Cryomedia additive agents and their effects on cryopreserved cells.
\r\n\tThis book aims to present an overview of the current status of nanofibers, fabrication and recent trends in the fabrication of nanofibers, and functional nanofibers and applications of nanofibers in various fields including environmental, bio-sensing, drug delivery, catalysis, and medical. The book hopes to provide a piece of up-to-date information about the mentioned topics and fundamental knowledge necessary for the advanced study in the field of nanofibers and their applications, making it interesting to research students, scientists, engineers, and material scientists.
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He has done Ph.D. and post doctorate in the field of Material Science (Nanoscience). His research interests include fabrication of nanomaterials and their structural, optical, magnetic, and electrical characterizations. He has authored more than 100 research articles and published 10 books. Presently, he is the Editor-in-Chief of ‘Journal of Materials, Processing and Design\\' and \\'The Nucleus\\'. He is also the Executive Editor of \\'International Journal of Nano Studies and Technology\\'. 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Gomes and Marisa P. Sarria",coverURL:"https://cdn.intechopen.com/books/images_new/5884.jpg",editedByType:"Edited by",editors:[{id:"146466",title:"Prof.",name:"Andreia",surname:"Ferreira de Castro Gomes",slug:"andreia-ferreira-de-castro-gomes",fullName:"Andreia Ferreira de Castro Gomes"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"71258",title:"Cryomedia Formula: Cellular Molecular Perspective",doi:"10.5772/intechopen.91382",slug:"cryomedia-formula-cellular-molecular-perspective",body:'Cryopreservation is one of the most effective techniques that widely used for preserving living cells and organs in research and therapeutic industries [1]. The principle of cryopreservation is to protect cells from the application of super low temperature and ice crystal formation by using media that consist of antifreezing or cryoprotective (CPA) substances such as; glycerol, dimethylsulfoxide (DSMO) or trehalose. The expansion in clinical experiments for medical applications revealed the limitations of utilizing the conventional CPAs which resulting sub-optimal cell quality. This is attributed to the detrimental effects of conventional CPAs and their molecular interactions that compromise cell quality. The new research areas and advanced techniques significantly increase the demand of better cryopreservation to maintain the quality and functionality of cells and tissues.
Current trends in cryopreservation are actively focusing on identifying a safe and effective alternative CPAs to substitute or support the conventional agents. In addition, there are various cell types valuable for investigation and medical development and their different biological profile and functional mechanisms required customizing cryopreservation. However, there are limiting number of studies addressing the influence of the cryomedia formulation on the global proteomic and biomolecular profile of the cells.
Cryomedia formulation usually encompass of cryoprotective agents (CPAs) and carrier media prepared at or close to cell isotonic concentrations to provide support to cells at low temperature [2]. It also may contain salts, pH buffers, osmotic agents, nutrients, antioxidants or apoptosis inhibitors [2]. There are about 56 CPAs commonly used for different cell cryopreservation [3, 4]. Glycerol and dimethylsulfoxide (DMSO) are the most common CPAs used in cryomedia formula. CPAs are classified based on the permeability through cell membrane into, permeable (pCPA), and impermeable (ipCPA).
pCPAs are generally small, non-ionic molecules that are highly soluble in water, even at low temperatures. They can pass through cellular membranes and equilibrate within the cytoplasm in exchange for intracellular water during dehydration without over dehydrating the cell [5]. They become solid at a lower temperature than water freezing point and subsequently suppress ice crystal formation [6] and mitigate cellular physical damage that could occur in cellular compartments and membranes. Moreover, pCPAs reduce salt-induced stress by dissolving solute and reducing concentrations in the remaining water fraction intracellularly until the cell is cooled to a sufficiently low temperature [6, 7]. pCPA permeability is controlled by their viscosity and the membrane properties of the cell itself [8, 9]. The latter mentioned is variable between different cell types as well as the varying ages of cells [10, 11]. Most of the pCPAs are polyols, such as glycerol and dimethylsulfoxide (DMSO), which are prominence in cryopreservation. Many successful cryopreserving protocols utilized these compounds for their high efficiency in compare to others such as methanol and ethylene glycol [6].
ipCPAs are large molecules usually comprised of long chains of polymers that are unable to permeate through cellular membranes. They are water soluble and thought to increase the osmolarity around cells, which result in cellular dehydration and reduce ice crystal formation intracellularly [12]. The combination of high concentrations of ipCPAs and fast cooling promotes vitrification and stabilizing cellular proteins and membranes [13, 14]. Their protective mechanism is based on preventing ice formation extracellularly as well as intracellular through dehydration [15]. There are several classes of ipCPAs, such as certain forms of sugars, macromolecules, and polymers. Sugars are classified based on their chemical structure into: mono-, di- and poly-saccharides (glucose, trehalose, and raffinose, respectively). A number of these sugars are permeable (e.g., glucose) and others are impermeable (e.g., trehalose). Sugars have garnered unique interest over the last decades. They have been found to protect protein activity and reduce thermal denaturing heat capacity of chemicals [16, 17, 18, 19, 20, 21], which leads to protein stabilization. In particular, trehalose has been identified as a universal protein stabilizer and been involved in many freezing and desiccation studies [18, 19, 20].
Depending on the freezing mode (slow cooling or vitrification), the concentrations of CPAs are variant in the solution. For instance, slow freezing mode requires less CPA concentration than vitrification. Choosing the optimum concentration of CPA in combination with the cooling rate is crucial for successful cryopreservation [20]. For instance, when preserving human ovarian tissue following slow freezing protocol, DMSO is used with initial concentration of 7.5% and gradually increased to 12.5%. Whereas preserving the same tissue using the vitrification protocol, 20% DMSO is needed [21]. Different tissues and cells, however, demonstrate different responses to the cryopreservation approaches and CPAs. Therefore, the selection of the appropriate protocol and CPA is subject to the cryopreservation empirical success of the desired cells or tissues.
An accurate assessment of cryopreserved cells or tissues considering the viability and functionality is paramount to determine the quality and reliability of the cryopreservation protocol and solution. In the past, classical parameters, such as survival rate or motility, were the only quality measurements [22, 23]. With the evolution in technologies and developed assays, scientists can obtain more information surrounding the level of stress that heralds cellular death cascades and dynamic changes that impact cryopreserved cells’ function and morphology.
Nowadays, there are a wide range of viability assays available; however, selecting the appropriate assay mainly depends on cell types to avoid inaccurate measurement. For instance, the measurement of LDH leakage in media can be used for membrane integrity assessment because of its reliability and easy performance. It is an applicable measurement in single cells as well as tissues and organs [24]. Conversely, using fluorescent probes for viability measurement is suitable for many cells excluding hepatocytes, because of their detoxification activity with respect to probes that influencing the accuracy of the measurement [25].
The emergence of developed technologies, such as genomics, transcriptomics, proteomics, and metabolomics (collectively termed OMICs), has provided a comprehensive profile of cryopreserved cells, including their stressed and compromised biological pathways, which may help designing protocols or solutions in order to modulate the damaged pathways. So far, the majority of OMICs applications in cryopreservation are limited to reproduction medicine and plants [26, 27], such as in human sperm characterization post-thaw [28]. The deep analysis OMICs stresses the importance of adopting such analytical approach in researches aiming at advancing cryopreservation and biobanking for better CTMPs outcome [25].
Introducing CPAs in high concentration (molars) is accompanied with non-specific adverse effects such as osmotic stress and cell dehydration [29] that also could induce the oxidative stress [30]. This can cause severe cell damage; for instance, increasing the concentration of DMSO, glycerol, and 1,2-propanediol is linked with the production of non-enzymatic formaldehyde [31], a cytotoxic compound that contributes to cell death [32]. The long exposure duration of cells to high concentration of CPA also harm cell development, as reported when exposing bovine blastocytes to a high concentration of ethylene glycol over 10 min [33]. Likewise, introducing a high concentration of propanediol to mouse zygotes was found to have a similar damaging effect on cell development to that observed in bovine blastocytes [34].
The high concentration of CPA accumulated intracellularly has a detrimental effect on cells. In cryopreserved human mesenchymal stem cells (hMSC), it has a significant effect on cellular viability, filamentous actin distribution, intracellular pH, and mitochondria aggregation [35]. It has also been found to cause abnormal spindles and morphology in human oocytes, which can potentially influence their viability post-cryopreservation [36]. Similarly, CPA causes a serious alteration in mammalian sperm viability, physiological properties, protein phosphorylation patterns [37], and can lethally damage enzymatic activity and DNA [38]. However, osmotic stress factors and associated cell shock cannot be decoupled since they interact with each other, though the resultant effects can be reversed or limited to a certain extent by minimizing exposure time, accelerating freezing and thawing speeds, and gradually diluting CPAs in cells [39], which can increase post-thaw cell viability. These types of reported damages are considered non-specific since it is not limited to specific CPA identity. However, the molecular interaction of CPAs is more closely linked to the permeable CPAs, as they are able to interact with the cell compartments and biomolecules [30].
CPA toxicity effect can be either reversible (e.g., osmotic shock and cellular shrinkage) [40, 41] or irreversible. Notably, cryopreservation protocols involving short exposure times to CPAs can reverse the induced damages. Nevertheless, irreversible damage is common in cells lacking self-renewal or repair mechanisms, such as RBCs [42] and embryonic stem cells [43, 44].
Oxidative stress occurs during cryopreservation, mainly when adding CPAs to cells [45]. The increased oxidative stress results in more ROS production [46], which leads to a disequilibrium between the generated ROS and the cellular antioxidant capacity within the redox pathway. A decrease in cellular-reduced glutathione (GSH) content was observed during the freezing step of sperm [47], indicating that oxidative damage occurs during the initial steps of cryopreservation. Consequently, increased ROS production results in lipid peroxidation [48], DNA instability [49], protein oxidation [50], overall dysfunctional cells, and low survival rates [47, 49]. Oxidative stress has been observed when applying glycerol [51], DMSO [52], and trehalose [50] to cells.
Cells naturally have a dynamic and complex system involving active biomolecules that respond distinctly to all forms of environmental stressors, including CPA media and temperature alterations. The cells’ response to stressors involves complex biomolecular events influencing their fate. Measuring the survival rate of thawed cells is a classical parameter that is not precise when determining the efficacy of cryopreservation. This is because during the recovery period, a decrease in cellular viability occurs in different cell types [53]. This is attributed to the activation of apoptosis machinery post-thaw [54]. Xu et al. [53] reported that exposing cells to DMSO and freezing conditions activate apoptosis through extrinsic and intrinsic pathways, including caspase-8, caspase-9, and p53. Some CPAs have different mechanisms, yet they lead to the same lethal results. Propylene glycol (ProH), for instance, reduced cell viability via increasing intracellular calcium to a cytotoxic level [55].
Furthermore, the cryopreservation affects cells’ biomarkers [56]. It alters the proteome profile of cells, which in some cases can bring about changes in cellular metabolism, function, and structure [57]. In previous work, there is often no clear demarcation between the effect of CPAs and the cryopreservation protocol itself. However, the exact effect of CPAs can be investigated in an experiment if cell viability and functionality are analyzed before freezing.
Considering the aforementioned limitations in cryomedia formula, many active studies investigated the efficacy of additive agents to improve the cryomedia and modulate the resultant damages in cryopreserved cells (Table 1). Additive agents have variable effects on different cells. This was evidently observed in number of cases such as; quercetin, glutathione, and ascorbic acid [58]. On other hand, some other demonstrated similar efficient antioxidant protection effect on several cells (e.g., curcumin) [58]. Notably, many protective factors share their antioxidant protection effects at different concentrations (e.g., hyaluronan and glutamine [59, 60]) that commonly include reducing oxidative stress on lipid and proteins and improve viability rate.
Additive agents | Example | Concentration | Cell types | Molecular and biological effects |
---|---|---|---|---|
Antioxidants | Resveratrol [61] Salidroside [62] | 15 μM 200 μM | Human sperms Red blood cells | Decrease DNA fragmentation through activating AMP-activated protein kinase (AMPK) Increase glutathione reductase (GR) activity and cells stability post thawing Reduce hemolysis, lactate dehydrogenase activity and protect protein and lipid from oxidation damage |
Proteins | Type III anti-freezing proteins [63] Sericin [64] | 0.8 mg/ml 5% | Human carcinoma cells Human sperm | Increase cells recovery post-thawing Increase cells motility Decreased DNA fragmentation |
Enzymes | Catalase [65] | 40 μl/ml | Mice spermatogonia stem cells | Reduce apoptosis and ROS production Increase viability |
Vitamins | Vit E [66] | 100–200 μmol | Human sperm | Increase motility |
Anti-apoptotic drugs | Sphingosine-1- and Z-VAD-FMK [67] | 10 μM | Ovarian sheep | Preserve primordial follicular density, with normal morphology and improved proliferation |
Cryomedia additive agents and their effects on cryopreserved cells.
In our published studies, the discovery of the protection potent of salidroside and nigerose was exceptional on nucleated as well as anucleated hematopoietic cells [RBCs and human leukemia cells (HL-60)] in various cryomedia formulae and freezing modes. The efficacy of these compounds was evidenced at low concentrations (200–300 μM) of salidroside and nigerose, respectively. The effect of the additive compounds was determined by analyzing both the biomolecular and proteomic profiles of the survival cells [58]. First, we examined the effect of salidroside in standard cryo-solutions (glycerol and trehalose), which are commonly used for the RBCs biopreservation, using RBCs [62]. When comparing the survival cells rate, RBCs cryopreserved in solutions contained salidroside showed higher survival rate in compare to those cryopreserved in standard cryomedia alone. On biomolecular level, salidroside improved the intracellular activity of glutathione reductase (GR), the active enzyme in the redox pathway. In addition, it reduced the level of stress resultant from freeze-thaw process, as it was measured by intracellular lactate dehydrogenase (LDH) activity [68]. Moreover, it protected RBC proteins against oxidative damages [62]. Further investigation on human leukemia cells (HL-60) using salidroside in 2% DMSO and fetal bovine serum cryosolution demonstrated similar protection effects to what have been seen in RBCs [62, 68]. Additionally, it protected lipid against oxidative stress. In the same study, we used nigerose for comparison, which showed similar protection effect on the biomolecular profile of the cells.
On top of the biological profile of cryopreserved cells, proteomic analysis revealed the specific and unique modulation effect of additive agents on compromised biological pathways [68]. Each compound was observed to have a demonstrably unique effect on the proteome pattern of cryopreserved HL-60 cells. Nigerose was strongly engaged with cell maintenance, energetic, and metabolic pathways, whereas salidroside influenced proteins associated with DNA binding and nuclear activities. Both overlapped with regards to influencing proteins associated with redox pathways. Moreover, the damaging effects of classical cryomedia were modulated by the reformulated media comprising the novel protective agents. The protective mechanisms of the compounds on the proteomic level were strongly compatible with the biochemical analysis of the cells cryosurvival rate and their resistance to stressors [68]. This has shed the light over the potency of specific effectiveness of additive agents in the cryosolution and their specific applications for preserving different cells and tissues for pharmaceutical and clinical applications.
Understanding of the protective mechanisms of cryomedia ingredients along with identifying powerful protective compounds to enhance cryomedia performance is highly demanded. Due to the wide range of preserved cells and tissues, designing the appropriate cryosolution with suitable protocol is beneficial. In fact, these are particularly important for CTMP industries and end-users at clinics, such as those with cancer and diabetes or requiring blood transfusion, organ transplantation, and infertility treatments.
One of the most common and well recognized technologies in modern medicine is ultrasonography. Its use has been used in many medical fields, and new methods and devices using ultrasound are frequently emerging. While there have been many recent advancements, ultrasound has a rich history dating back centuries.
Some consider the earliest investigation into ultrasound beginning with the ancient Greeks [1]. Pythagoras invented the sonometer to study music; Boethius compared the waves generated by dropping a pebble into water to sound waves.
In 1880, French scientists and brothers, Pierre and Jacques Curie, discovered piezoelectricity [2]. When certain materials (such as some crystals) are exposed to an electric field they undergo mechanical changes. The reverse is also true: when piezoelectric materials have mechanical force exerted on them they generate an electric charge. Thus, these crystals can both transmit and receive sound. Such piezoelectric devices are the basis of ultrasound transducers [3]. When voltage is applied to the transducer sound waves are emitted; when the reflected waves are picked up by the transducer, they generate voltage. This electrical signal can then be processed to produce an “image” based on the reflected sound waves.
While the Curie brothers discovered the piezoelectric effect in the 1880s, it wasn’t utilized for ultrasonography until a few decades later. One of their students, Paul Langevin, was commissioned by the French government to develop technology to detect enemy submarines [2]. His studies became the base for what was to later become known as sound navigation ranging (SONAR), which was developed extensively in World War II [4, 5].
Later, ultrasound started to see use in medicine. Karl Dussik used it to study brain tissue; George Ludwig used ultrasound to help detect gallstones [2]. In 1951, John Wild and John Reid built the first B-mode scanner [6]. B-mode (brightness-mode) scanners are what is most often thought of when one refers to ultrasound. B-mode produces a two-dimensional reconstructed image of internal body structures based on reflected sound waves. The first commercially available handheld B-mode scanner was produced in 1963 [6]. Around the same time, many researchers started looking into Doppler applications with ultrasound to detect blood flow as well.
The 1960s and 1970s proved to be a time of rapid development for the use of ultrasound in medicine [2, 6]. Its application in cardiology and obstetrics and gynecology became more widespread. The field of medical sonography continued to grow, and various societies and institutions dedicated to medical sonography began to emerge.
In the 1950s, ultrasound was first used in ophthalmology and optometry [7, 8]. These early explorations aimed at using measurements of the depth and velocity of sound waves in the eye to try to distinguish various conditions. This type of ultrasonography is known as A-scanning (amplitude scanning). Further advances in the 1960s used A-mode scanning to better measure the length of the eye, distances within the eye [9], and visual axis of the eye [10]. The axial A-scanning was also used to help determine intraocular lens power [11]. Diagnostic A-scan works by emitting a sonic pulse and measuring the time and amplitude of the echo. This information can then be interpreted to give information on the number of interfaces the wave has passed through. For example, the waveform produced from passing through normal structures of the eye versus a hemangioma versus a more solid lesion will all look different [7]. While A-scanning can give important data on some basic characteristics of the eye, such as lens power and length of the eye, it does not produce a visual re-creation of internal ocular structures. Because of this, its use is somewhat limited and must be combined with B-scan (Figures 1 and 2).
A diagram illustrating the various sources of reflected waves within the eye and how they would appear on the view screen.
Example of an ultrasound probe designed specifically for ophthalmic use.
Another valuable application of ultrasound is the use of Doppler flow ultrasonography. Doppler was a physicist and astronomer in the mid 1800s who demonstrated that blue stars appeared that color because they were moving toward the observer while red stars appeared red because they were moving away from the observer [12]. This became known as the Doppler effect, and held true not only for electromagnetic waves but also acoustic waves; thus, the Doppler effect can be applied to ultrasound to help measure the magnitude and direction of flow.
Doppler found early application in cardiology, where the evaluation of flow was obvious [13, 14]. Doppler ultrasound soon found other applications, though. It proved useful in monitoring and measuring peripheral vasculature [15, 16, 17, 18]. This proved useful for applications such as detecting tumor neovascularization [19, 20] and evaluation for ectopic pregnancy [21].
Doppler ultrasonography was also shown to be useful in estimating the degree of stenosis in vasculature, which proved especially useful in evaluating carotid artery disease [22]. This is of special interest because the ophthalmic artery is the first branch of the internal carotid artery, and disease affecting the internal carotid has the propensity to affect ocular vasculature either indirectly secondary to decreased flow or directly through embolism of atherosclerotic plaque [23]. Doppler was also shown to be helpful in diagnosis and evaluation of open angle glaucoma [24]. Doppler has also been used in ophthalmology to evaluate the ocular fundus [25] and flow through the retinal artery [26].
While A-mode scanning and Doppler flow ultrasound had their specific uses, B-mode scanning was explored more broadly. Devices became more accessible with handheld transducers and options to attach to a TV screen for viewing [27, 28]. In addition, differently shaped probes were developed to aid in imaging for different surface areas or structures [29]. B-mode proved an invaluable tool for evaluating intraocular foreign bodies, masses, hemorrhage, retinal detachment, and congenital abnormalities. It was first pioneered in ophthalmology Baum and Greenwood [30], and the first ocular specific probe was produced by Bronson [27].
One of the first ways B-mode ultrasonography was used in ophthalmology was for evaluation of an opaque eye [30]. For example, if there is a potential foreign body in the eye and there is clouding of the cornea or lens, the object may not be able to be observed directly. Furthermore, if it is radio-opaque, it will not be visible via X-ray. This makes ultrasound an excellent modality for evaluation in such instances. Another advantage is that ultrasonographic evaluation of the eye can be done bedside with the need for minimal equipment, making it ideal for traumatic evaluation [31, 32, 33].
B-mode was also used early on for evaluation of intraocular tumors [34, 35, 36]. Similar to the above example of foreign object location, if there is a soft tissue lesion that is in a position not easily visible by direct ophthalmoscopy or if there is clouding of anterior structures, such that a direct view is not possible, B-mode ultrasonography can aide in evaluation of intraocular soft tissue masses (Figure 3). This has been especially explored in the setting of diagnosing choroidal melanoma [37]. B-mode scanning can help ascertain the position, size, and height of ocular melanoma.
Examples of images from B-scan ultrasound. (A) An image of choroidal hemorrhage. Areas of hemorrhage indicated by (a). (B) Choroidal melanoma (b) with associated superior retinal detachment (arrow). (C) A diabetic patient with vitreous retinal traction. Area of vitreous hemorrhage shown by (c); single arrow indicates posterior hyaloid membrane; double arrow shows point of fibrovascular adhesion. (D) Ciliary melanoma (d) invading into the anterior chamber; arrow indicates cornea while star shows the location of the iris. Images used courtesy of Ellex.
While early scans for evaluation of intraocular masses were focused on identifying presence and location, later research focused on better quantifying such tumors [38, 39]. By taking serial cross-sectional scans over the shape of the tumor, the shape and volume of the tumor could be estimated [40, 41]. This helped to improve evaluation and characterization of intraocular masses and guided radioactive plaque placement.
Despite the many applications of ultrasonography for ocular evaluation, there are some limitations. The depth of penetration of ultrasonic waves is inversely proportional to the frequency used [42]; a transducer using a 10 MHz frequency can penetrate 50 mm, where as a system using 60 MHz frequency will only penetrate 5 mm. Furthermore, the image resolution is limited by the frequency used, with higher frequency systems achieving higher resolution images. Ultrasonic imaging using high frequencies has been termed “ultrasonic microscopy” or “ultrasonic backscatter microscopy” (UBM) [43, 44]. Such systems use very high frequency waves (60-100 MHz) to achieve high resolution images at depths in the 4 mm range. This technique is ideal for high resolution imaging the anterior chamber, ciliary body and its structures, as well as parts of the peripheral retina [45]. Because images can get distorted at the close interface between the transducer and the object being imaged, eye-cup devices are used to create an offset distance between the transducer and the surface of what is being imaged (Figure 4) [42].
The 2D image is an example of B-scan ultrasonography of a human eye. The lower section of the image shows a superimposed A-scan for comparison.
The use of high frequency ultrasonic biomicroscopy has been applied in several ways. One study used UBM for tracking corneal changes related to the laser-assisted in-situ keratomileusis (LASIK) procedure [46]. 50 MHz scanning was used to map the cornea before and after LASIK. They showed that this technique was an accurate and feasible way to track changes in corneal shape and thickness following LASIK. Another application of ultrasonic biomicroscopy was the characterization of the lens [47]. By being able to better characterize the natural lens, more accurate synthetic lenses can be produced (Figure 5). A similar approach was used to better categorize the ciliary body [48]. In contrast to anterior structures such as the cornea and lens can be easily evaluated via direct visualization with methods such as slit lamp examination, the ciliary body is obscured from direct visual view. Because of this, UBM is an ideal modality for evaluation of ciliary body pathology, small tumors in particular [49, 50, 51]. UBM has also been useful in identifying structural morphologies contributing to glaucoma, such as iris plateau syndrome [52]. These examples illustrate how high frequency, high resolution ultrasonic biomicroscopy can practically be applied to ocular evaluation and how this imaging can change practice and drive innovation.
Example of UBM for visualization of anterior structures. Cornea indicated by (a), iris by (b), and ciliary body by (c). This technique can be used for measurements for intraocular lens placement, or to assess ciliary body tumors which would not be directly visible. Image used courtesy of Ellex.
Despite advances in high resolution imaging and faster B-scanning, there were still limitations. One is that ultrasonic evaluation can be time consuming and the quality of the exam is dependent of how skillful the examiner is. Another limitation is that traditional B-mode scanning can only evaluate structures directly opposite to the probe and cannot provide information in the XY plane without 3D reconstruction. While 3D reconstruction was shown to be possible [40, 41] and could give information about the XY planes, such imaging required time consuming serial acquisition of small 2D image slices and subsequent reconstruction. This meant that while 3D reconstruction is possible, it is too cumbersome to be used frequently in the average clinical setting. Much research went into designing systems that were able to scan faster to produce more reliable 3D reconstructions of ocular models [53, 54]. This included using array systems that were able to image at both high frequencies and traditional frequencies to obtain a reconstruction with as much detail as possible at multiple depths [54]. As opposed to traditional A-mode and B-mode transducers which have a single piezoelectric element (and thereby a fixed depth of focus), arrays use a transducer with many elements (over 100) in each transducer head [55].
Despite the valuable information that can be gained from an array system that utilizes both traditional and high frequency waves, such systems have not been readily adopted [56, 57]. One potential reason for the underutilization of high frequency systems may be because of the equipment constraints. Because array transducers require over 100 piezoelectric parts aligned in close proximity within a relatively small transducer head, the production is technically challenging [55, 58]. Moreover, the frequency generated is inversely proportional to the size and spacing of the piezoelectric elements [58]: lower frequency systems can use larger more widely spaced piezoelectric crystals.
A combined low and high frequency array system called the Vevo 2100 (VisualSonics, Toronto, Ontario, Canada) has been tested for use in ophthalmologic evaluation and shows promise [55]. This system uses an array transducer with 256 elements in each transducer head. By utilizing an array system, most of the imaged field can be kept in focus, allowing high-resolution real-time imaging. This system has two linear array transducer probes: a 25 MHz probe and a 50 MHz probe (Figure 6). This system generates 3D reconstructions in seconds utilizing a mechanical motor for efficient scanning. A scan using the 50 MHz probe yields high definition data on the anterior eye structures, while a second scan using the 25 MHz probe will produce data on the posterior segment and orbit. Each scan with 3D reconstruction takes about 10 seconds. An initial evaluation would be a scan with the 50 MHz transducer to assess anterior structures followed by a scan using the 25 MHz probe to image the rest of the eye. By choosing an appropriate transducer and frequency, the whole eye can efficiently be scanned, and a 3D reconstruction generated either from a specific area or the whole eye in less than 1 minute. In addition to generation of 3D reconstructions (Figures 7 and 8), this system can perform several other scanning functions, including B-mode, M-mode, PW Doppler, Color Doppler, Power Doppler, Tissue Doppler, Contrast Mode, and Photoacoustic Imaging.
(A) Picture of the Vevo 2100 system (VisualSonics, Toronto, Ontario, Canada). (B) Picture of the 25 MHz and 50 MHz probe utilized by this system. Used with permission from Gholam A. Peyman.
The lower panels depict 2D slices of a pig eye in different planes obtained with the 25 MHz probe. The upper image is a 3D perspective. Used with permission from Gholam A. Peyman.
Example of 3D reconstruction of a pig eye with anterior lens injury. Various planes are shown. Used with permission from Gholam A. Peyman.
One important application of this system is in emergency evaluation of traumatic eye injuries. Traditional hand-held B-mode evaluation is an excellent tool for detecting foreign bodies but is not without risk. Extreme caution must be used when scanning an injured eye, which must be considered as a potential open globe. It is critical to avoid placing pressure and extruding intraocular contents through a penetrating wound. This system can perform a mechanized scan through a closed eyelid with a coupling medium. There is decreased risk of causing further injury to the eye. This feature, combined with the high-resolution 3D reconstruction, will provide detailed information in seconds on the extent of ocular injury, presence and position of a foreign body (Figure 9).
This image shows the presence of a linear foreign body (A) and secondary acoustic shadowing (B). The images were generated using Matlab (Mathworks, Natick, MA) and a point-and-click technique in which an object is found in one plane, clicked on, and automatically images in the two other planes are generated. used with permission from Gholam A. Peyman.
This system is also useful for routine clinical outpatient evaluation. The 50 MHz transducer can provide a very high level of detail on eyelid, including Meibomian glands (Figure 10), and anterior ocular structures: cornea, iris, ciliary body, and lens. Because of the very high level of resolution, it could be a powerful tool for assessing Meibomian gland disease. By using this scan routinely, small tumors or lesions behind the iris or in the choroid may be detected early, before they caused visual distortion or metastases. Small retinal detachments at the peripheral retina can also be detected using this transducer. This may be very helpful in children with retinal detachment.
Image obtained from a scan using the 50 MHz transducer. Structures visible, including the Meibomian glands, are labeled. used with permission from Gholam A. Peyman.
In summary, the 50 MHz transducer can provide fine detail of anterior structures, a subsequent scan using the 25 MHz transducer can provide information on the remainder of the eye. This is useful for assessment in the setting of trauma, additionally it can also provide valuable information on retinal detachment, size and location of intraocular masses, and information on the optic nerve head drusen or edema. Because this system can provide simultaneous B-mode scanning and Doppler flow imaging, both anatomic assessment and evaluation of vascular disease of the optic nerve head can be performed. Doppler flow imaging can also be utilized to assess other vascular diseases, such as temporal arteritis (Figure 11).
Example of color Doppler image showing flow through temporal artery. Obtained using 50 MHz transducer. Used with permission from Gholam A. Peyman.
Overall, the Vevo 2100 system (VisualSonics, Toronto, Ontario, Canada) potentially is a powerful tool for emergency and routine evaluation of ophthalmic pathology. By utilizing an array system in the transducer head, real-time images well focused throughout the scan may be obtained. The mechanical scanning function can produce 3D models in seconds with decreased risk of expulsion of ocular contents following trauma. These 3D reconstructions provide information in planes traditional B-scanning methods cannot assess. The level of detail produced using this system can provide information on a wide array of ocular disease, from Meibomian gland, evaluation of intraocular tumors or foreign bodies, vascular diseases affecting the optic nerve head, glaucomatous cupping, drusen and optic nerve edema as well as orbital tumors (Figure 12 and Table 1).
Example of an ultrasound image showing the correct placement of an Ahmed valve for treatment of glaucoma. The cornea is visible (a) with the tip of the valve in the anterior chamber (b) with the iris labeled (d). The main tubing (c) lies within the sclera (e).
Clinical uses of ocular ultrasonography | |
---|---|
Use | Clinical comment |
Intraocular foreign bodies (see Figure 9) |
|
Intraocular tumors (see Figure 3B) |
|
Vascular disease |
|
Ciliary body pathology (see Figure 5) |
|
Structural changes in glaucoma (see Figure 12) |
|
Evaluation of an opaque eye (see Figure 3A) |
|
Examples of clinical applications of ultrasonic evaluation in ophthalmology.
Ultrasound is an established diagnostic imaging modality. There are many systems which are relatively small with handheld probes. No ionizing radiation is used, but the image resolution can be limited compared to other visualization modalities. Advances have allowed high resolution imaging possible, especially of the anterior segment with the ability to create 3D reconstructions of ocular tissues and foreign bodies to aid in diagnosis and management of many disorders. Doppler flow can be an invaluable tool in the real time diagnosis of vasculopathies. However, 3D systems with rapid scan acquisition are not yet readily available.
The authors thank Global Retina Institute for providing funding and for Ellex, Inc for use of ultrasound images.
No author has a conflict of interest related to this work.
SONAR | sound navigation imaging |
UBM | ultrasonic backscatter microscopy |
LASIK | laser-assisted in-situ keratomileusis |
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Since no superfluid has exact zero viscosity, we analyze the consequences of SQS’s viscosity on light propagation, and we show that a static Universe could be possible, by solving a modified Navier-Stokes equation. Indeed, Hubble’s law may actually refer to tired light, though described as energy loss due to SQS’s nonzero viscosity instead of Compton scattering, bypassing known historical problems concerning tired light. We see that SQS’s viscosity may also account for the Pioneer anomaly. Our evaluation gives a magnitude of the anomalous acceleration aP = −HΛc = −8.785°10−10 ms−2. Here, HΛ is the Hubble parameter loaded by the cosmological constant Λ. 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The applications of this research cover many related fields, such as biotechnology and medicine, where, for example, Bioinformatics contributes to faster drug design, DNA analysis in forensics, and DNA sequence analysis in the field of personalized medicine. Personalized medicine is a type of medical care in which treatment is customized individually for each patient. Personalized medicine enables more effective therapy, reduces the costs of therapy and clinical trials, and also minimizes the risk of side effects. Nevertheless, advances in personalized medicine would not have been possible without bioinformatics, which can analyze the human genome and other vast amounts of biomedical data, especially in genetics. The rapid growth of information technology enabled the development of new tools to decode human genomes, large-scale studies of genetic variations and medical informatics. 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Recently, bioinspired systems have been successfully employing biomechanics to develop and improve assistive technology and rehabilitation devices. The research topic "Bioinspired Technology and Biomechanics" welcomes studies reporting recent advances in bioinspired technologies that contribute to individuals\' health, inclusion, and rehabilitation. 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