Quantitative Phase Imaging in Biomedicine
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REVIEW ARTICLE https://doi.org/10.1038/s41566-018-0253-x Quantitative phase imaging in biomedicine YongKeun Park1,2,3, Christian Depeursinge4,5 and Gabriel Popescu 6* Quantitative phase imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unla- belled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. As the images represent quantitative maps of optical path length delays introduced by the specimen, QPI provides an objective measure of morphology and dynamics, free of variability due to contrast agents. Owing to the tremen- dous progress witnessed especially in the past 10–15 years, a number of technologies have become sufficiently reliable and translated to biomedical laboratories. Commercialization efforts are under way and, as a result, the QPI field is now transi- tioning from a technology-development-driven to an application-focused field. In this Review, we aim to provide a critical and objective overview of this dynamic research field by presenting the scientific context, main principles of operation and current biomedical applications. maging biological cells and tissues is central to biological research will wash out the image altogether due to multiple scattering. Both and medical diagnosis. Following its four-century-old history, of these extremes result in poor image contrast. Single cells and thin microscopy has become the most commonly used tool in medi- tissue slices belong to the first category: the scattered light they gen- I 1 cine and biology . However, despite significant breakthroughs, opti- erate is orders of magnitude weaker than the incident light. This cal imaging of biological specimens remains an active research field, class of specimens is referred to as ‘phase objects’, as they affect sig- aiming to exceed current spatial and temporal resolution, contrast, nificantly only the phase of the incident field and not the amplitude. penetration depth, molecular specificity and quantitative capabili- To render such structures visible, one solution was to convert them ties. Quantitative phase imaging (QPI)2 is emerging as a powerful, into ‘amplitude objects’ using various stains or fluorescent tags6,7. label-free approach to imaging cells and tissues, especially because Today, the gold standard of histopathology is performing manual it combines qualities found in microscopy, holography and light- diagnosis on stained, several-micrometre-thick tissue slices, while scattering techniques: nanoscale sensitivity to morphology and fluorescence microscopy is the main imaging tool in cell biology6. dynamics, 2D, 3D and 4D (that is, time-resolved tomography) However, while stains and tags offer high-contrast imaging with non-destructive imaging of completely transparent structures, and molecular specificity, they are often qualitative and sample-prep- quantitative signals based on intrinsic contrast. Here, we review the aration-dependent, while photobleaching and phototoxicity limit context in which the QPI field developed, the main principles of fluorescent imaging of live cells. Furthermore, the use of exogenous operation, and representative basic and clinical science applications. labelling agents, such as fluorescent proteins or dyes, may alter the We end with a summary and outlook of the field. normal physiology of cells and, furthermore, labelled cells cannot In 1873, the first theory of image formation in an optical micro- be re-injected into the human body. These limitations may hinder scope was established by Abbe3. The idea that imaging is formed advances in neuroscience, stem cell research and immunotherapy, by the superposition of waves emerging at different angles from to name just a few. a sample was new and powerful: it established that the image is a A major advance in intrinsic contrast imaging occurred in the (complicated) interferogram. The result also implied that a micro- 1930s, when Zernike invented a technique capable of imaging phase scope could not resolve objects smaller than half the wavelength, objects with high contrast and without the need for tagging8. The when using propagating waves. This resolution dogma remained principle of Zernike’s phase contrast microscopy builds on Abbe’s essentially unchallenged for more than a century, until Hell and understanding of imaging as an interference process. Specifically, Wichmann’s paper in 1994 (ref. 4). Therefore, the other crucial met- the incident and scattered fields are treated as, respectively, the ref- ric of imaging, contrast, can be credited with the tremendous devel- erence and object wave of an interferometer (see, for example, ref. 9, opments in microscopy over its long history1. Contrast defines how page 472). Thus, the total field, U, has the form: clearly a subject of interest is distinguished from the background. ix(,y) For a biological specimen, the thickness and refractive index inho- Ux(,yU)[=∣ ∣≈eUϕ ∣∣1(+ ixϕ ,)y ] (1) mogeneity determine how much light scattering it produces. It is scattering rather than absorption that determines the ultimate con- where ϕ(x,y) is the phase delay map of a sample. Note that the inten- trast of the image, as in the visible spectrum most cells and tissues sity image of such an object, ∣∣U 2, exhibits little spatial modulation, do not absorb significantly5. Too little scattering from the specimen that is, no contrast. To boost the contrast of the resulting interfero- makes it challenging to reveal the structure from an overwhelming gram, and thus the image, Zernike added a further π/2 phase shift incident light background, while too strong a scattered component between the two. The additional π/2 shift places the scattered field 1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. 2KAIST Institute for Health Science and Technology, Daejeon, Republic of Korea. 3Tomocube Inc., Daejeon, Republic of Korea. 4Microvision and Microdiagnostic Group (SCI STI CHD), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. 5Laboratory for Cellular Imaging and Energetics, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia. 6Departments of Electrical and Computer Engineering and Bioengineering, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA. *e-mail: [email protected] 578 NATURE PHOTONICS | VOL 12 | OCTOBER 2018 | 578–589 | www.nature.com/naturephotonics NATURE PHOTONICS REVIEW ARTICLE in antiphase with respect to the incident field, such that the phase mechanical instability of the scanning process. To eliminate scan- contrast image field becomes: ning, researchers devoted efforts towards developing full-field QPI methods, in which the phase map across a field of view is −ϕ (,xy) UxPC (,yU){=∣ ∣+1[iiϕ (,xy)]} ≈∣Ue∣ (2) retrieved simultaneously. Due to the additional π /2 phase shift, the resulting phase contrast Full-field QPI and figures of merit field converts the phase into amplitude modulation (the complex The full-field methods can be grouped into two classes, according exponential was converted into a real exponential). Currently, phase to the phase shifting (Fig. 1a) and off-axis (Fig. 1b) holography contrast microscopes also balance the power of the two fields by geometry from which they originate. Unlike in holography, where attenuating the incident field. These simple modifications provided the goal is to record a mixed phase and amplitude signal that is the microscope with the ability to visualize in high detail live, unla- then used to reconstruct the image of the object, QPI extracts the belled cells and other transparent objects. Today, the phase contrast phase map associated with the object, decoupled from the inten- microscope is broadly used in most cell biology laboratories. sity information. In the first type of approach, QPI images can be In 1948, when Gabor proposed using an optical approach to cor- obtained directly in the focal plane by in-line geometries used in rect spherical aberrations in electron micrographs, perhaps he did combination with temporal phase shifting, in which the time delay not anticipate that holography would become an area of research of the reference is controllably incremented, and several corre- in itself, most productively used in the optical region of the spec- sponding intensity images are recorded (Fig. 1a). The irradiance trum10. Gabor showed that recording the intensity of the light at the detector is: emerging from an object at an out-of-focus plane incorporates both amplitude and phase information about the field at the image plane. When a recording film is illuminated by the same incident wave, the Ix(,yI)(=+01Ix,)yI++2(01Ix,)yxcos[ωτ ϕ (,y)] (3) image of the object is recreated at a certain distance away from the film. The appearance of the 3D light distribution in space via paral- where I0 and I1 are the irradiances for the reference phase wave and lax generated a broadly spread confusion that holography is synony- object field, respectively, ω is the angular frequency of the optical mous with 3D imaging. As we show in this Review, holography data field and τ is the time delay between the two waves. In general, the can be used to obtain the 3D structure of a transparent object, but a phase image ϕ can be determined from four intensity measure- single hologram is not sufficient