Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)¹, where the optical irradiation is focused to the diffraction limit to achieve cellular¹ or even subcellular² level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy³ or with optical-scattering-based OCT⁴ (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology^{5, 6}, ophthalmology^{7, 8}, vascular biology⁹, and dermatology¹⁰. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.Download video file.^{(52M, mov)}     

Interaction of radiofrequency and microwave radiation with living systems

Summary Human health aspects and biological effects of radio frequency (RF) and microwave radiation have been in the focus of research efforts in the last decade. An understanding of the interaction mechanisms between such radiation and living systems is essential in interpreting experimental results and assessing potential health hazards.A comprehensive review of basic biophysical interaction mechanisms between RF and microwaves in the frequency range between 10 MHz and 300 GHz and biological systems is provided in this paper. The interactions at various levels of organization of a living organisms such as molecular, cellular and macroscopic are discussed.     

Possible mechanisms of synaptic plasticity modulation by extremely low-frequency magnetic fields

Understanding the biological mechanisms by which extremely low-frequency (ELF, < 300 Hz) magnetic fields (MFs) interact with human brain activity is an active field of research. Such knowledge is required by international agencies providing guidelines for general public and workers exposure to ELF MFs (such as ICNIRP, the International Commission on Non-Ionizing Radiation Protection). The identification of these interaction mechanisms is extremely challenging, since the effects of ELF MF exposure need to be monitored and understood at very different spatial (from micrometers to centimeters) and temporal (from milliseconds to minutes) scales. One possibility to overcome these issues is to develop biophysical models, based on the systems of mathematical equations describing the electric or metabolic activity of the brain tissue. Biophysical models of the brain activity offer the possibility to simulate how the brain tissue interacts with ELF MFs, in order to gain new insights into experimental data, and to test novel hypotheses regarding interaction mechanisms. This paper presents novel hypotheses regarding the effects of power line (60 Hz in North America) MFs on human brain activity, with arguments from biophysical models. We suggest a hypothetic chain of events that could bridge MF exposure with detectable effects on human neurophysiology. We also suggest novel directions of research in order to reach a convergence of biophysical models of brain activity and corresponding experimental data to identify interaction mechanisms.     

Sensory adaptation

Adaptation occurs in a variety of forms in all sensory systems, motivating the question: what is its purpose? A productive approach has been to hypothesize that adaptation helps neural systems to efficiently encode stimuli whose statistics vary in time. To encode efficiently, a neural system must change its coding strategy, or computation, as the distribution of stimuli changes. Information theoretic methods allow this efficient coding hypothesis to be tested quantitatively. Empirically, adaptive processes occur over a wide range of timescales. On short timescales, underlying mechanisms include the contribution of intrinsic nonlinearities. Over longer timescales, adaptation is often power-law-like, implying the coexistence of multiple timescales in a single adaptive process. Models demonstrate that this can result from mechanisms within a single neuron.     

Solution NMR views of dynamical ordering of biomacromolecules

Background

To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

Quantum dots find their stride in single molecule tracking

Thirteen years after the demonstration of quantum dots as biological imaging agents, and nine years after the initial commercial introduction of bioconjugated quantum dots, the brightness and photostability of the quantum dots has enabled a range of investigations using single molecule tracking. These materials are being routinely utilized by a number of groups to track the dynamics of single molecules in reconstituted biophysical systems and on living cells, and are especially powerful for investigations of single molecules over long timescales with short exposure times and high pointing accuracy. New approaches are emerging where the quantum dots are used as 'hard-sphere' probes for intracellular compartments. Innovations in quantum dot surface modification are poised to substantially expand the utility of these materials.     

Multi-scale molecular photoacoustic tomography of gene expression

Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ～1.0 mm and ～0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 μm and an axial resolution of 15 μm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.     

Towards biological applications of nanophotonic tweezers

Optical trapping (synonymous with optical tweezers) has become a core biophysical technique widely used for interrogating fundamental biological processes on size scales ranging from the single-molecule to the cellular level. Recent advances in nanotechnology have led to the development of ‘nanophotonic tweezers,’ an exciting new class of ‘on-chip’ optical traps. Here, we describe how nanophotonic tweezers are making optical trap technology more broadly accessible and bringing unique biosensing and manipulation capabilities to biological applications of optical trapping.     

On the use of <Emphasis Type="Italic">Pichia pastoris</Emphasis> for isotopic labeling of human GPCRs for NMR studies

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and ¹³C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.     

The Impacts of Prepared Plasma-Activated Medium (PAM) Combined with Doxorubicin on the Viability of MCF-7 Breast Cancer Cells: A New Cancer Treatment Strategy

Background:For many years, the chemotherapeutic agent doxorubicin (DOX) has been used to treat various cancers; however, DOX initiates several critical adverse effects. Many studies have reported that non-thermal atmospheric pressure plasma can provide novel, but challenging, treatment strategies for cancer patients. To date, tissues and cells have been treated with plasma-activated medium (PAM) as a practical therapy. Consequently, due to the harmful adverse effects of DOX, we were motivated to elucidate the impact of PAM in the presence of DOX on MCF-7 cell proliferation.Methods:MTT assay, N-acetyl-L-cysteine (NAC) assay, and flow cytometry analysis were utilized in this research.Results:The results demonstrated that 0.45 µM DOX combined with 3-min PAM significantly induced apoptosis (p< 0.01) through intracellular ROS generation in MCF-7 when compared with 0.45 µM DOX alone or 3-min PAM alone. In contrast, after treatment with 0.45 µM DOX plus 4-min PAM, cell necrosis was increased. Hence, DOX combined with 4-min PAM has cytotoxic effects with different mechanisms than 4-min PAM alone, in which the number of apoptotic cells increases.Conclusion:Although further investigations are crucial, low doses of DOX plus 3-min PAM could be a promising strategy for cancer therapy. The findings from this research may offer advantageous and innovative clinical strategies for cancer therapy using PAM.Key Words: Apoptosis, Breast cancer lymphedema, Doxorubicin, Plasma-activated medium (PAM), Necrosis     

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.      

Biophysical mechanisms: a component in the weight of evidence for health effects of power-frequency electric and magnetic fields

Comparatively high exposures to power-frequency electric and magnetic fields produce established biological effects that are explained by accepted mechanisms and that form the basis of exposure guidelines. Lower exposures to magnetic fields (< 1 microT average in the home) are classified as "possibly carcinogenic" on the basis of epidemiological studies of childhood leukemia. This classification takes into consideration largely negative laboratory data. Lack of biophysical mechanisms operating at such low levels also argues against causality. We survey around 20 biophysical mechanisms that have been proposed to explain effects at such low levels, with particular emphasis on plausibility: the principle that to produce biological effects, a mechanism must produce a "signal" larger than the "noise" that exists naturally. Some of the mechanisms are impossible, and some require specific conditions for which there is limited or no evidence as to their existence in a way that would make them relevant to human exposure. Others are predicted to become plausible above some level of field. We conclude that effects below 5 microT are implausible. At about 50 microT, no specific mechanism has been identified, but the basic problem of implausibility is removed. Above about 500 microT, there are established or likely effects from accepted mechanisms. The absence of a plausible biophysical mechanism at lower fields cannot be taken as proof that health effects of environmental electric and magnetic fields are impossible. Nevertheless, it is a relevant consideration in assessing the overall evidence on these fields.     

Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: recent developments in self-referencing amperometry for physiological sensing

Ultimately, advances in genomics, proteomics and metabolomics will be realized by combining these approaches with biophysical sensors for understanding the functional and structural (physiological) aspects of sub-cellular systems (cytomics). Therefore, the emergence of the new fields of cytomics and physiomics will require new technologies to probe the functional realm of living cells. While amperometric sensors have been used, their sensitivity and reliability are significantly improved through the development of new strategies and data acquisition systems for the operation of the sensors. This includes the application of the principles of the vibrating or self-referencing microsensor to the operation of amperometric sensors. The development of self-referencing amperometry (SRA) is significant because it effectively converts static concentration sensors into dynamic biophysical sensors that directly monitor physiological flux. SRA has been developed for analytes such as O2, NO, H2O2 and ascorbate. These sensors have been validated against non-biological microscopic flux sources that were theoretically modeled, before being applied to biological research. This new sensor technology has been shown, through research in a wide variety of biological and biomedical research projects, to be an important new tool in the arsenal of the cell biologist. SRA technology has been adapted through SRA-H2O2 and SRA-NADH sensors, for electrochemically coupled enzyme based self-referencing biosensors (SRB) for glucose, glutamate and ethanol. These developments in self-referencing sensor technologies offer great promise in extending electroanalytical chemistry and biosensor technologies from the micro to the nanoscale where researchers can study physiology at the sub-cellular and organellar levels.     

Electromagnetic information transfer through aqueous system

Several beneficial effects of the electromagnetic information transfer through aqueous system (EMITTAS) procedure have previously been reported in vitro. The clinical potential of this procedure has also started to be evaluated. Information flow in biological systems can be investigated through chemical and molecular approaches or by a biophysical approach focused on endogenous electrodynamic activities. Electromagnetic signals are endogenously generated at different levels of the biological organization and, likely, play an active role in synchronizing internal cell function or local/systemic adaptive response. Consequently, each adaptive response can be described by its specific electromagnetic pattern and, therefore, correlates with a unique and specific electromagnetic signature. A biophysical procedure synchronously integrating the EMITTAS procedure has already been applied for the treatment of articular pain, low-back pain, neck pain and mobility, fluctuating asymmetry, early-stage chronic kidney disease, refractory gynecological infections, minor anxiety and depression disorders. This clinical strategy involves a single treatment, since the EMITTAS procedure allows the patient to continue his/her own personal treatment at home by means of self-administration of the recorded aqueous system. A significant and long-lasting improvement has been reported, showing a potential beneficial use of this biophysical procedure in the management of common illnesses in an efficient, effective and personalized way. Data from recent studies suggest that aqueous systems may play a key role in providing the basis for recording, storing, transferring and retrieving clinically effective quanta of biological information. These features likely enable to trigger local and systemic self-regulation and self-regeneration potential of the organism.     

The need for co-product allocation in the life cycle assessment of agricultural systems—is “biophysical” allocation progress?

Purpose

Several new “biophysical” co-product allocation methodologies have been developed for LCA studies of agricultural systems based on proposed physical or causal relationships between inputs and outputs (i.e. co-products). These methodologies are thus meant to be preferable to established allocation methodologies such as economic allocation under the ISO 14044 standard. The aim here was to examine whether these methodologies really represent underlying physical relationships between the material and energy flows and the co-products in such systems, and hence are of value.

Methods

Two key components of agricultural LCAs which involve co-product allocation were used to provide examples of the methodological challenges which arise from adopting biophysical allocation in agricultural LCA: (1) the crop production chain and (2) the multiple co-products produced by animals. The actual “causal” relationships in these two systems were illustrated, the energy flows within them detailed, and the existing biophysical allocation methods, as found in literature, were critically evaluated in the context of such relationships.

Results and discussion

The premise of many biophysical allocation methodologies has been to define relationships which describe how the energy input to agricultural systems is partitioned between co-products. However, we described why none of the functional outputs from animal or crop production can be considered independently from the rest on the basis of the inputs to the system. Using the example of manure in livestock systems, we also showed why biophysical allocation methodologies are still sensitive to whether a system output has economic value or not. This sensitivity is a longstanding criticism of economic allocation which is not resolved by adopting a biophysical approach.

Conclusions

The biophysical allocation methodologies for various aspects of agricultural systems proposed to date have not adequately explained how the physical parameters chosen in each case represent causal physical mechanisms in these systems. Allocation methodologies which are based on shared (but not causal) physical properties between co-products are not preferable to allocation based on non-physical properties within the ISO hierarchy on allocation methodologies and should not be presented as such.

     

Long-timescale simulation methods

The outstanding challenges in computer simulations of biological macromolecules are related to their complexity. Part of the complexity of biological systems concerns their physical size. Enumerating atoms ranging from a few in small signal molecules to the millions of particles in biological complexes is an obvious example of biological hierarchy. Another aspect is the extremely broad range of timescales of life science processes (many orders of magnitude); this adds another dimension of complexity. This extended range of timescales may even be observed for a single biomolecular process. Consider, for example, the R to T transition in hemoglobin. The complete conformational change occurs in tens of microseconds. However, the system has more than one timescale. Considerable activity occurs on a range of timescales before the final event (heme relaxation, picoseconds; tertiary relaxation, nanoseconds; ligand escape from the protein matrix and rebinding, hundreds of nanoseconds and so on). Whereas the basic time-step of atomically detailed simulations is about a femtosecond, it is not difficult to find molecular processes in biology that span more than ten orders of magnitude of relevant times, making the straightforward simulation of these events very difficult. Several techniques have been developed in recent years to address these problems.