Calorimetry is a powerful label-free technique for characterizing biochemical interactions. However, conventional calorimeters are limited by large sample requirements and low throughput, relegating their use to a limited number of high-value measurements. To increase the throughput and sensitivity of calorimetry, we have developed a novel microfluidic calorimeter that uses optical methods to measure the temperature change caused by reactions occurring in sub-nanoliter droplets. In this calorimeter, a microfluidic system creates a mixed droplet of reactants, a thermochromic liquid crystal (TLC) reporter converts the temperature change to a spectral shift, and a sensitive optical detector measures the spectral shift. Experimental measurements of the temperature change induced in droplets by the exothermic binding of EDTA to Ca2+ show good agreement with a thermal multiphysics model. Our ongoing work to improve the microfluidic mixing of reactants and increase the temperature resolution of the calorimeter has yielded a temperature resolution for this calorimeter of 2.4 mK, which corresponds to an energy resolution of 16 nJ. This resolution is on the same order as commercial isothermal titration calorimeter (ITC) systems and 10-fold better than most nanocalorimeters.
A novel hyperspectral imaging sensor is demonstrated that can enable breakthrough applications of hyperspectral imaging in domains not previously accessible. Our technology consists of a planar hyperspectral encoder combined with a traditional monochrome image sensor. The encoder adds negligibly to the sensor’s overall size, weight, power requirement, and cost (SWaP-C); therefore, the new imager can be incorporated wherever image sensors are currently used, such as in cell phones and other consumer electronics. In analogy to Fourier spectroscopy, the technique maintains a high optical throughput because narrow-band spectral filters are unnecessary. Unlike conventional Fourier techniques that rely on Michelson interferometry, our hyperspectral encoder is robust to vibration and amenable to planar integration. The device can be viewed within a computational optics paradigm: the hardware is uncomplicated and serves to increase the information content of the acquired data, and the complexity of the system, that is, the decoding of the spectral information, is shifted to computation. Consequently, system tradeoffs, for example, between spectral resolution and imaging speed or spatial resolution, are selectable in software. Our prototype demonstration of the hyperspectral imager is based on a commercially-available silicon CCD. The prototype encoder was inserted within the camera’s ~1 cu. in. housing. The prototype can image about 49 independent spectral bands distributed from 350 nm to 1250 nm, but the technology may be extendable over a wavelength range from ~300 nm to ~10 microns, with suitable choice of detector.
A compact and fast-response wavelength monitor is described that can determine the wavelength of individual laser pulses with a resolution of a few pm. It combines a position-sensitive photo detector with an optical coating that converts the wavelength information of the incident light into a spatial intensity distribution on the photo detector. Differential read-out of the photo detector is used to determine the centroid of this distribution. Wavelength change between individual laser pulses is detected as a shift of the centroid of the spatial light distribution on the detector. The wavelength monitor is demonstrated with results from a wavelength-tunable fiber laser that can produce randomly accessible sequences of laser pulses.
We are developing a continuous glucose monitor for subcutaneous long-term
implantation. This detector contains a double chamber Fabry-Perot-etalon that
measures the differential refractive index (RI) between a reference and a
measurement chamber at 850 nm. The etalon chambers have wavelength
dependent transmission maxima which dependent linearly on the RI of their
contents. An RI difference of ▵n=1.5·10-6 changes the spectral position of a
transmission maximum by 1pm in our measurement. By sweeping the
wavelength of a single-mode Vertical-Cavity-Surface-Emitting-Laser (VCSEL)
linearly in time and detecting the maximum transmission peaks of the etalon we
are able to measure the RI of a liquid. We have demonstrated accuracy of
▵n=±3.5·10-6 over a ▵n-range of 0 to 1.75·10-4 and an accuracy of 2% over a ▵nrange
of 1.75·10-4 to 9.8·10-4. The accuracy is primarily limited by the reference
The RI difference between the etalon chambers is made specific to glucose by
the competitive, reversible release of Concanavalin A (ConA) from an
immobilized dextran matrix. The matrix and ConA bound to it, is positioned
outside the optical detection path. ConA is released from the matrix by reacting
with glucose and diffuses into the optical path to change the RI in the etalon.
Factors such as temperature affect the RI in measurement and detection
chamber equally but do not affect the differential measurement. A typical
standard deviation in RI is ±1.4·10-6 over the range 32°C to 42°C. The detector
enables an accurate glucose specific concentration measurement.
Accurate measurements of aqueous glucose concentrations have been made in a double-chamber Fabry-Pérot etalon that can be miniaturized for subcutaneous implantation to determine the concentration of glucose in interstitial fluid. In general, optical approaches to glucose detection measure light intensity, which in tissue varies due to inherent scattering and absorption. In our measurements, we compare the spectral positions of transmission maximums in two adjunct sections of an etalon in order to determine the refractive index difference between these sections and therefore we can tolerate large changes in intensity. With this approach, we were able to determine aqueous glucose concentrations between 0 mg/dl and 700 mg/dl within the precision of our reference measurement (±2.5 mg/dl or 2% of the measurement value). The use of reference cavities eliminates interference due to temperature variations, and we show the temperature independence over a temperature range of 32°C to 42°C. Furthermore, external filters eliminate interference from large molecule contaminants.
We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast
and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through
a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets,
resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8x8 multi
anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each
column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two
scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly
shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral
information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and
orange peel cells.
We have developed a new descanned parallel (32-fold) pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.
Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.
In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-106/cm3 and 20-106/cm3, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.
Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2•107cm3. Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.