contrast is the first factor to be concerned with for optical imaging. By now, fluorescent imaging has provided the highest contrast due to the filtering out of excitation light. Organic dyes, quantum dots, and fluorescent protein are the three most widely used fluorescent labeling agents. Fluorescent proteins, which won the novel prize for chemistry in 2008, provide a genetic way for labeling so that fluorescent imaging with live cells, organelles, and even live animals becomes possible.

High‐end microscopes fall into two categories: widefield microscopes and point scanning microscopes (Figure 1.1). A wide‐field microscope takes imaging by a camera and usually has high speed and high photon efficiency. Typical examples are the Tirf‐microscope, structured illumination microscope, and single‐molecule localization super‐resolution microscope. The point‐scanning microscope takes imaging by fast scanning the excitation laser beam or sample, usually at a lower speed but with higher axial sectioning capability. Typical examples include laser scanning confocal microscopes, two‐photon microscopes, and STED super‐resolution.

Figure 1.1 (a) Comparison of wide‐field microscopy. (b) Point‐scanning microscopy.

Source: Chong Chen.

This chapter mainly introduces the advance of the wide‐field fluorescent microscope in the last ten years. We first introduce the methods to improve the optical sectioning and the resolution by structured illumination, then introduce the methods by light sheet illumination. The optical principle, setup, image processing method will be introduced in each section. The chapter ends with a prospect for future development.

1.2 Optical Sectioning by Structured Illumination

1.2.1 Optical Section in Wide‐Field Microscopy

The optical section in microscopy defines its capability to resolve structure axially. In an epi‐fluorescent microscope, the entire sample space is illuminated, and all of the excited fluorescence signals collected by the objective can go into the array detector. Consequently, when the sample goes out of focus, its image becomes blurred, but the signals do not disappear. This problem presents a significant hindrance in wide‐field microscopy.

In optical microscopy, the depth of focus is how far the sample plane can move while the specimen remains in perfect focus. The numerical aperture of the objective lens is the main factor that determines the depth of focus (D):

where λ is the wavelength of the fluorescent light, n is the refractive index of the medium [usually air (1.000) or immersion oil (1.515)], NA is the numerical aperture of the objective lens. The variable e is the smallest distance that can be resolved by a detector that is placed in the image plane of the microscope objective, whose lateral magnification is M. For a high‐end fluorescent microscope with NA 1.4 and a 100× magnification objective, the depth of focus is on the order of 500 to 700 nm, dependent on the fluorescent wavelength. This depth of field defines the best optical section capability for an epi‐fluorescence microscope.

Figure 1.2 (a) Sketch of the depth of field. (b) The defocused signals cause blur of the focused image.

Source: Chong Chen.

However, when imaging samples with a thickness larger than the microscope's depth‐of‐focus, the sample's out of focus plane is also excited and forms a defocus image at the camera plane (Figure 1.2). The superimposition of these defocus images lowers the contrast of the camera's captured image and practically lowers the axial resolution. The imperfections in the microscope's optics and the scattering of fluorescence signals by the sample itself make the situation even worse. So, the priority demand to improve the wide‐field microscope's performance lies in eliminating the out‐of‐focus signal, yielding better optical sectioning capability.

1.2.2 Principle of Optical Section with Structured Illumination

In a laser scanning confocal microscope, the out‐of‐focus light is rejected using a pinhole. In a wide‐field microscope, no pinhole can be used since it uses a array detector. One way to reject the out‐of‐focus signals is by using structured illumination. By utilizing a grating or a digital mirror device (DMD), stripe patterns were projected on the image plane so that a structured illumination was created to excite the fluorescence molecules within the focus plane. The actual focus range of the stripes can be made very sharp if the proper period of the grating is used. Out of focus, the strip patterns become uniform, which will generate a nonmodulated background. Therefore, the image formed by the microscope will consist of striped in‐focus features superposed with uniformly illuminated out‐of‐focus features. A postprocessing algorithm could reject this background afterward, thus obtaining a better optical section capability.

So, to obtain optical sectioning with structured illumination, two factors are needed: optical instrumentation to create structured illumination and the optical section reconstruction algorithm. These two aspects will be discussed in the following section.

1.2.3 Methods for Generating Create Structured Illumination

For structured illumination, the excitation light field needs to be patterned, and the pattern needs to be shifted or rotated to capture all sample information. Several methods have been developed for this purpose.

1. The fluorescence grating imager inserts a grid into the field diaphragm plane of a fluorescence microscope (Figure 1.3). The shift of the grid projection can be achieved by translating the grate or tilting a plane‐parallel glass plate located directly behind the grid. This method requires very little modification to the microscope, so it is easy to implement and low cost. Zeiss, Inc. uses the technique in their APOTOME equipment for 3D imaging [1]. However, the image speed is limited by the mechanical movement of the grate or the glass plate. Generally, 10 frames per second could be obtained, which is not fast enough for some subcellular dynamics imaging. Another drawback is that the period of a grate is fixed, so one has to change the grid if one wants to use a different period.

   Figure 1.3 Structured illumination with grating for optical section.

   Source: Chong Chen.

   Figure 1.4 Structured illumination with digital mirror device.

   Source: Chong Chen.

2. To avoid mechanical translation, a DMD was introduced to project fringe patterns on the sample plane [2]. The DMD is a micro‐electro‐mechanical system (MEMS) consisting of a few hundred thousand tiny switchable mirrors with two stable mirror states (such as, −12° and +12°). When a micromirror is set at +12° toward the illumination, it is referred to as an “on” state. Similarly, at the position of −12° it is referred to as the “off” state. The mirrors are highly reflective and have a higher refreshing speed and a broader spectral response (Figure 1.4). However, since DMD is a reflective device and has to be positioned at 12° in the light path, the optical layout using DMD is more complex than using grate [3–6].
3. Structured illumination could also be realized using an LED array as a light source, as implemented by V. Poher et al. [7]. The microstructured light source is an InGaN LED consisting of 120 side‐by‐side and individually addressable microstripe elements. Each stripe of the device is 17 microns wide and 3600 microns long, with a center‐to‐center spacing between stripes of 34 microns, giving an overall diode structure size of 3.6 × 4.08 mm. A dedicated electrical driver was constructed to allow arbitrary combinations of the stripes to be driven...