To analyze the age dependence of the longitudinal modulus of the crystalline lens in vivo using Brillouin scattering data in healthy subjects.
Brillouin scans were performed along the crystalline lens in 56 eyes from 30 healthy subjects aged from 19 to 63 years. Longitudinal elastic modulus was acquired along the sagittal axis of the lens with a transverse and axial resolution of 4 and 60 μm, respectively. The relative lens stiffness was computed, and correlations with age were analyzed.
Brillouin axial profiles revealed nonuniform longitudinal modulus within the lens, increasing from a softer periphery toward a stiffer central plateau at all ages. The longitudinal modulus at the central plateau showed no age dependence in a range of 19 to 45 years and a slight decrease with age from 45 to 63 years. A significant intersubject variability was observed in an age-matched analysis. Importantly, the extent of the central stiff plateau region increased steadily over age from 19 to 63 years. The slope of change in Brillouin modulus in the peripheral regions were nearly age-invariant.
The adult human lens showed no measurable age-related increase in the peak longitudinal modulus. The expansion of the stiff central region of the lens is likely to be the major contributing factor to age-related lens stiffening. Brillouin microscopy may be useful in characterizing the crystalline lens for the optimization of surgical or pharmacological treatments aimed at restoring accommodative power.
Brillouin imaging of turbid biological tissues requires an effective rejection of the background noise due to elastic scattering of probe laser light. We have developed a narrowband spectral notch filter based on a pair of a free-space Fabry-Perot etalon and a mirror. The etalon filter in a 4-pass configuration is able to suppress elastically-scattered laser light with a high extinction ratio of > 40 dB and transmit inelastically-scattered light in a frequency shift range of 2-14 GHz with only 2 dB insertion loss. We also describe a simple etalon that enables us to use semiconductor diode laser sources for Brillouin microscopy by removing spontaneous emission noise. Using a clinically-viable Brillouin microscope employing these filters, we demonstrate the first Brillouin confocal imaging of the sclera and conjunctiva of the porcine eye.
The goal of brain tumor surgery is to maximize tumor removal without injuring critical brain structures. Achieving this goal is challenging as it can be difficult to distinguish tumor from nontumor tissue. While standard histopathology provides information that could assist tumor delineation, it cannot be performed iteratively during surgery as freezing, sectioning, and staining of the tissue require too much time. Stimulated Raman scattering (SRS) microscopy is a powerful label-free chemical imaging technology that enables rapid mapping of lipids and proteins within a fresh specimen. This information can be rendered into pathology-like images. Although this approach has been used to assess the density of glioma cells in murine orthotopic xenografts models and human brain tumors, tissue heterogeneity in clinical brain tumors has not yet been fully evaluated with SRS imaging. Here we profile 41 specimens resected from 12 patients with a range of brain tumors. By evaluating large-scale stimulated Raman imaging data and correlating this data with current clinical gold standard of histopathology for 4,422 fields of view, we capture many essential diagnostic hallmarks for glioma classification. Notably, in fresh tumor samples, we observe additional features, not seen by conventional methods, including extensive lipid droplets within glioma cells, collagen deposition in gliosarcoma, and irregularity and disruption of myelinated fibers in areas infiltrated by oligodendroglioma cells. The data are freely available in a public resource to foster diagnostic training and to permit additional interrogation. Our work establishes the methodology and provides a significant collection of reference images for label-free neurosurgical pathology.
Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.
Biomedical optics is a broadly interdisciplinary field at the interface of optical engineering, biophysics, computer science, medicine, biology, and chemistry, helping us understand light-tissue interactions to create applications with diagnostic and therapeutic value in medicine. Implementation of biomedical optics tools and principles has had a notable scientific and clinical resurgence in recent years in the neurosurgical community. This is in great part due to work in fluorescence-guided surgery of brain tumors leading to reports of significant improvement in maximizing the rates of gross-total resection. Multiple additional optical technologies have been implemented clinically, including diffuse reflectance spectroscopy and imaging, optical coherence tomography, Raman spectroscopy and imaging, and advanced quantitative methods, including quantitative fluorescence and lifetime imaging. Here we present a clinically relevant and technologically informed overview and discussion of some of the major clinical implementations of optical technologies as intraoperative guidance tools in neurosurgery.
To test whether increased intereye retinal nerve fiber layer (RNFL) asymmetry may be indicative of glaucoma. To determine the best statistical methods and intereye RNFL cutoffs for differentiating between normal and glaucoma subjects to better alert clinicians to early glaucomatous damage.
Sixty-six primary open-angle glaucoma (OAG) and 40 age-matched normal subjects had both eyes imaged at the Massachusetts Eye and Ear Infirmary with a commercially available spectral-domain optical coherence tomography (OCT) machine. Statistical methodologies were used to find cutoffs that achieved the best sensitivities and specificities for differentiating OAG from normal subjects.
Intereye RNFL asymmetry for global average, all quadrants, and all sectors was significantly greater in OAG than normal subjects. Intereye RNFL asymmetry for global average showed the greatest statistical difference (P<0.001) between OAG (23.64 ± 14.90 μm) and normal eyes (3.58 ± 3.96 μm), with 6.60 times greater asymmetry in OAG eyes. The inferior quadrant showed the second greatest difference, with 3.91 times greater asymmetry in OAG eyes. Using a statistically determined cutoff of 6.0 μm as abnormal, intereye RNFL asymmetry for global average achieved a sensitivity of 74.24% and specificity of 90% in differentiating between normal and OAG subjects, achieving a better combination of sensitivity and specificity than intereye RNFL asymmetry of any quadrant or sector.
Intereye RNFL asymmetry may be a useful clinical OCT measurement to provide quantitative assessment of early glaucomatous damage. Newly developed algorithms for intereye RNFL asymmetry may improve the ability to detect glaucoma.
Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1 mW of input optical power, and they required two objective lenses to form a 90° scattering geometry necessary for polarization coupling by shear phonons. Here, we demonstrate a confocal Brillouin microscope capable of detecting both shear and longitudinal phonons with improved speeds and with a single objective lens. Brillouin scattering spectra were measured from polycarbonate, fused quartz, and borosilicate in 1-10 s at an optical power level of 10 mW. The elastic constants, phonon mean free path and the ratio of the Pockels coefficients were determined at microscopic resolution.
The mechanical stability of the cornea is critical for maintaining its normal shape and refractive function. Here, we report an observation of the mechanical resonance modes of the cornea excited by sound waves and detected by using phase-sensitive optical coherence tomography. The cornea in bovine eye globes exhibited three resonance modes in a frequency range of 50-400 Hz. The vibration amplitude of the fundamental mode at 80-120 Hz was ~8 µm at a sound pressure level of 100 dB (2 Pa). Vibrography allows the visualization of the radially symmetric profiles of the resonance modes. A dynamic finite-element analysis supports our observation.
Corneal mechanical strength is critical to withstanding intraocular pressure and maintaining normal shape. In keratoconus, the mechanical stability is compromised,which may lead to progressive morphological changes. Therefore, a noninvasive technique capable of accurately measuring the mechanical properties of the cornea may help us understand the mechanism of keratoconus development and improve detection and intervention in keratoconus. We previously developed Brillouin microscopy based on light scattering from inherent acoustic waves in tissuesand showed that this technique can provide quantitative estimates of local longitudinal modulus,which correlate to the Young and/or shear moduli of the cornea. Using a clinically viable instrument, for the first time, to our knowledge, we mapped the elastic modulus of normal and keratoconic corneas in vivo. We found distinctive biomechanical features that differentiate normal and keratoconic corneas and therefore have the potential to serve as diagnostic metrics for keratoconus.
To determine the diagnostic capability of spectral-domain optical coherence tomography (SD OCT) peripapillary retinal thickness (RT) measurements from 3-dimensional (3D) volume scans for primary open-angle glaucoma (POAG).
setting: Institutional. study population: 156 patients (89 POAG and 67 normal subjects). observation procedures: One eye of each subject was included. SD OCT peripapillary RT values from 3D volume scans were calculated for 4 quadrants of 3 different sized annuli. Peripapillary retinal nerve fiber layer (RNFL) thickness values were also determined. main outcome measures: Area under the receiver operating characteristic curve (AUROC) values, sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios.
The top 5 RT AUROCs for all glaucoma patients and for a subset of early glaucoma patients were for the inferior quadrant of outer circumpapillary annulus of circular grid (OCA) 1 (0.959, 0.939), inferior quadrant of OCA2 (0.945, 0.921), superior quadrant of OCA1 (0.890, 0.811), inferior quadrant of OCA3 (0.887, 0.854), and superior quadrant of OCA2 (0.879, 0.807). Smaller RT annuli OCA1 and OCA2 consistently showed better diagnostic performance than the larger RT annulus OCA3. For both RNFL and RT measurements, best AUROC values were found for inferior RT OCA1 and OCA2, followed by inferior and overall RNFL thickness.
Peripapillary RT measurements from 3D volume scans showed excellent diagnostic performance for detecting both glaucoma and early glaucoma patients. Peripapillary RT values have the same or better diagnostic capability compared to peripapillary RNFL thickness measurements, while also having fewer algorithm errors.
Although substantial evidence has established that microglia and astrocytes play a key role in the establishment and maintenance of persistent pain in animal models, the role of glial cells in human pain disorders remains unknown. Here, using the novel technology of integrated positron emission tomography-magnetic resonance imaging and the recently developed radioligand (11)C-PBR28, we show increased brain levels of the translocator protein (TSPO), a marker of glial activation, in patients with chronic low back pain. As the Ala147Thr polymorphism in the TSPO gene affects binding affinity for (11)C-PBR28, nine patient-control pairs were identified from a larger sample of subjects screened and genotyped, and compared in a matched-pairs design, in which each patient was matched to a TSPO polymorphism-, age- and sex-matched control subject (seven Ala/Ala and two Ala/Thr, five males and four females in each group; median age difference: 1 year; age range: 29-63 for patients and 28-65 for controls). Standardized uptake values normalized to whole brain were significantly higher in patients than controls in multiple brain regions, including thalamus and the putative somatosensory representations of the lumbar spine and leg. The thalamic levels of TSPO were negatively correlated with clinical pain and circulating levels of the proinflammatory citokine interleukin-6, suggesting that TSPO expression exerts pain-protective/anti-inflammatory effects in humans, as predicted by animal studies. Given the putative role of activated glia in the establishment and or maintenance of persistent pain, the present findings offer clinical implications that may serve to guide future studies of the pathophysiology and management of a variety of persistent pain conditions.
Biomechanics – the study of the relationship between forces and function in living organisms – is thought to play a critical role in a significant number of ophthalmic disorders. This is not surprising, as the eye is a pressure vessel that requires a delicate balance of forces to maintain its homeostasis. Over the past few decades, basic science research in ophthalmology mostly confirmed that ocular biomechanics could explain in part the mechanisms involved in almost all major ophthalmic disorders such as optic nerve head neuropathies, angle closure, ametropia, presbyopia, cataract, corneal pathologies, retinal detachment, and macular degeneration. Translational biomechanics in ophthalmology, however, is still in its infancy. It is believed that its use could make significant advances in diagnosis and treatment. Several translational biomechanics strategies are already emerging, such as corneal stiffening for the treatment of keratoconus, and more are likely to follow. This review aims to cultivate the idea that biomechanics plays a major role in ophthalmology and that its clinical translation, lead by collaborative teams of clinicians and biomedical engineers, will benefit our patients. Specifically, recent advances and future prospects in corneal, iris, trabecular meshwork, crystalline lens, scleral and lamina cribrosa biomechanics are discussed.
Most techniques measuring corneal biomechanics in vivo are biased by side factors. We demonstrate the ability of optical coherence tomographic (OCT) vibrography to determine corneal material parameters, while reducing current prevalent restrictions of other techniques (such as intraocular pressure (IOP) and thickness dependency). Modal analysis was performed in a finite-element (FE) model to study the oscillation response in isolated thin corneal flaps/eye globes and to analyse the dependency of the frequency response function on: corneal elasticity, viscoelasticity, geometry (thickness and curvature), IOP and density. The model was verified experimentally in flaps from three bovine corneas and in two enucleated porcine eyes using sound excitation (100-110 dB) together with a phase-sensitive OCT to measure the frequency response function (range 50-510 Hz). Simulations showed that corneal vibration in flaps is sensitive to both, geometrical and biomechanical parameters, whereas in whole globes it is primarily sensitive to corneal biomechanical parameters only. Calculations based on the natural frequency shift revealed that flaps of the posterior cornea were 0.8 times less stiff than flaps from the anterior cornea and cross-linked corneas were 1.6 times stiffer than virgin corneas. Sensitivity analysis showed that natural vibration frequencies of whole globes were nearly independent from corneal thickness and IOP within the physiological range. OCT vibrography is a promising non-invasive technique to measure corneal elasticity without biases from corneal thickness and IOP.
Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo.
We obtained eight tissue samples from healthy donor corneas used in Descemet's stripping endothelial keratoplasty (DSEK) and 10 advanced keratoconic corneas from patients undergoing deep anterior lamellar keratoplasty (DALK). Within 2 hours after surgery, a confocal Brillouin microscope using a monochromatic laser at 532 nm was used to map the Brillouin frequency shifts of the corneas.
The mean Brillouin shift in the anterior 200 μm of the keratoconic corneas at the cone was measured to be 7.99 ± 0.10 GHz, significantly lower than 8.17 ± 0.06 GHz of the healthy corneas (P < 0.001). The Brillouin shift in the keratoconic corneas decreased with depth from the anterior toward posterior regions with a steeper slope than in the healthy corneas (P < 0.001). Within keratoconic corneas, the Brillouin shift in regions away from the apex of the cone was significantly higher than within the cone region (P < 0.001).
Brillouin measurements revealed notable differences between healthy and keratoconic corneas. Importantly, Brillouin imaging showed that the mechanical loss is primarily concentrated within the area of the keratoconic cone. Outside the cone, the Brillouin shift was comparable with that of healthy corneas. The results demonstrate the potential of Brillouin microscopy for diagnosis and treatment monitoring of keratoconus.
To determine the retinal nerve fiber layer (RNFL) thickness at which visual field (VF) damage becomes detectable and associated with structural loss.
Retrospective cross-sectional study.
Eighty-seven healthy and 108 glaucoma subjects (1 eye per subject) were recruited from an academic institution. All patients had VF examinations (Swedish Interactive Threshold Algorithm 24-2 test of the Humphrey Visual Field Analyzer 750i) and spectral-domain optical coherence tomography RNFL scans. Comparison of RNFL thickness values with VF threshold values showed a plateau of VF threshold values at high RNFL thickness values and then a sharp decrease at lower RNFL thickness values. A broken stick statistical analysis was used to estimate the tipping point at which RNFL thickness values are associated with VF defects. The slope for the association between structure and function was computed for data above and below the tipping point.
The mean RNFL thickness value that was associated with initial VF loss was 89 μm. The superior RNFL thickness value that was associated with initial corresponding inferior VF loss was 100 μm. The inferior RNFL thickness value that was associated with initial corresponding superior VF loss was 73 μm. The differences between all the slopes above and below the aforementioned tipping points were statistically significant (P < .001).
In open-angle glaucoma, substantial RNFL thinning or structural loss appears to be necessary before functional visual field defects become detectable.
We report the design, characterization, and validation of an optimized simultaneous color and near-infrared (NIR) fluorescence rigid endoscopic imaging system for minimally invasive surgery. This system is optimized for illumination and collection of NIR wavelengths allowing the simultaneous acquisition of both color and NIR fluorescence at frame rates higher than 6.8 fps with high sensitivity. The system employs a custom 10-mm diameter rigid endoscope optimized for NIR transmission. A dual-channel light source compatible with the constraints of an endoscope was built and includes a plasma source for white light illumination and NIR laser diodes for fluorescence excitation. A prism-based 2-CCD camera was customized for simultaneous color and NIR detection with a highly efficient filtration scheme for fluorescence imaging of both 700- and 800-nm emission dyes. The performance characterization studies indicate that the endoscope can efficiently detect fluorescence signal from both indocyanine green and methylene blue in dimethyl sulfoxide at the concentrations of 100 to 185 nM depending on the background optical properties. Finally, we performed the validation of this imaging system in vivo during a minimally invasive procedure for thoracic sentinel lymph node mapping in a porcine model.
Efficient transfer of sound by the middle ear ossicles is essential for hearing. Various pathologies can impede the transmission of sound and thereby cause conductive hearing loss. Differential diagnosis of ossicular disorders can be challenging since the ossicles are normally hidden behind the tympanic membrane (TM). Here we describe the use of a technique termed optical coherence tomography (OCT) vibrography to view the sound-induced motion of the TM and ossicles simultaneously. With this method, we were able to capture three-dimensional motion of the intact TM and ossicles of the chinchilla ear with nanometer-scale sensitivity at sound frequencies from 0.5 to 5 kHz. The vibration patterns of the TM were complex and highly frequency dependent with mean amplitudes of 70-120 nm at 100 dB sound pressure level. The TM motion was only marginally sensitive to stapes fixation and incus-stapes joint interruption; however, when additional information derived from the simultaneous measurement of ossicular motion was added, it was possible to clearly distinguish these different simulated pathologies. The technique may be applicable to clinical diagnosis in Otology and to basic research in audition and acoustics.
Purpose: To determine the effects of age, sex, and race on the retinal nerve fiber layer (RNFL) in the normal human eye as measured by the spectral domain optical coherence tomography (SD-OCT) Spectralis machine (Heidelberg Engineering).
Methods: Peripapillary SD-OCT RNFL thickness measurements were determined in normal subjects seen at a university-based clinic. One randomly selected eye per subject was used for analysis in this cross-sectional study. Multiple regression analysis was applied to assess the effects of age, sex, ethnicity, and mean refractive error on peripapillary RNFL thickness. Results are expressed as means±SD wherever applicable.
Results: The study population consisted of 190 healthy participants from 9 to 86 years of age. Of the 190 participants, 62 (33%) were men, 125 (66%) Caucasians, 26 (14%) African Americans, 14 (7%) Hispanics, 16 (8%) Asians, and 9 (5%) other races. The mean RNFL thickness for the normal population studied was 97.3±9.6 µm. Normal RNFL thickness values follow the ISNT rule with decreasing RNFL thickness values starting from the thickest quadrant inferiorly to the thinnest quadrant temporally: inferior quadrant (126±15.8), superior quadrant (117.2±16.13), nasal quadrant (75±13.9), and temporal quadrant (70.6±10.8 µm). Thinner RNFL measurements were associated with older age (P<0.001); being Caucasian, versus being either Hispanic or Asian (P=0.02 and 0.009, respectively); or being more myopic (P<0.001). For every decade of increased age, mean RNFL thickness measured thinner by approximately 1.5 µm (95% confidence interval, 0.24-0.07). Comparisons between ethnic groups revealed that Caucasians had mean RNFL values (96±9.2 µm) slightly thinner than those of Hispanics (102.9±11 µm; P=0.02) or Asians (100.7±8.5 µm; P=0.009). African Americans RNFL values (99.2±10.2 µm) were not significantly different when compared with Caucasians. There was no relationship between RNFL thickness and sex.
Conclusions: The thickest RNFL measurements were found in the inferior quadrant, followed by the superior, nasal, and temporal quadrants (ISNT rule applied to the RNFL). Thinner RNFL measurements were associated with older age and increasing myopia. Caucasians tend to have thinner RNFL values when compared with Hispanics and Asians. SD-OCT analysis of the normal RNFL showed results similar to time domain OCT studies.
Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.
We demonstrate sub-Rayleigh limit imaging of an object via speckle illumination. Imaging beyond the conventional Rayleigh limit is achieved by illuminating the object with pseudothermal light that exhibits a random speckle pattern. An object image is reconstructed from the second-order correlation measurement and the resolution of the image, which exceeds the Rayleigh limit, is shown to be related to the size of the speckle pattern that is tied to the lateral coherence length of the pseudothermal light.