In the field of in vitro fertilisation (IVF), there is a need to understand how cell properties of the cumulus oocyte complex may be used to predict successful pregnancy and live birth rates post-IVF. Here we used optical tweezers for the first time to measure the viscosity of the cumulus cell matrix surrounding the oocyte (egg). This study aimed to determine whether the viscosity of the cumulus cell matrix – prior to fertilisation – is reflective of subsequent embryo developmental potential and indicative of pregnancy success.
Measurements were performed using a 1µm diameter silica probe particle trapped by a focused 1064nm laser. We benchmarked the accuracy of the system by measuring the viscosity of glycerol with varying mass fractions. Viscosity measurements of the cumulus cell matrix were performed in isolation from both the cumulus cells and the oocyte. This showed that the viscosity of cumulus matrix was significantly higher when sampled from oocytes with a higher developmental potential (in vivo matured) compared to those of lower quality (in vitro matured).
Medical imaging is advancing rapidly through the development of novel laser sources and non-linear imaging methodologies. These developments are boosting deep tissue imaging allowing researchers to study diseases deep in the body enabling early diagnosis and better treatment. To help with the testing and optimization of these imaging systems and to aid in this process of deep tissue imaging, it's important to have robust, stable and reproducible standards and phantoms. Herein we present the design and fabrication of robust, multi-layered, hydrogel-based standards. The hydrogel used is a double network hydrogel consisting of two interpenetrating networks agarose and polyacrylamide. Thin layers of tough double network hydrogels are stacked to form multilayered depth standards having modality specific signaling markers embedded in between. Standard design and assembly ensured long term stability and easy transport. These proved useful in-depth imaging studies, utilizing multiple imaging modalities, including one photon fluorescence (1PEF), two photon fluorescence (2PEF), coherent anti-Stokes Raman imaging (CARS) and second harmonic generation imaging (SHG).
SignificanceSkin color affects light penetration leading to differences in its absorption and scattering properties. COVID-19 highlighted the importance of understanding of the interaction of light with different skin types, e.g., pulse oximetry (PO) unreliably determined oxygen saturation levels in people from Black and ethnic minority backgrounds. Furthermore, with increased use of other medical wearables using light to provide disease information and photodynamic therapies to treat skin cancers, a thorough understanding of the effect skin color has on light is important for reducing healthcare disparities.AimThe aim of this work is to perform a thorough review on the effect of skin color on optical properties and the implication of variation on optical medical technologies.ApproachPublished in vivo optical coefficients associated with different skin colors were collated and their effects on optical penetration depth and transport mean free path (TMFP) assessed.ResultsVariation among reported values is significant. We show that absorption coefficients for dark skin are ∼6% to 74% greater than for light skin in the 400 to 1000 nm spectrum. Beyond 600 nm, the TMFP for light skin is greater than for dark skin. Maximum transmission for all skin types was beyond 940 nm in this spectrum. There are significant losses of light with increasing skin depth; in this spectrum, depending upon Fitzpatrick skin type (FST), on average 14% to 18% of light is lost by a depth of 0.1 mm compared with 90% to 97% of the remaining light being lost by a depth of 1.93 mm.ConclusionsCurrent published data suggest that at wavelengths beyond 940 nm light transmission is greatest for all FSTs. Data beyond 1000 nm are minimal and further study is required. It is possible that the amount of light transmitted through skin for all skin colors will converge with increasing wavelength enabling optical medical technologies to become independent of skin color.
SignificanceRapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques.AimWe aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth.ApproachStandards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers.ResultsThese standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and 1240 μm, respectively. Once fabricated, the phantoms were found to be stable for many months.ConclusionsThe ability to image at depth, the phantom’s robustness and flexible layered structure, and the ready incorporation of “optical markers” make these ideal depth standards for the validation of a variety of imaging modalities.
We present a bespoke new instrument for micro-mechanical sensing and imaging of 3D cell cultures. Our instrument combines optical tweezers and multiplane imaging for 3D optical microrheology, with light sheet microscopy for 3D fluorescence imaging. This integrated system allows us to visualise cells in their 3D environment while also mapping the physical properties of the extracellular matrix local to the cells. Such a holistic approach will allow for a wide range of fundamental questions in the life sciences to be addressed by effectively sensing the micro-world from a cell’s perspective.
This talk will introduce a novel, recently developed, instrument that combines light sheet microscopy, optical trapping and multiplane imaging in a single platform capable of imaging 3D cultures of live cell over long time courses and mapping to these images the local mechanical properties of the material surrounding the cells. The presentation will highlight the wide-range of samples this instrument can study and discuss the analysis involved to evaluate the rheological properties of the material when either the trapping potential or the samples are non-isotropic.
We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluorescence or second harmonic signals as merit factors. We present here several strategies to overcome photobleaching issues associated with lengthy optimization routines by adapting the search algorithm and the experimental methodology. Optimizations were performed on extrinsic fluorescent dyes, fluorescent beads loaded into organotypic tissue cultures and the intrinsic second harmonic signal of these cultures. We validate the approach of using these preoptimized mirror shapes to compile a robust look-up table that can be applied for imaging over several days and through a variety of tissues. In this way, the photon exposure to the fluorescent cells under investigation is limited to imaging. Using our look-up table approach, we show signal intensity improvement factors ranging from 1.7 to 4.1 in organotypic tissue cultures and freshly excised mouse tissue. Imaging zebrafish in vivo, we demonstrate signal improvement by a factor of 2. This methodology is easily reproducible and could be applied to many photon starved experiments, for example fluorescent life time imaging, or when photobleaching is a concern.
Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy
laboratories and facilities. The force applied by the laser beam on the trapped object can be accurately determined
allowing any external forces acting on the trapped object to be deduced. We aim to design a series of experiments that
use an optical trap to measure and quantify the interaction force between immune cells. In order to cause minimum
perturbation to the sample we plan to directly trap T cells and remove the need to introduce exogenous beads to the
sample. This poses a series of challenges and raises questions that need to be answered in order to design a set of effect
end-point experiments. A typical cell is large compared to the beads normally trapped and highly non-uniform – can we
reliably trap such objects and prevent them from rolling and re-orientating? In this paper we show how a spatial light
modulator can produce a triple-spot trap, as opposed to a single-spot trap, giving complete control over the object’s
orientation and preventing it from rolling due, for example, to Brownian motion. To use an optical trap as a force
transducer to measure an external force you must first have a reliably calibrated system. The optical trapping force is
typically measured using either the theory of equipartition and observing the Brownian motion of the trapped object or
using an escape force method, e.g. the viscous drag force method. In this paper we examine the relationship between
force and displacement, as well as measuring the maximum displacement from equilibrium position before an object
falls out of the trap, hence determining the conditions under which the different calibration methods should be applied.
Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three
dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system’s optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.
We present a viability study of optically trapped live T cell hybridomas. T cells form an important part of the
adaptive immune response system which is responsible for fighting particular pathogens or diseases. The cells of
interest were directly trapped by a laser operating at a wavelength of 1064 nm and their viability measured as a
function of time. Cell death was monitored using an inverted fluorescent microscope to observe the uptake by the
cell of the fluorescent dye propidium iodide. Studies were undertaken at various laser powers and beam profiles.
There is a growing interest in optically trapping immune cells and this is the first study that investigates the
viability of a T cell when trapped using a conventional optical trapping system. In such experiments it is crucial
that the T cell remains viable and trapping the cell directly means that any artefacts due to a cell-bead interface
are removed. Our motivation behind this experiment is to use optical tweezers to gain a greater understanding
of the interaction forces between T cells and antigen presenting cells. Measuring these interactions has become
important due to recent theories which indicate that the strength of this interaction may underlie the activation
of the T-cell and subsequent immune response.
The interactions between T cells and antigen-presenting cells (APCs) are crucial in triggering a successful antigenspecific,
adaptive immune response leading to protection against a particular pathogen or disease. At present very little is
known about the magnitudes of the forces involved in these interactions. We present results showing for the first time
that optical tweezers can be used to measure these cell-cell interaction forces. We were able to see a significant
difference in the force distributions taken with and without antigen, as evidenced by a Mann-Whitney U-test. The T cells
of interest were trapped directly and no exogenous beads were added to the sample. Interaction forces between T cells
and APCs in the presence of specific antigen ranged from 0-6.5 pN, whereas, when the specific antigen was absent the
interaction forces ranged from 0-1.5 pN. The accuracy of the system will be discussed in terms of how we tracked the
position of the optically trapped cell and the methods we used to minimise cell roll.
We present results demonstrating for the first time that an optically trapped bead can be used as a local probe to measure
the variation in the viscoelastic properties of the vitreous humor of a rabbit eye. The Brownian motion of the optically
trapped bead was monitored on a fast CCD camera on the millisecond timescale. Analysis of the bead trajectory provides
local information about the viscoelastic properties of the medium surrounding the particle. Previous, bulk, methods for
measuring the viscoelastic properties of the vitreous destroy the sample and allow only a single averaged measurement to
be taken per eye. Whereas, with our approach, we were able to observe local behaviour typical of non-Newtonian and
gel-like materials, along with the homogenous and in-homogeneous nature of different regions of the dissected vitreous
humor. The motivation behind these measurements is to gain a better understanding of the structure of the vitreous
humor in order to design effective drug delivery techniques. In particular, we are interested in methods for delivering
drug to the retina of the eye in order to treat sight threatening diseases such as age related macular degeneration.
We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the
image brightness and quality at increased optical penetration depths in biological material. The principle of the
technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect
optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in
multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free
biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3rd order non-linear optical
process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in
tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a
deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit
determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of
implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant
increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the
penetration depth beyond 200 μm.
The Brownian dynamics of an optically trapped water droplet is investigated across the transition from over to
under-damped oscillations. The spectrum of position fluctuations evolves from a Lorentzian shape typical of overdamped
systems (beads in liquid solvents), to a damped harmonic oscillator spectrum showing a resonance peak.
In this later under-damped regime, we excite parametric resonance by periodically modulating the trapping
power at twice the resonant frequency. We also derive from Langevin dynamics an explicit numerical recipe
for the fast computation of the power spectra of a Brownian parametric oscillator. The obtained numerical
predictions are in excellent agreement with the experimental data.
In the 1970s, Jones demonstrated a photon drag by showing that the translation of a window caused a slight displacement
of a transmitted light beam. Similarly he showed that a spinning medium slightly rotated the polarization state. Rather
than translating the medium, the speed of which is limited by mechanical considerations, we translate the image and
measure its lateral delay with respect to a similar image that has not passed through the window. The equivalence, or
lack of it, of the two frames is subtle and great care needs to be taken in determining whether or not similar results are to
be obtained.
Sample induced optical aberrations in slices of rat brain tissue have been corrected with a deformable membrane mirror.
The aberration correction required by the DMM was determined using a genetic algorithm with the intensity at a point in
the sample as a fitness value. We show that by optimising on the intensity of a single point in the sample we are able to
improve the axial resolution across the whole field of view of the image at a fixed sample depth. The ratio between the
corrected axial resolution and the diffraction limited resolution is on average 2.7 for a 50 μm thick rat brain tissue sample
and 12 for a 380 μm thick sample across the whole field of view. The uncorrected ratio being 4.1 and 15.5 respectively.
Using a single aberration correction per depth, compared to a point-by-point aberration correction, will significantly
decrease scan times and therefore reduce photobleaching and phototoxic effects enabling more rapid microscopy with
active aberration correction.
Confocal techniques allow the user to achieve optically sectioned images with significantly enhanced axial and improved lateral resolution compared to widefield methods. Unfortunately, as one images more deeply within a sample, sample induced aberrations lead to a significant reduction in image resolution and contrast. Using adaptive optic techniques, we report on the effectiveness of a number of algorithms for removing sample induced aberrations. The viability and efficiency at a number of fitness parameters used in the optimisation routines is also considered.
We present a method for aberration correction in a confocal microscope that successfully combines both a spatial light modulator and a deformable membrane mirror. An active locking technique is used that benefits from the fast update rate of the deformable membrane mirror and the large effective stroke of the spatial light modulator. Concentrating on defocus, we were able to track 'best focus' over a distance of 80 μm with a lock RMS precision of 57 nm. In principle, this technique can be applied to any Zernike mode or aberration that can be accurately reproduced on the deformable membrane mirror.
Many applications of laser tweezers rely on the accurate measurement of the transverse or axial trapping force. We have concentrated on the transverse trapping force and the most common method used to measure it, applying a viscous drag force. A trapped sphere was subjected to a viscous drag force via a Stokesian flow. The flow was achieved by oscillating the sample stage at a constant speed of 750 microns/second. A Zeiss oil-immersion (N.A. equals 1.3) objective was used to focus a 1064 nm Nd:YVO4 laser beam in order to trap 6 microns diameter polystyrene spheres suspended in distilled water. The minimum power needed to hold the particle in the trap at a particular viscous drag force was then measured. The influence of trap depth, oscillation amplitude used and particle concentration have been investigated, in particular the effects caused by the characteristics of the function used to create the oscillation. The minimum laser power needed to trap a sphere was found to increase with a rise in oscillation amplitude. The velocity profile through the fluid, the rotation of the trapped particle and the effect of interactions with other particles is considered when explaining these effects.
The transverse force of an optical trap is usually measured by equating the trapping force to the viscous drag force applied to the trapped particle according to Stokes' Law. Under normal conditions, the viscous drag force on a trapped particle is proportional to the fluid velocity of the medium. In this paper we show that an increase of particle concentration within the medium affects force measurements. In order to trap the particle, 1064 nm light from a Nd:YVO4 laser was brought to a focus in a sample slide, of thickness around 380 microns, by using an inverted Zeiss microscope objective, with NA equals 1.3. The slide was filled with distilled water containing 6 micron diameter polystyrene spheres. Measurements were taken at a fluid velocity of 0.75 microns/sec, achieved by moving the sample stage with a piezo-electric transducer whilst a particle was held stationary in the trap. The laser power required to hold a sphere at different trap depths for various concentrations was measured. Significant weakening of the trap was found for concentrations >0.03% solids by weight, becoming weaker for higher trap depths. These results are explained in terms of aberrations, particle-particle interactions and distortion of the beam due to particle-light interactions.
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