Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
The term biophotonics denotes a combination of biology and photonics, with photonics being the science and technology of generation, manipulation, and detection of photons, quantum units of light. Photonics is related to electronics and photons. Photons play a central role in information technologies, such as fiber optics, the way electrons do in electronics.
Photoacoustic imaging or optoacoustic imaging is a biomedical imaging modality based on the photoacoustic effect. Non-ionizing laser pulses are delivered into biological tissues and part of the energy will be absorbed and converted into heat, leading to transient thermoelastic expansion and thus wideband ultrasonic emission. The generated ultrasonic waves are detected by ultrasonic transducers and then analyzed to produce images. It is known that optical absorption is closely associated with physiological properties, such as hemoglobin concentration and oxygen saturation. As a result, the magnitude of the ultrasonic emission, which is proportional to the local energy deposition, reveals physiologically specific optical absorption contrast. 2D or 3D images of the targeted areas can then be formed.
Molecular imaging is a field of medical imaging that focuses on imaging molecules of medical interest within living patients. This is in contrast to conventional methods for obtaining molecular information from preserved tissue samples, such as histology. Molecules of interest may be either ones produced naturally by the body, or synthetic molecules produced in a laboratory and injected into a patient by a doctor. The most common example of molecular imaging used clinically today is to inject a contrast agent into a patient's bloodstream and to use an imaging modality to track its movement in the body. Molecular imaging originated from the field of radiology from a need to better understand fundamental molecular processes inside organisms in a noninvasive manner.
Functional imaging is a medical imaging technique of detecting or measuring changes in metabolism, blood flow, regional chemical composition, and absorption.
Modeling photon propagation with Monte Carlo methods is a flexible yet rigorous approach to simulate photon transport. In the method, local rules of photon transport are expressed as probability distributions which describe the step size of photon movement between sites of photon-matter interaction and the angles of deflection in a photon's trajectory when a scattering event occurs. This is equivalent to modeling photon transport analytically by the radiative transfer equation (RTE), which describes the motion of photons using a differential equation. However, closed-form solutions of the RTE are often not possible; for some geometries, the diffusion approximation can be used to simplify the RTE, although this, in turn, introduces many inaccuracies, especially near sources and boundaries. In contrast, Monte Carlo simulations can be made arbitrarily accurate by increasing the number of photons traced. For example, see the movie, where a Monte Carlo simulation of a pencil beam incident on a semi-infinite medium models both the initial ballistic photon flow and the later diffuse propagation.
Biological imaging may refer to any imaging technique used in biology. Typical examples include:
Acoustic microscopy is microscopy that employs very high or ultra high frequency ultrasound. Acoustic microscopes operate non-destructively and penetrate most solid materials to make visible images of internal features, including defects such as cracks, delaminations and voids.
Thermoacoustic imaging was originally proposed by Theodore Bowen in 1981 as a strategy for studying the absorption properties of human tissue using virtually any kind of electromagnetic radiation. But Alexander Graham Bell first reported the physical principle upon which thermoacoustic imaging is based a century earlier. He observed that audible sound could be created by illuminating an intermittent beam of sunlight onto a rubber sheet. Shortly after Bowen's work was published, other researchers proposed methodology for thermoacoustic imaging using microwaves. In 1994 researchers used an infrared laser to produce the first thermoacoustic images of near-infrared optical absorption in a tissue-mimicking phantom, albeit in two dimensions (2D). In 1995 other researchers formulated a general reconstruction algorithm by which 2D thermoacoustic images could be computed from their "projections," i.e. thermoacoustic computed tomography (TCT). By 1998 researchers at Indiana University Medical Center extended TCT to 3D and employed pulsed microwaves to produce the first fully three-dimensional (3D) thermoacoustic images of biologic tissue [an excised lamb kidney ]. The following year they created the first fully 3D thermoacoustic images of cancer in the human breast, again using pulsed microwaves. Since that time, thermoacoustic imaging has gained widespread popularity in research institutions worldwide. As of 2008, three companies were developing commercial thermoacoustic imaging systems – Seno Medical, Endra, Inc. and OptoSonics, Inc.
The photoacoustic Doppler effect is a type of Doppler effect that occurs when an intensity modulated light wave induces a photoacoustic wave on moving particles with a specific frequency. The observed frequency shift is a good indicator of the velocity of the illuminated moving particles. A potential biomedical application is measuring blood flow.
Ultrasound-modulated optical tomography (UOT), also known as Acousto-Optic Tomography (AOT), is a hybrid imaging modality that combines light and sound; it is a form of tomography involving ultrasound. It is used in imaging of biological soft tissues and has potential applications for early cancer detection. As a hybrid modality which uses both light and sound, UOT provides some of the best features of both: the use of light provides strong contrast and sensitivity ; these two features are derived from the optical component of UOT. The use of ultrasound allows for high resolution, as well as a high imaging depth. However, the difficulty of tackling the two fundamental problems with UOT have caused UOT to evolve relatively slowly; most work in the field is limited to theoretical simulations or phantom / sample studies.
Preclinical imaging is the visualization of living animals for research purposes, such as drug development. Imaging modalities have long been crucial to the researcher in observing changes, either at the organ, tissue, cell, or molecular level, in animals responding to physiological or environmental changes. Imaging modalities that are non-invasive and in vivo have become especially important to study animal models longitudinally. Broadly speaking, these imaging systems can be categorized into primarily morphological/anatomical and primarily molecular imaging techniques. Techniques such as high-frequency micro-ultrasound, magnetic resonance imaging (MRI) and computed tomography (CT) are usually used for anatomical imaging, while optical imaging, positron emission tomography (PET), and single photon emission computed tomography (SPECT) are usually used for molecular visualizations.
Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.
Multi-spectral optoacoustic tomography (MSOT), also known as functional photoacoustic tomography (fPAT), is an imaging technology that generates high-resolution optical images in scattering media, including biological tissues. MSOT illuminates tissue with light of transient energy, typically light pulses lasting 1-100 nanoseconds. The tissue absorbs the light pulses, and as a result undergoes thermo-elastic expansion, a phenomenon known as the optoacoustic or photoacoustic effect. This expansion gives rise to ultrasound waves (photoechoes) that are detected and formed into an image. Image formation can be done by means of hardware or computed tomography. Unlike other types of optoacoustic imaging, MSOT involves illuminating the sample with multiple wavelengths, allowing it to detect ultrasound waves emitted by different photoabsorbing molecules in the tissue, whether endogenous or exogenous. Computational techniques such as spectral unmixing deconvolute the ultrasound waves emitted by these different absorbers, allowing each emitter to be visualized separately in the target tissue. In this way, MSOT can allow visualization of hemoglobin concentration and tissue oxygenation or hypoxia. Unlike other optical imaging methods, MSOT is unaffected by photon scattering and thus can provide high-resolution optical images deep inside biological tissues.
Super-resolution photoacoustic imaging is a set of techniques used to enhance spatial resolution in photoacoustic imaging. Specifically, these techniques primarily break the optical diffraction limit of the photoacoustic imaging system. It can be achieved in a variety of mechanisms, such as blind structured illumination, multi-speckle illumination, or photo-imprint photoacoustic microscopy in Figure 1.
Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by , which is much faster than that of two-photon microscopy by . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.
Deep learning in photoacoustic imaging combines the hybrid imaging modality of photoacoustic imaging (PA) with the rapidly evolving field of deep learning. Photoacoustic imaging is based on the photoacoustic effect, in which optical absorption causes a rise in temperature, which causes a subsequent rise in pressure via thermo-elastic expansion. This pressure rise propagates through the tissue and is sensed via ultrasonic transducers. Due to the proportionality between the optical absorption, the rise in temperature, and the rise in pressure, the ultrasound pressure wave signal can be used to quantify the original optical energy deposition within the tissue.
A specific branch of contrast-enhanced ultrasound, acoustic angiography is a minimally invasive and non-ionizing medical imaging technique used to visualize vasculature. Acoustic angiography was first developed by the Dayton Laboratory at North Carolina State University and provides a safe, portable, and inexpensive alternative to the most common methods of angiography such as Magnetic Resonance Angiography and Computed Tomography Angiography. Although ultrasound does not traditionally exhibit the high resolution of MRI or CT, high-frequency ultrasound (HFU) achieves relatively high resolution by sacrificing some penetration depth. HFU typically uses waves between 20 and 100 MHz and achieves resolution of 16-80μm at depths of 3-12mm. Although HFU has exhibited adequate resolution to monitor things like tumor growth in the skin layers, on its own it lacks the depth and contrast necessary for imaging blood vessels. Acoustic angiography overcomes the weaknesses of HFU by combining contrast-enhanced ultrasound with the use of a dual-element ultrasound transducer to achieve high resolution visualization of blood vessels at relatively deep penetration levels.
Photoacoustic flow cytometry or PAFC is a biomedical imaging modality that utilizes photoacoustic imaging to perform flow cytometry. A flow of cells passes a photoacoustic system producing individual signal response. Each signal is counted to produce a quantitative evaluation of the input sample.
Ultrasound-switchable fluorescence (USF) imaging is a deep optics imaging technique. In last few decades, fluorescence microscopy has been highly developed to image biological samples and live tissues. However, due to light scattering, fluorescence microscopy is limited to shallow tissues. Since fluorescence is characterized by high contrast, high sensitivity, and low cost which is crucial to investigate deep tissue information, developing fluorescence imaging technique with high depth-to-resolution ratio would be promising.. Recently, ultrasound-switchable fluorescence imaging has been developed to achieve high signal-to-noise ratio (SNR) and high spatial resolution imaging without sacrificing image depth.
