Composition-tunable nanocrystals are fluorescent nanoparticles with a uniform particle size and with adjustable optical
characteristics. When used for optical labeling of biomolecular targets these and other nanotechnology solutions have
enabled new approaches which are possible because of the high optical output, narrow spectral signal, consistent
quantum efficiency across a broad emission range and long lived fluorescent behavior of the nanocrystals. When
coupled with spectral imaging the full potential of multiplexing multiple probes in a complex matrix can be realized.
Spectral imaging can be used to improve sensitivity of narrowband fluorophores through application of chemometric
image processing techniques used to reduce the influence of autofluorescence background.
Composition-tunable nanocrystals can be complexed together to form nanoclusters which have the advantage of
significantly stronger signal and therefore a higher sensitivity. These nanoclusters can be targeted in biomolecular
systems using standard live-cell labeling and immunohistochemistry based techniques. Composition-tunable
nanocrystals and nanoclusters have comparable mass and brightness across a wide emission range. This enables the
production of nanocrystal-based probes that have comparable reactivity and sensitivity over a large color range.
We present spectral imaging results of antibody targeted nanocrystal cluster labeling of target proteins in cultured cells
and a Western blot experiment. The combination of spectral imaging with the use of clusters of nanocrystals further
improves the sensitivity over either of the approaches independently.
Apoptosis, also known as programmed cell death, is a process in which cells initiate a series of events to trigger their
own demise. Normal cells use this mechanism in the regulation of their life cycle. On the contrary, abnormal or cancer
cells have lost the ability to regulate themselves by this process. Because of this, there is much interest in the study of
the apoptotic process. Currently, there are many commercial assays available to detect apoptosis in cells, most of which
are fluorescence based. Limitations of such fluorescent assays lead to arbitrary or inclusive results.
Raman spectroscopy is a powerful technique that yields specific molecular information on samples under study. The
Raman spectra obtained from cell samples are very complex, yet the differences in the complex Raman spectra analyzed
using chemometric techniques can identify chemical and physiological information about cells. Furthermore, Raman
spectroscopy is a sensitive, rapid, reagentless, low-cost technique, making it a superior alternative to traditional
fluorescence based apoptosis assays.
In this study, we have employed Raman spectroscopy and Raman chemical imaging, along with chemometric
techniques, to distinguish apoptotic cells from non-apoptotic cells in two prostate cancer cell lines, PC3 and LnCAP.
Initial results indicate that Raman spectra of apoptotic and non-apoptotic cells are different in both cell lines.
Furthermore, chemometric analysis of the data shows that the spectra separate into two distinct populations, apoptotic
and non-apoptotic. Traditional fluorescence based apoptotic assays confirm the results. This work provides ample
evidence that Raman spectroscopy is a valuable tool in biomedical imaging.
Raman spectroscopy is a powerful technique for rapid, non-invasive and reagentless analysis of materials, including
biological cells and tissues. Raman Molecular Imaging combines high molecular information content Raman
spectroscopy and digital full field imaging to enable the investigation of cells and tissues. We have conducted widefield
imaging using a new class of birefringent liquid crystal tunable filter that provides high throughput over an extended
wavelength range. This tool has been applied to investigate the linkage between reagentless spectral imaging in tissue
and cells and standard reagent based approaches. In this report, we describe Raman imaging data on a clinical tissue
sample and cultured cells. The results demonstrate the sensitivity of Raman Molecular Imaging and fluorescence
spectral imaging to molecular differences in biological systems laying the foundation for the eventual use of this
approach as a biological research and clinical diagnostic tool.
Raman spectroscopy is a powerful technique for rapid, non-invasive and reagentless analysis of materials, including biological cells. In many samples of biological origin, laser illumination leads to luminescence in addition to Raman scattering. This luminescence will often dissipate after prolonged laser exposure. A common practice is to allow a sample to "photobleach" prior to acquisition of a high quality Raman spectrum. In an effort to automate data acquisition on such samples we are investigating an automated means of quantifying photobleaching and acquiring Raman spectra after photobleaching. We present results of a comparison between an automated approach to acquiring a spectrum on a sample with dynamic luminescence and a manual approach taken by a trained spectroscopist. This component of smart biomedical sensor technology will help allow high quality spectral data to be acquired reproducibly to potentially aid non-spectroscopists with application of Raman spectroscopic approaches.
Contamination of drinking water with pathogenic microorganisms such as Cryptosporidium has become an increasing concern in recent years. Cryptosporidium oocysts are particularly problematic, as infections caused by this organism can be life threatening in immunocompromised patients. Current methods for monitoring and analyzing water are often laborious and require experts to conduct. In addition, many of the techniques require very specific reagents to be employed. These factors add considerable cost and time to the analytical process. Raman spectroscopy provides specific molecular information on samples, and offers advantages of speed, sensitivity and low cost over current methods of water monitoring.
Raman spectroscopy is an optical method that has demonstrated the capability to identify and differentiate microorganisms at the species and strain levels. In addition, this technique has exhibited sensitivities down to the single organism detection limit. We have employed Raman spectroscopy and Raman Chemical Imaging, in conjunction with chemometric techniques, to detect small numbers of oocysts in the presence of interferents derived from real-world water samples. Our investigations have also indicated that Raman Chemical Imaging may provide chemical and physiological information about an oocyst sample which complements information provided by the traditional methods. This work provides evidence that Raman imaging is a useful technique for consideration in the water quality industry.
Chemical imaging is a powerful technique combining molecular spectroscopy and digital imaging for rapid, non-invasive and reagentless analysis of materials, including biological cells and tissues. Raman chemical imaging is suited to the characterization of molecular composition and structure of biomateials at submicron spatial resolution (< 250 nm). As a result, Raman imaging has potential as a routine tool for the assessment of cells and subcellular components. In this presentation, we discuss Raman chemical imaging and spectroscopy of single human cells obtained from a culture line. Rapid three dimensional Raman imaging is shown using deconvolution to improve image quality.
In recent years researchers have made significant progress in understanding the physics of fluorescence in highly scattering materials such as tissues in the near-infrared. We have quantitatively verified a model which describes fluorescence in ideal (homogeneous and infinite) tissue-like media. Given the quantitative accuracy of this model, one can use measurements of the fluorescence of a tissue with a homogeneous distribution of fluorophore to obtain the quantum yield, lifetime of the probe, and the absorption and scattering coefficients of the tissue at the fluorescent wavelength. We demonstrate that this can be done with a simple measurement of the photon density as a function of source- detector separation at the excitation and emission wavelengths. To verify our approach we present the lifetime, quantum yield of the fluorescent probe (rhodamine B), and the absorption and scattering coefficients of the medium at the emission peak wavelength that are obtained by a fit of the model to experimental measurements.
In the near-infrared spectral region (700 - 900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the dc fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable dc fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in rhodamine concentration on the order of 1% using the dc fluorescence intensity. This dc fluorescence sensitivity is characterized experimentally at two concentrations (55 and 360 nM) and over a range of source-detector separations. Other aspects of the sensitivity are discussed over a large range of concentrations using a fluorescence photon migration model.
Our research is aimed at the development of a frequency-domain instrument for conducting non-invasive, real-time, near-infrared, optical tomography of tissue in vivo. Our goal is to reconstruct a spatial map of the optical properties of a strongly scattering medium in a semi-infinite-geometry sampling configuration. Specifically, we focus our attention on the absorption coefficient ((mu) a) and the reduced scattering coefficient ((mu) s') of the medium. We have developed a frequency- domain measurement protocol (which we call precalibrated), which permits one to recover the values of (mu) a and (mu) s' of a uniform tissue-like phantom from a measurement at a single source-detector separation and a single modulation frequency. It requires a preliminary reference measurement on a calibration sample of known optical properties before the measurement on the investigated sample. This approach is in principle rigorous only in macroscopically homogeneous media. We have verified that the equations valid for uniform media can still be applied to yield qualitative information on the optical nature of the inhomogeneity if the effect of macroscopic inhomogeneities on the measured phase and intensity is not too large. In vitro measurements on turbid media containing scattering and absorbing homogeneities, with optical properties very similar to the background medium, gave encouraging results. We plan to implement this measurement protocol in a multisource, multidetector instrument for optical tomography.
Near-IR optical tomography is thwarted by the highly scattering nature of light propagation in tissue. We propose a weighted back-projection method to produce a spatial map of an optical parameter which characterized the investigated medium. We have studied the problem of the choice of the back-projection weight function for the absorption coefficient ((mu) a) and for the reduced scattering coefficient ((mu) s') of tissuelike phantoms. Working in frequency-domain optical imaging, we have initially approached the problem of quantifying the effect caused by a small absorbing defect embedded in the medium on the measured DC intensity, AC amplitude, and phase. The collection of DC, AC, and phase changes during a 1 mm step raster scan of the absorbing defect provides information on the photon path distributions and, in general, on the probed spatial region when DC, AC, and phase are, respectively, the measured parameters. We report experimentally determined weight functions for (mu) a and (mu) s'. They indicate that absorption and scattering maps can significantly differ in terms of resolution.
Near infrared optical imaging is emerging as a potentially important imaging modality, because it offers real time data access, portability, cost-effectiveness, and the relatively safe use of non-ionizing radiation. Reconstruction of images by optical tomography is complicated by the diffusive character of light propagation in optically heterogeneous tissue. The spatial volume element probed by the light path between the light source and optical detector is rather wide and depends on a variety of experimental and instrumental factors. We have published an optical image of the hand in air based on photon density wave distribution characteristics, using both steady-state (intensity) and frequency-domain (phase and modulation) experimental conditions. Since then, we have developed new instrumentation, better measurement protocols, some reconstruction algorithms and a more complete theoretical understanding of photon diffusion in both homogeneous and heterogeneous media. We have now performed frequency-domain measurements (at a modulation frequency of 160 MHz with 760 nm near infrared light) with the hand immersed in a scattering fluid (the infinite geometry arrangement). The advantages of our current approach include the spectroscopic resolution of physiologically interesting tissue regions, greater spatial resolution, the generation of absorption and reduced scattering coefficient maps of the image, rapid data acquisition, real time simultaneous display of the experimental parameters and calculated optical parameters and the possibility of obtaining some tomographic reconstruction.
In this paper, we present a series of measurements made with a portable frequency-domain near-infrared tissue spectrometer (OMNIA). This is the first application of the OMNIA in a clinical setting. All of the measurements presented here were taken in vivo, most were on human subjects. We report the results of three experiments: (1) A simple ischemia/plethysmography experiment, which indicates ability of the instrument to noninvasively, continuously monitor the hemoglobin saturation of a limb. (2) A survey of hemoglobin saturation in patients with peripheral vascular disease. (3) An animal experiment to demonstrate the correlation of our instrument readings with results from established techniques for measuring hemoglobin saturation. We measured the absorption and reduced scattering coefficients of the tissue at two wavelengths (715 nm and 850 nm). From the absorption coefficients, we calculated the concentrations of oxygenated and deoxygenated hemoglobin ([HbO2] and [Hb]), which immediately yield the hemoglobin saturation (Y) and the total blood volume (T) in the tissue. Our preliminary results indicate some of the potential of the instrument and the areas for future improvement of it.
We have designed and constructed a near-infrared spectrometer for the non-invasive optical study of biological tissue. This instrument works in the frequency-domain and employs multiple source-detector distances to recover the absorption coefficient ((mu) (alpha )) and the reduced scattering coefficient ((mu) s') of tissue. The light sources are eight light emitting diodes (LEDs) whose intensities are modulated at a frequency of 120 MHz. Four LEDs emit light at a peak wavelength of 715 nm ((lambda) 1), while the other four LEDs emit at a peak wavelength of 850 nm ((lambda) 2). From the frequency-domain raw data of phase, dc intensity, and ac amplitude obtained from each one of the eight light sources, which are located at different distances from the detector fiber, we calculate (mu) (alpha ) and (mu) s' at the two wavelengths (lambda) 1 and (lambda) 2. The concentrations of oxy- and deoxy-hemoglobin, and hence hemoglobin saturation, are then derived from the known extinction coefficients of oxy- and deoxy-hemoglobin at (lambda) 1 and (lambda) 2. The statistical error in the measurement of the optical coefficients due to instrument noise is about 1 - 2%. The accuracy in the determination of the absolute value of the optical coefficients is within 10 - 20%. Preliminary results obtained in vivo on the forearm of a volunteer during an ischemia measurement protocol are presented.
We have designed a multisource frequency-domain spectrometer for the optical study of biological tissues. Eight multiplexed, intensity-modulated LEDs are employed as the light sources. Four of them emit light at a peak wavelength of 715 nm (λ1); the other four, 850 nm (λ2). The frequency of intensity modulation is 120 MHz. This instrument measures the frequency-domain parameters phase, dc intensity, and ac amplitude at the two wavelengths λ1 and λ2 and for different distances between light source and detector. From these frequency-domain raw data, the absolute values of the absorption and reduced scattering coefficients of tissue at λ1 and λ2 are obtained. The oxy- and deoxyhemoglobin concentrations, and hence the hemoglobin saturation, are then analytically derived from the molar extinction coefficients. Acquisition times as short as hundreds of milliseconds provide real-time monitoring of the measured parameters. We performed a systematic test in vitro to quantify the precision and accuracy of the instrument reading. We also report in vivo measurements. This spectrometer can be packaged as a compact portable unit.
We studied the ability to detect small absorbing objects embedded in a highly scattering medium. Absorbing spheres of varying size, from 0.8 mm to 6.8 mm radius, submerged in a solution of highly scattering, low absorbing liquid: skim milk, were studied in a trans- illumination geometry. Groups of more than one sphere and a single circular disk, with radius identical to that of one of the spheres, were also studied. Single linear raster scans in the plane of the sphere, with the spheres centered between the source and detector, were made. Data was taken in the frequency-domain, yielding profiles of the objects in each of the three measurable quantities: dc intensity, phase, and modulation. The diffraction pattern form the sphere differed from that of the disk, demonstrating a volume effect associated with photon diffusion. The diffraction pattern of multiple spheres differed from that of single spheres.
High frequency, intensity-modulated light waves are attenuated and phase-shifted by the absorption and scattering properties of highly scattering media, such as tissue. The simultaneous measurement of the average light intensity, modulation amplitude, and phase- shift at a fixed distance from a sinusoidally modulated light source, permits a quantitative determination of the absolute values of the absorption and scattering coefficients from a frequency-domain scan. Our studies have established the range of modulation frequencies that give the highest sensitivity to changes of the optical parameters in model systems. We have measured the optical absorption spectra of dyes suspended in highly scattering media. These spectra match those found in non-scattering media. This frequency-domain approach provides a simple method to perform quantitative spectroscopy in highly scattering media.
KEYWORDS: Modulation, Interference (communication), Phase shift keying, Sensors, Lamps, Signal to noise ratio, Signal detection, Phase measurement, Xenon, Analog electronics
In frequency domain fluorometry, as well as all other spectroscopic techniques, the noise ultimately limits the sensitivity of the instrument and the precision of the measurement. The analysis of the sources of noise in different instruments has revealed that the noise is due to a number of different instrumental factors rather than photon statistics. The ultimate goal is to eliminate those factors to achieve a situation in which the limit is the detector intrinsic noise. We have developed a system, based on digital signal processing, in which the influence of several spurious noise sources has been reduced. A study of the range of cross-correlation frequencies used to obtain the best signal-to-noise ratio is presented.
Imaging of thick tissue has been an area of active research during the past several years. Among the methods proposed to deal with the high scattering of biological tissues, the time resolution of a short light probe traversing a tissue seems to be the most promising. Time resolution can be achieved in the time domain using correlated single photon counting techniques or in the frequency domain using phase resolved methods. We have developed a CCD camera system which provides ultra high time resolution on the entire field of view. The phase of the photon diffusion wave traveling in the highly turbid medium can be measured with an accuracy of about one degree at each pixel. The camera has been successfully modulated at frequencies on the order of 100 MHz. At this frequency, one degree of phase shift corresponds to about 30 ps maximum time resolution. Powerful image processing software displays in real time the phase resolved image on the computer screen.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.