Gregory H. Bearman

High-Q whispering-gallery mode sensor in liquids

Optical sensing of biomolecules on microfabricated glass surfaces requires surface coatings that minimize nonspecific binding while preserving the optical properties of the sensor. Microspheres with whispering-gallery (WG) modes can achieve quality factor (Q) levels many orders of magnitude greater than those of other WG-based microsensors: greater than 1010 in air, and greater than 109 in a variety of solvents, including methanol, H2O and phosphate buffered saline (PBS). The presence of dyes that absorb in the wavelength of the WG excitation in the evanescent zone can cause this Q value to drop by almost 3 orders of magnitude. Silanization of the surface with mercapto-terminal silanes is compatible with high Q (>109), but chemical cross-linking of streptavidin reduces the Q to 105-106 due to build-up of a thick, irregular layer of protein. However, linkage of biotin to the silane terminus preserves the Q at a ~2x107 and yields a reactive surface sensitive to avidin-containing ligands in a concentration-dependent manner. Improvements in the reliability of the surface chemistry show promise for construction of an ultrasensitive biosensor.

Sensitive imaging of spectrally overlapping flourochromes using the LSM 510 META

Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.

Archeological Applications of Advanced Imaging Techniques

Utilizing techniques developed by NASA, the imaging of texts on papyri, parchment, pottery, or plaster can capture much information invisible to the naked eye. Multi-spectral imaging and computer manipulation of digital images enhances legibility of obscured Dead Sea Scroll texts and faded ostraca alike.

Volume holographic spectral imaging

We report on a volume holographic imaging spectrometer (VHIS) system which allows retrieval of a scenes two-dimensional spatial information as well as its spectral information. This is performed using a transmission volume hologram and a simple rotary scanning mechanism. The system has the advantages of high spectral and spatial resolutions and the potential of single-shot, four-dimensional (3D spatial plus 1D spectral) imaging by recording multiple volume holograms in the same material. Also, due to the transmission diffraction geometry, the system automatically eliminates the stray excitation light from the captured signal. We give theoretical analysis of the performance and experimental demonstration using fluorescent CdSe/ZeS quantum dots. The measured quantum dots spectra agree well with the spectra obtained using a conventional spectrometer.

An all-reflective computed tomography imaging spectrometer

The computed tomographic imaging spectrometer (CTIS) is a passive non-scanning instrument which simultaneously records a scenes spectral content as well as its 2-D spatial. Simultaneously implies a time frame limited only by the frame rate and signal-to-noise of the imaging device. CTIS accomplishes this by feeding incident scene radiation through a computer generated hologram (CGH) in Fourier space. The resulting dispersion pattern is recorded on a conventional pixilated imager and is stored on a local computer for post processing using iterative reconstruction techniques. A virtual 3-D datacube is constructed with one dimension in terms of energy weights for each wavelength band. CTIS is ideal for observing rapidly varying targets and has found use in military, bio-medical and astronomical applications. For the first time we have built an entirely reflective design based on the popular Offner reflector using a computer generated hologram formed on a convex mirror surface. Furthermore, a micro electro-mechanical system (MEMS) has been uniquely incorporated as a dynamic field stop for smart scene selection. Both the MEMS and reflective design are discussed. The CTIS multiplexes spatial and spectral information, so the two quantities are interdependent and adjustments must be made to the design in order to allow adequate sampling for our given application. Optical aberrations arising from a tilted image plane are alleviated through design optimization.

Multispectral imager for the agricultural user

Imaging spectrometers have recently moved out of the spaceflight environment, in which they were developed, to a host of other applications. Some of these new uses include the graphics and printing industry, process control, bio-medicine, clinical diagnostics and agriculture. For any of these applications, new approaches are necessary to design compact, portable instruments that can be easily and reliably calibrated. This paper presents one such implementation of an imaging spectrometer suitable for field use.

Video-rate spectral imaging system for fluorescence microscopy

We describe fluorescence spectral-imaging results with the computed-tomography imaging spectrometer (CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. This system is also optimal for simultaneously monitoring changes in spectral characteristics of multiple probes from different locations within the same sample. This advantage will provide additional information about the physiological changes in function form populations of cells which respond in a heterogeneous manner. The results presented in this paper consist of proof-of-concept imaging results from the CTIS in combination with two different systems of fore- optics. In the first configuration, raw image data were collected using the CTIS coupled to an inverted fluorescence microscope. The second configuration combined the CTIS with a confocal microscope equipped with a fiber-optic imaging bundle, previously for in vivo imaging. Image data were collected at frame rates of 15 frame per second and emission spectra were sample at 10-nm intervals with a minimum of 29 spectral bands. The smallest spatial sampling interval presented in this paper is 0.7 micrometers .

In vivo skin chromophore mapping using a multimode imaging dermoscope (SkinSpec)

We introduce a multimode dermoscope (SkinSpectTM) we developed for early detection of melanoma by combining fluorescence, polarization and hyperspectral imaging. Acquired reflection image datacubes were input to a wavelength-dependent linear model to extract the relative contributions of skin chromophores at every pixel. The oxy-hemoglobin, deoxy hemoglobin, melanin concentrations, and hemoglobin oxygen saturation by the single step linear least square fitting and Kubelka-Munk tissue model using cross polarization data cubes were presented. The comprehensive data obtained by SkinSpect can be utilized to improve the accuracy of skin chromophore decomposition algorithm with less computation cost. As an example in this work, the deoxy-hemoglobin over-estimation error in highly pigmented lesion due to melanin and deoxy hemoglobin spectral cross talk were analyzed and corrected using two-step linear least square fitting procedure at different wavelength ranges. The proposed method also tested in skin with underlying vein area for validating the proof of concept.

High-speed spectral imager for imaging transient fluorescence phenomena

We describe fluorescence spectral-imaging results with the microscope computed-tomography imaging spectrometer ((mu) CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, (mu) CTIS can be used to image dynamic phenomena. The results presented in this paper consist of imaging results using static targets consisting of 1 micrometers and 6 micrometers fluorescing microspheres. The emission spectra were sampled at a 10-nm interval between 430 nm and 710 nm. The smallest spatial sampling interval presented in this paper is 1.7 micrometers . Image data were collected in integration times of 16 msec.

Video-rate spectral imaging of fluorescence phonomena

We describe fluorescence spectral-imaging results with the microscope computed-tomography imaging spectrometer ((mu) CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the (mu) CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. The result presented in this paper consists of proof-of-concept imaging result using two static targets. The first is composed of 6-micrometers fluorescing microspheres and the second consists of rat sinusoid epithelial cells loaded with 0.5-micrometers fluorescing microspheres. Image data were collected in integration times of 16 msec, comparable to video frame rate integration times. The emission spectra were sampled at 10-nm intervals between 420 nm and 710 nm. The smallest spatial sampling interval presented in this paper is 1.7 micrometers .