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Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22μV/Pa and its noise equivalent pressure (NEP) of 11.4Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

Electric-field induced physical phenomena, such as thermal, mechanical and electrochemical dynamics, may be the driving mechanism behind bioeffects observed in mammalian cells during exposure to nanosecond-duration electric pulses (nsEP) in-vitro. Correlating a driving mechanism to a biological response requires the experimental measurement and quantification of all physical dynamics resulting from the nsEP stimulus. A passive and electromagnetic interference (EMI) immune sensor is required to resolve these dynamics in high strength electric fields. The probe beam deflection technique (PBDT) is a passive and EMI immune optical method for quantifying and imaging refractive index gradients in liquids and gases, both dynamic and static, with nanosecond temporal resolution. In this work, a probe beam deflection imaging system was designed to acquire 2-D time-lapse images of thermal/mechanical dynamics resulting from monopolar and bipolar nsEP stimulus.

Fluorescent lifetime imaging is an optical technique that facilitates imaging molecular interactions and cellular functions. Because the excited lifetime of a fluorophore is sensitive to its local microenvironment,1, 2 measurement of fluorescent lifetimes can be used to accurately detect regional changes in temperature, pH, and ion concentration. However, typical state of the art fluorescent lifetime methods are severely limited when it comes to acquisition time (on the order of seconds to minutes) and video rate imaging. Here we show that compressed ultrafast photography (CUP) can be used in conjunction with fluorescent lifetime imaging to overcome these acquisition rate limitations. Frame rates up to one hundred billion frames per second have been demonstrated with compressed ultrafast photography using a streak camera.3 These rates are achieved by encoding time in the spatial direction with a pseudo-random binary pattern. The time domain information is then reconstructed using a compressed sensing algorithm, resulting in a cube of data (x,y,t) for each readout image. Thus, application of compressed ultrafast photography will allow us to acquire an entire fluorescent lifetime image with a single laser pulse. Using a streak camera with a high-speed CMOS camera, acquisition rates of 100 frames per second can be achieved, which will significantly enhance our ability to quantitatively measure complex biological events with high spatial and temporal resolution. In particular, we will demonstrate the ability of this technique to do single-shot fluorescent lifetime imaging of cells and microspheres.

Short infrared laser pulses have many physiological effects on cells including the ability to stimulate action potentials in neurons. Here we show that short infrared laser pulses can also reversibly block action potentials. Primary rat hippocampal neurons were transfected with the Optopatch2 plasmid, which contains both a blue-light activated channel rhodopsin (CheRiff) and a red-light fluorescent membrane voltage reporter (QuasAr2). This optogenetic platform allows robust stimulation and recording of action potential activity in neurons in a non-contact, low noise manner. For all experiments, QuasAr2 was imaged continuously on a wide-field fluorescent microscope using a Krypton laser (647 nm) as the excitation source and an EMCCD camera operating at 1000 Hz to collect emitted fluorescence. A co-aligned Argon laser (488 nm, 5 ms at 10Hz) provided activation light for CheRiff. A 200 mm fiber delivered infrared light locally to the target neuron. Reversible action potential block in neurons was observed following a short infrared laser pulse (0.26-0.96 J/cm2; 1.37-5.01 ms; 1869 nm), with the block persisting for more than 1 s with exposures greater than 0.69 J/cm2. Action potential block was sustained for 30 s with the short infrared laser pulsed at 1-7 Hz. Full recovery of neuronal activity was observed 5-30s post-infrared exposure. These results indicate that optogenetics provides a robust platform for the study of action potential block and that short infrared laser pulses can be used for non-contact, reversible action potential block.

Remote chemically specific detection of trace impurities in the atmosphere from distances on the order of kilometers is an important problem from both an environmental and a national defense viewpoint. A new scheme is discussed consisting of the remote generation of a backward propagating stimulated Raman pulse. This pulse is then used to drive a coherent anti-Stokes Raman scattering scheme, resulting in a strong chemically specific signal propagating back to the detector.

We recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) to identify membrane disruption events in live cells. This technique was used to detect nanoporation in the plasma membrane following nanosecond pulsed electric field (nsPEF) exposure. It has been hypothesized that similar poration events could be induced by the thermal gradients generated by infrared (IR) laser energy. Optical pulses are a highly desirable stimulus for the nervous system, as they are capable of inhibiting and producing action potentials in a highly localized but non-contact fashion. However, the underlying mechanisms involved with infrared neural stimulation (INS) are not well understood. The ability of our method to non-invasively measure membrane structure and transmembrane potential via Two Photon Fluorescence (TPF) make it uniquely suited to neurological research. In this work, we leverage our technique to understand what role membrane structure plays during INS and contrast it with nsPEF stimulation. We begin by examining the effect of IR pulses on CHO-K1 cells before progressing to primary hippocampal neurons. The use of these two cell lines allows us to directly compare poration as a result of IR pulses to nsPEF exposure in both a neuron-derived cell line, and one likely lacking native channels sensitive to thermal stimuli.

Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a “threshold” point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (

Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

Recently, infrared light has been shown to both stimulate and inhibit excitatory cells. However, studies of infrared light for excitatory cell inhibition have been constrained by the use of invasive and cumbersome electrodes for cell excitation and action potential recording. Here, we present an all optical experimental design for neuronal excitation, inhibition, and action potential detection. Primary rat neurons were transfected with plasmids containing the light sensitive ion channel CheRiff. CheRiff has a peak excitation around 450 nm, allowing excitation of transfected neurons with pulsed blue light. Additionally, primary neurons were transfected with QuasAr2, a fast and sensitive fluorescent voltage indicator. QuasAr2 is excited with yellow or red light and therefore does not spectrally overlap CheRiff, enabling imaging and action potential activation, simultaneously. Using an optic fiber, neurons were exposed to blue light sequentially to generate controlled action potentials. A second optic fiber delivered a single pulse of 1869nm light to the neuron causing inhibition of the evoked action potentials (by the blue light). When used in concert, these optical techniques enable electrode free neuron excitation, inhibition, and action potential recording, allowing research into neuronal behaviors with high spatial fidelity.

The underlying mechanism(s) responsible for nanoporation of phospholipid membranes by nanosecond pulsed electric fields (nsEP) remains unknown. The passage of a high electric field through a conductive medium creates two primary contributing factors that may induce poration: the electric field interaction at the membrane and the shockwave produced from electrostriction of a polar submersion medium exposed to an electric field. Previous work has focused on the electric field interaction at the cell membrane, through such models as the transport lattice method. Our objective is to model the shock wave cell membrane interaction induced from the density perturbation formed at the rising edge of a high voltage pulse in a polar liquid resulting in a shock wave propagating away from the electrode toward the cell membrane. Utilizing previous data from cell membrane mechanical parameters, and nsEP generated shockwave parameters, an acoustic shock wave model based on the Helmholtz equation for sound pressure was developed and coupled to a cell membrane model with finite-element modeling in COMSOL. The acoustic structure interaction model was developed to illustrate the harmonic membrane displacements and stresses resulting from shockwave and membrane interaction based on Hooke’s law. Poration is predicted by utilizing membrane mechanical breakdown parameters including cortical stress limits and hydrostatic pressure gradients.

Nanosecond pulsed electric fields (nsPEF) cause the formation of small pores, termed nanopores, in the membrane of cells. Current nanoporation models treat nsPEF exposure as a purely electromagnetic phenomenon, but recent publications showing pressure transients, ROS production, temperature gradients, and pH waves suggest the stimulus may be physically and chemically multifactorial causing elicitation of diverse biological conditions and stressors. Our research group's goal is to quantify the breadth and participation of these stressors generated during nsPEF exposure and determine their relative importance to the observed cellular response. In this paper, we used advanced imaging techniques to identify a possible source of nsPEF-induced acoustic shock waves. nsPEFs were delivered in an aqueous media via a pair of 125 μm tungsten electrodes separated by 100 μm, mirroring our previously published cellular exposure experiments. To visualize any pressure transients emanating from the electrodes or surrounding medium, we used the Schlieren imaging technique. Resulting images and measurements confirmed that mechanical pressure waves and electrode-based stresses are formed during nsPEF, resulting in a clearer understanding of the whole exposure dosimetry. This information will be used to better quantify the impact of nsPEF-induced acoustic shock waves on cells, and has provided further evidence of non-electrical-field induced exposures for elicitation of bioieffects.

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm−1. Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium., Unlike conventional lasers that require a uniform resonant cavity to operate, random lasers use a highly disordered gain medium in which scattering is dominant. Hokr et al. report Raman lasing from a bulk three-dimensional disordered medium whose intensity exceeds that of other random lasers by many orders of magnitude.

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.

The time-temperature effects of laser radiation exposure are investigated as a function of wavelength. Here, we report the thermal response of bulk tissue as a function of wavelength from 700 to 1064 nm. Additionally, Monte Carlo simulations were used to verify the thermal response measured and predict damage thresholds based on the response.

Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to or ientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di-4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.

Despite 30 years of research, the mechanism behind the induced breakdown of plasma membranes by electrical pulses, termed electroporation, remains unknown. Current theories treat the interaction between the electrical field and the membrane as an entirely electrical event pointing to multiple plausible mechanisms. By investigating the biophysical interaction between plasma membranes and nanosecond electrical pulses (nsEP), we may have identified a non-electric field driven mechanism, previously unstudied in nsEP, which could be responsible for nanoporation of plasma membranes. In this investigation, we use a non-contact optical technique, termed probe beam deflection technique (PBDT), to characterize acoustic shockwaves generated by nsEP traveling through tungsten wire electrodes. We conclude these acoustic shockwaves are the result of the nsEP exposure imparting electrohydraulic forces on the buffer solution. When these acoustic shockwaves occur in close proximity to lipid bilayer membranes, it is possible that they impart a sufficient amount of mechanical stress to cause poration of that membrane. This research establishes for the first time that nsEP discharged in an aqueous medium generate measureable pressure waves of a magnitude capable of mechanical deformation and possibly damage to plasma membranes. These findings provide a new insight into the longunanswered question of how electric fields cause the breakdown of plasma membranes.

The study of living cells may require advanced imaging techniques to track weak and rapidly changing signals. Fundamental to this need is the recent advancement in camera technology. Two camera types, specifically sCMOS and EM-CCD, promise both high signal-to-noise and high speed (>100 fps), leaving researchers with a critical decision when determining the best technology for their application. In this article, we compare two cameras using a live-cell imaging test case in which small changes in cellular fluorescence must be rapidly detected with high spatial resolution. The EM-CCD maintained an advantage of being able to acquire discernible images with a lower number of photons due to its EM-enhancement. However, if high-resolution images at speeds approaching or exceeding 1000 fps are desired, the flexibility of the full-frame imaging capabilities of sCMOS is superior.

The optical damaging potential of near-infrared radiation was investigated as a function of wavelength. Higher incident powers can be afforded by using longer wavelength excitation leading to deeper imaging depths.

Second Harmonic Generation (SHG) microscopy is an effective tool for studying the order of symmetry-breaking interfaces, such as lipid bilayers. Recently, we have shown that disruptions in the interfacial nature of the membrane can be studied with the addition of lipophilic SHG probes, specifically Di-4-ANEPPDHQ (Di-4). In addition to the conformational information provided by the SHG signal, this particular probe can be used to determine lipid phase and transmembrane voltage through the dye's fluorescence behavior. We now strengthen our technique by changing the polarity of the incident laser beam from linear to circular polarization. The modification provides SHG signal around the circumference of the cell in every optical slice, while maintaining a sufficient signal-to-noise ratio. Utilizing nanosecond pulsed electric fields (nsPEFs) of varying pulse widths and intensities as a tool for provoking spatially and temporally minute perturbations in the cell membrane, we observe membrane poration on the nano-scale. To verify our results, we adapted a previously-published electrical membrane model to map the theoretical angular area affected by these nsPEFs. The tests further establish the effectiveness of SHG probes, in our case Di-4, as a tool for observing subtle membrane alterations and document the extent to which electric fields of varying pulse parameters affect the cellular membrane.

We explore the feasibility of generating strong coherent anti-Stokes Raman scattering (CARS) signal in the backward direction.

We demonstrate the first experimental evidence of efficient random Raman lasing in a highly scattering powdered medium. Monte Carlo simulations were used to guide the experiment and provide support for experimental findings.

Terahertz time-domain spectroscopy (THz-TDS) is an effective technique to probe the intermolecular and collective vibrational modes of biological macromolecules at THz frequencies. To date, the vast majority of spectroscopic studies have been performed on dehydrated biomolecular samples. Given the fact that all biochemical processes occur in aqueous environments and water is required for proper protein folding and function, we hypothesize that valuable information can be gained from spectroscopic studies performed on hydrated biomolecules in their native conformation. In this study, we used a THz-TDS system that exploits photoconductive techniques for THz pulse generation and freespace electro-optical sampling approaches for detection. We used the THz spectrometer to measure the time-dependent electric field of THz waves upon interaction with water, phosphate buffered saline (PBS), and collagen gels. By comparing these waveforms with references, we simultaneously determined each sample's index of refraction (n) and absorption coefficients (μa) as a function of frequency. Our data show that the properties we measure for the water, PBS and collagen are comparable to those reported in the literature. In the future, we plan to examine the effect that both temperature and pH have on the optical properties of other biological macromolecules. Studies will also be performed to compare our results to those generated using molecular dynamics simulations.

For the first time, we demonstrate traveling wave stimulated Raman scattering, which allows arbitrary direction of the generated signal towards the target.

Stimulated Raman scattering (SRS) is a powerful tool for obtaining background-free chemical information about a material without extrinsic labeling. Background-free spectra are particularly important in the fingerprint region (~800 and 1800 cm(-1)) where peaks are narrow, closely-spaced, and may be in abundance for a particular chemical. We demonstrate a method for obtaining SRS spectra using a single femtosecond laser oscillator. A photonic crystal fiber is used to create a supercontinuum to provide a range of Stokes shifts from ~300 to 3400 cm(-1). This SRS approach provides for collection capabilities that are easily modified between obtaining broadband spectra and single-frequency images.

Highly sensitive, miniature biosensors are desired for the development of new techniques for biological and environmental analyte sensing. One potential approach uses the detection of optical resonances, known as Whispering Gallery Modes (WGMs), from quantum dot embedded polystyrene microspheres. These modes arise from the total internal reflection of the quantum dot emission light within the high index polystyrene microsphere, to produce narrow spectral peaks, which are sensitive to refractive changes in the immediate vicinity of the microsphere surface. The high refractometric sensitivity of the WGMs in these microspheres offers potential for remote detection of molecules adsorbed onto or bound to the microsphere surface without the need for direct coupling of the light via an optical fiber. The sensitivity of these modes has been shown to exceed the theoretical sensitivity of a homogeneous microsphere, using a Mie theory model. This enhancement is believed to be due to the embedded layer of quantum dots at the surface of the microspheres. A model was developed to demonstrate that the embedded QDs could be modeled as a high index outer layer to explain the observed WGM spectra and explore the sensitivity of the modes. In this work, we extend this idea to multiple layers to model the effects of protein adsorption or binding to the surface. The theoretical results are shown to provide a close fit to our previous experimental results.

New methods of biological analyte sensing are needed for development of miniature biosensors that are highly sensitive and require minimal sample preparation. One technique employs optical resonances, known as whispering gallery modes (WGMs), in spherical or cylindrical microstructures. The spectral positions of these resonant modes are very sensitive to the local refractive index and spectral shifts may be used to sense changes in the index. To excite these WGMs and enable remote excitation, quantum dots are embedded in polystyrene microspheres to serve as local light sources. Using a simple continuous wave excitation optical system, these sensors are demonstrated by monitoring the wavelength shift of multiple resonant modes as bulk index of refraction is changed in ethanol-water mixtures. The potential for targeted biosensing is explored through addition of a protein that adsorbs to the microsphere surface, thrombin, and one that does not, bovine serum albumin (BSA). The thrombin produced a spectral shift that was much larger than that due to the bulk index change. The BSA produced a significantly smaller shift that was slightly larger than the expected shift due to bulk index change. Most likely due to the thin, high index layer of quantum dots, microsensor response in all cases demonstrated increased sensitivity over theoretical predictions.

The overall goal for this project is the development and study of a quantitative fluorescence sensor for in vivo detection of β-amyloid (Aβ), the primary protein component of senile plaques in Alzheimer's disease (AD). Toward achieving that goal a Monte Carlo simulation has been developed to model photon propagation through the human head and a phantom model of the human head has been built and tested. In both cases a four layer model that included the skin, skull, fluorescent biosensor, and gray matter was used. A sensitivity study was performed to investigate the influence on the fluorescent output intensity of changes in concentration of the sensor. The results show that the fluorescent output intensity is detectable with a reasonable fluorescent sensor concentration and increases nearly linearly with increases in fluorescent concentration in the sensor. These results imply that the sensor would be detectable through the head using a reasonable optical system. The overall results are being used to aid in the design of the fluorescent sensor and the optical system for early detection of AD.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy is explored as a tool for obtaining micro-scale thermal measurements. A single femtosecond oscillator is used to pump a photonic crystal fiber to provide the broadband Stokes pulse. The CARS signals from the broad OH-stretching modes between 3000 and 3600 cm(-1) are shown to correlate with temperature with an accuracy of ± 1°C for water and ± 1.5°C for phosphate-buffered saline. Local variation of temperature is mapped on a microscopic level, using black-dyed microspheres as thermal sources.

Whispering gallery modes (WGMs) have been demonstrated in polystyrene microspheres embedded with quantum dots (QDs) in the periphery to serve as local light sources for the remote excitation of low Q-factor microsphere sensors. These sensors were shown to display varied WGM spectra and refractometric sensitivity exceeding that predicted for a homogeneous microsphere. To explain these differences from the basic Mie scattering theory, the QDs have been modeled as a thin high index outer layer. This model has been shown to provide a good fit of the observed WGM spectra, with calculated values for the QD layer refractive index and penetration depth falling within our observed estimates. The possible enhancement in sensitivity for these microspheres has also been determined.