From optical microscopes to electron microscopes, human efforts to observe the microscopic world have never stopped. As a fast and non-destructive characterization method, optical microscopy can obtain image information of micron-scale observation objects and has a wide range of applications; electron microscopy uses high-speed moving electrons as a medium, and “sees” smaller observations through electron beams. sample. But whether it is an optical microscope or an electron microscope, what is obtained is only a flat image. Digital holographic microscopy is a novel approach to optical 3D imaging.
interference of light
From torches to electric lamps, the use of light by humans has always appeared in the development of civilization. But the understanding of the nature of light is not so smooth. As early as the 17th century, Newton proposed in his Optics that light is a flow of particles moving at high speed, but this statement could not explain some phenomena seen in everyday life. During the same period, the Dutch physicist Huygens proposed the wave theory of light, but due to the imperfect theory and Newton’s influence, the theory was not recognized by the public.
In 1801, Thomas Young performed the famous double-slit interference experiment. He irradiated a beam of monochromatic light on two parallel and sufficiently narrow slits, and the screen behind the slits displayed several bright and dark fringes, a phenomenon known as interference. The wave theory of light can better explain the interference phenomenon. Think of light as ripples on the water, undulating up and down as it spreads out into the distance. It can be seen from the principle analysis that when the light shines on the slit, two coherent beams are actually formed, and their distances to any point on the screen are not the same. When the two beams meet at this point, their peaks and The trough locations are not consistent. When the two are superimposed, the offset and enhancement of their fluctuations form light and dark stripes. In daily life, interference phenomena can be seen everywhere, such as colorful fringes on soap bubbles, which is the reason for the thin film interference of light.
The history of holography
Light is an electromagnetic wave that propagates with a phase that vibrates back and forth. However, unlike the water surface ripples that can be easily observed, the phase change of light is too fast, reaching the order of 1014 Hz, while the recording frequency of conventional cameras is only tens to hundreds of hertz, and it is too late to record the process of phase change. Therefore, usually only the average value of the amplitude of the light field, that is, the light intensity, can be obtained, but its phase information cannot be obtained.
The interference fringes can reflect the phase information of light, and the method of inversely deriving the phase information of the light field according to the interference phenomenon is holography. Holography is translated from English holography. As the name suggests, it means to obtain all the information of light at the same time, including light intensity and phase information. It was first proposed by D. Gabor, a Hungarian-born physicist at Imperial College London in 1947 . He found that when an object is irradiated with monochromatic light, the phase information of the light can be retained on the recording medium by using the phenomenon of interference, and the light field information of the object can be regained when the recording medium is irradiated with the same light. Gabor recorded the first hologram using chemical film and developed the technique of holography, for which he won the 1971 Nobel Prize in Physics.
Holography was not conspicuous at the beginning of its appearance, because it did not work well in actual use. At that time, the mercury lamp as the light source had poor coherence, and the ultra-fine particle silver salt holographic dry plate required long-term exposure to record holographic information, and then a series of processes such as developing, fixing, and bleaching were required in the dark room to obtain a holographic image. . The extremely stable conditions required for holographic recording and the high cost of time and materials have limited the development of holography.
In the early holography, the object light and the reference light are in the same direction, which is coaxial holography. The hologram obtained by coaxial holography will generate a conjugate virtual image overlapping with the object image when restoring the light field information, so there is a big problem in the restoration of the phase. To solve this problem, in 1962, E. Leith and J. Upatnieks of the University of Michigan proposed off-axis holography . They extended the carrier frequency concept in communication theory to the airspace, and realized the separation between the real image and the conjugated virtual image. The off-axis holography introduces a slight inclination angle, so that the propagation directions of the object light and the reference light are not completely consistent, and the formed hologram can distinguish the object image and the conjugate virtual image in the spectrum, and then can avoid the superposition of the conjugate virtual image by filtering. , which solves the difficult phase calculation problem in the coaxial holographic method, and makes it possible to calculate the phase using a single holographic image.
In the 1960s, the emergence of laser generators provided a stable and highly coherent light source for holography, which greatly increased the upper limit of holography accuracy. On the other hand, the development of electronic imaging equipment and computers has created conditions for the digitization of holography. In 1967, JW Goodman and RW Lawrence recorded holographic images using a computer-controlled camera. Using a helium-neon laser as the light source, they recorded an eight-level grayscale image of 256 × 256 pixels with a camera, and used a computer algorithm to perform a two-dimensional Fourier transform operation on the obtained image. By comparing the results of digital calculations with images of purely optical methods, they confirmed the feasibility of using cameras and computers for digital recording and computational reconstruction of recorded objects, laying a solid foundation for the vigorous development of digital holographic computing in the future.
In 1994, U. Schnars and W. Jüptner realized the comprehensive digitization of holography, and the research of holography entered a new stage. They used a charge-coupled device (CCD) camera to record a dice holographically, and used a computer algorithm to digitally reconstruct the image of the dice’s light intensity. Compared with traditional optical holography, which needs to develop film in a dark room, digital holography has an overwhelming advantage in ease of use.
digital holographic microscope
Digital holographic microscope (digital holographic microscope) is an optical measurement device that uses the interference phenomenon of light to extract three-dimensional information of a sample. It combines optical microscopy imaging and digital holography technology to retain the phase information reflecting height lost during plane imaging in the interference fringes, which are recorded by the camera, and simulate the propagation of light in space through subsequent calculations. The light field in a specific three-dimensional space inside the target, thereby realizing quasi-three-dimensional imaging of the sample surface. Digital holographic microscopes have gradually expanded into a variety of applications in the years of development. The most mature applications are the tracking of three-dimensional motion of particles and the characterization and imaging of three-dimensional topography.
Particle tracking The
coaxial digital holographic microscope has the advantage of fast imaging and can track the three-dimensional movement of particles in space, which is difficult to achieve with traditional holographic technology. It first obtains the holographic image through the interference fringes formed by the interference between the scattered light of the sample particles and the surrounding blank area; . By judging the focus position, the coaxial digital holographic microscope can obtain the position information of the particle sample in space extremely accurately, and its precision can even break through the limitation of the diffraction limit
Because the coaxial digital holographic microscope does not need to carry out the sample preparation process that affects the biological activity, such as staining, it is especially suitable for studying the movement behavior of microorganisms and particles in the solution and the interface. The author’s research group used a coaxial digital holographic microscope to track the movement trajectory of Escherichia coli (the bacteria were cultured on a gradually degraded surface), observed and obtained parameters such as the movement direction, speed, and trend of Escherichia coli near the surface, and then The dynamic antifouling effect of this type of surface against microorganisms was analyzed . In addition, the research group also monitored the three-dimensional movement of E. coli in the electric field. By analyzing the differences in the movement states of E. coli in different periods of electric fields, the relationship between the adhesion behavior of E. coli and the period of the electric field is revealed, which provides a new idea for the use of electric fields for wastewater treatment and marine anti-fouling .
In addition to determining the position of the particles, the coaxial digital holographic microscope can also restore the size and shape of the particles, and do preliminary work for further analysis of the microscopic changes on the surface. In 2020, K. Snyder et al. applied holographic microscopy to study the binding of antibodies to antigens . They modified the surface of polystyrene microspheres with antigenic proteins, added the microspheres to solutions of two different antibodies, and fitted the holographic images based on the Lorenz-Mie light scattering theory. The changes in the diameter of the microspheres were obtained, and then the coverage of the antibody on the surface of the microspheres was inferred, which provided a reference for the analysis of antibody concentration and microscopic binding mechanism .
Traditional optical microscopes can only image planes and cannot obtain height information, while digital holographic microscopes can calculate the optical path difference by restoring the light field information, and then obtain the three-dimensional morphology of the sample. At the earliest, holography could only restore the light intensity to a certain extent and could not obtain the three-dimensional shape, because of the interference of the conjugate virtual image. To solve this interference problem, different optical design approaches have been proposed and developed individually.
The easiest to implement is the off-axis holography method proposed by Leith et al. With the improvement of algorithms and hardware conditions such as frequency domain filtering, the spatial accuracy of off-axis digital holographic microscopes has been greatly improved, reaching the nanometer level. In 2008, a Swiss research group used an off-axis digital holographic microscope to measure the surface topography of microsteps (about 8.9 nm in height) evaporated with chromium on a quartz substrate, and achieved high-precision three-dimensional topography observation  ]. They used two light sources with wavelengths of 657 nanometers and 680 nanometers respectively to observe, and found that the difference between the two in the reduction results was small, and the difference between the calculated cross-sectional height and the standard value obtained by other instrument standards was within the error range.
Since the off-axis digital holographic microscope can perform three-dimensional non-destructive observation of the dynamic process of the sample, it is suitable for the study of cell-related biological samples. For example, B. Rappaz et al. The division cycle of yeast cells was successfully observed . In addition, some researchers have used it to conduct three-dimensional imaging and research on various samples such as amoeba, HeLa cells, and human red blood cells. In conclusion, the off-axis digital holographic microscope can be widely used in the field of biological cell detection because it can restore the fine phase morphology of objects or surfaces.
In addition to off-axis holography, researchers have also proposed phase-shift holography. In 1969, R. Crane first clearly proposed the concept of phase-shift interferometry. Phase-shift digital holographic microscope is a device that combines holographic microscopy and phase-shift interference technology. It obtains the phase distribution of the light wavefront of the object to be measured through multiple interference images that carry a certain amount of phase shift. When in use, a series of holographic images are obtained by first modulating the reference light to change the phase difference between the coherent lights. After that, by calculating these holographic images, the phase information of the object light is obtained, and then the three-dimensional topography of the sample surface is obtained.
Since the phase-shift digital holographic microscope obtains three-dimensional topography based on multiple holographic images, it is necessary to image the stationary sample multiple times or use multiple cameras to image the same sample. The former is a more common practice, using a time-shifted digital holographic microscope. First, a camera is used for imaging under the condition that the phase difference moves with time. Images at different times form a set of phase-shifted holographic images, and then calculate 3D image of the sample. This type of device has a relatively simple optical path and flexible phase shift control, so it is relatively widely used in research. However, because the time-shifted digital holographic microscope requires the sample to remain unchanged before and after the phase shift, it is only suitable for static or quasi-static samples, such as electronic components. For the dynamic change process, some people have developed a digital holographic microscope with spatial phase shift, which uses multiple cameras or different positions of the same camera to record to obtain holographic images of the sample under different phase shifts at the same time. This device can realize dynamic observation, but it has high requirements on optical path design and hardware configuration. In 2004, Japanese scientist Y. Awatsuji and others added a phase-shift array in front of the camera through mask technology, which made the phase shift of each part on the same camera different, and realized multiple phase-shift holographic images. Also recorded .
The phase acquisition can also be achieved through the modulation of the light source. In 2019, Zhang He et al. published a paper on near-field Fourier stack imaging research , describing that they replaced the original uniform illumination with speckle illumination, and achieved the interference state through the relative motion between the speckle and the sample. Then, the light intensity and phase information of the sample are calculated by using multiple low-resolution images, and the resolution of the obtained images is greatly improved.
In addition to the modification of the imaging optical path, the development of algorithms to calculate the phase information of the light field in coaxial holography is also a research focus. As early as the 1970s, RW Gerchberg and WO Saxton of the University of Cambridge, UK, proposed the Gerchberg-Saxton algorithm (referred to as the GS algorithm) to recover the loss in the imaging process. phase information. On this basis, other researchers have developed improved methods such as the Fienup method. This type of method is different from the directly calculated holography mentioned above. It obtains phase information by iteratively approaching the real value by substituting the estimated value, which requires high computer computing power and image quality.
In recent years, with the improvement of camera imaging accuracy, computer algorithms and running speed, coaxial holography with simple structure and high stability has been paid attention to again. For example, a research group at the University of California in the United States introduced a light emitting diode (LED) array on the basis of a coaxial holographic microscope, and achieved super-resolution imaging through computer algorithms without adding a lens . They used the device to image red blood cells infected with the malaria parasite
As a fast quantitative three-dimensional imaging method, digital holographic microscopy enables us to realize real-time three-dimensional observation of particles and obtain the change process of their spatial three-dimensional information over time without excessive processing of the sample. At present, it has been widely used in scientific research fields such as particle characterization and biological sample detection.
Although holography is still affected by factors such as camera resolution and laser speckle, with the innovation and optimization of optical paths, the improvement of computing power, the vigorous development of image processing and deep learning algorithms, the improvement of laser modulation technology, and the accuracy of cameras And the breakthrough in acquisition speed, I believe in the near future, this technology will be more perfect. Combined with other technologies, it will bloom uniquely in research and detection in the microscopic field, and become a powerful tool for promoting scientific research.