We describe the fabrication of sub-100-nanometer-sized channels in a fused silica lab-on-a-chip device and experiments
that demonstrate detection of single fluorescently labeled proteins in buffer solution within the device with high signal
and low background. The fluorescent biomolecules are transported along the length of the nanochannels by
electrophoresis and/or electro-osmosis until they pass into a two-focus laser irradiation zone. Pulse-interleaved excitation
and time-resolved single-photon detection with maximum-likelihood analysis enables the location of the biomolecule to
be determined. Diffusional transport of the molecules is found to be slowed within the nanochannel, and this facilitates
fluidic trapping and/or prolonged measurements on individual biomolecules. Our goal is to actively control the fluidic
transport to achieve rapid delivery of each new biomolecule to the sensing zone, following the completion of
measurements, or the photobleaching of the prior molecule. We have used computer simulations that include
photophysical effects such as triplet crossing and photobleaching of the labels to design control algorithms, which are
being implemented in a custom field-programmable-gate-array circuit for the active fluidic control.
A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope
by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing
experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and
maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion
that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately
constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for
determining the position within the trap are also available for performing spectroscopic measurements, for applications
such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation
and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position
estimates and semi-empirical algorithms for trap control is discussed.
Recently, spatial light modulators (SLMs) have been used to generate polarization-engineered laser beams, such as
radially polarized doughnut modes, which may provide advantages for excitation of fluorophore dipoles in single-molecule
(SM) spectroscopy. Here we investigate the additional use of SLMs for spatially-dependent transformation of
the collected fluorescence field with a goal to improve the fidelity of three-dimensional molecular orientation
determination. Numerical calculations of a high numerical aperture single-molecule confocal microscope are presented
in which a SLM is placed in the back focal plane of the objective. The coherently imaged fluorescence undergoes
spatially-dependent phase and polarization transformation by the SLM, before it passes to a polarization beamsplitter,
and is subsequently focused onto two pinholes and single-photon avalanche photodiodes. We calculate the electric
vector field in the back focal plane of the objective using the Weyl representation and taking into account the forbidden
light emitted at angles above the critical angle of the cover glass-immersion fluid interface. The calculated electric field
is then subject to the spatially-dependent polarization change implemented by SLM. We numerically study the effects of
polarization control on the microscope sensitivity to molecule orientation. We also analyze the combined use of the
intensity and polarization information in the back focal plane of the SM microscope for single-molecule orientation
Optical technology is rapidly finding novel applications in several exiting bioanalytical, biological, and biomedical applications. Optical beams are increasingly used for bio-fluidic sample manipulation in BioMEMS devices replacing convectional mechanical, electrostatic, and electrokinetic methods. This paper presents novel multiphysics computational approach for modeling optical interaction with fluidic, thermal, mechanical, and biological processes. We present a model of optical manipulation of particles and biological cells with laser beams. Computational results are compared to available experimental data from laboratory experiments and from practical engineered optical bio microdevices. The modeling approach is demonstrated on selected specific applications of optical manipulation of micro spheres, micro cylinders, and optical manipulation and sorting of biological cells in microfluidic cytometers.
The application of the frequency domain and steady-state diffusive optical spectroscopy (DOS) and steady-state near infrared spectroscopy (NIRS) to diagnosis of the human lung injury challenges many elements of these techniques. These include the DOS/NIRS instrument performance and accurate models of light transport in heterogeneous thorax tissue. The thorax tissue not only consists of different media (e.g. chest wall with ribs, lungs) but its optical properties also vary with time due to respiration and changes in thorax geometry with contusion (e.g. pneumothorax or hemothorax). This paper presents a finite volume solver developed to model photon migration in the diffusion approximation in heterogeneous complex 3D tissues. The code applies boundary conditions that account for Fresnel reflections. We propose an effective diffusion coefficient for the void volumes (pneumothorax) based on the assumption of the Lambertian diffusion of photons entering the pleural cavity and accounting for the local pleural cavity thickness. The code has been validated using the MCML Monte Carlo code as a benchmark. The code environment enables a semi-automatic preparation of 3D computational geometry from medical images and its rapid automatic meshing. We present the application of the code to analysis/optimization of the hybrid DOS/NIRS/ultrasound technique in which ultrasound provides data on the localization of thorax tissue boundaries. The code effectiveness (3D complex case computation takes 1 second) enables its use to quantitatively relate detected light signal to absorption and reduced scattering coefficients that are indicators of the pulmonary physiologic state (hemoglobin concentration and oxygenation).
The response of the biological cells to optical manipulation in the bio-microfluidic devices is strongly influenced by the flow and motion inertia. There is a variety of microfluidic architectures in which both the cell-fluid interaction and the optical field are driving forces for segregation and manipulation of the cells. We developed a computational tool for analysis/optimization of these devices. The tool consists of two parts: an optical force library generator and the computational fluid dynamics solver with coupled optical force field. The optical force library can be computed for spherical and non-spherical objects of rotational symmetry and for complex optical fields. The basic idea of our method is to a) represent an incident optical field at the biological cell location as an angular spectrum of plane waves; b) compute the scattered field, being a coherent superposition of the scattered fields coming from each of the incident plane waves, with the powerful T-matrix method used to compute the amplitude matrix; c) use the incident and computed scattered fields to build a spatial map of optical forces exerted on biological cells at different locations in the optical beam coordinate system, and d) apply the library of optical forces to compute laser beam manipulation in microfluidic devices. The position and intensity of the optical field in the microfluidic device may be dynamic, thus optical forces in microfluidic device are based on the instantaneous relative location of the cell in the beam coordinate system. The cell is simulated by the macroparticle that undergoes mutual interactions with the fluid. We will present the exemplary applications of the code.
Matrices of binary micro-lenses monolithically integrated with the focal-place-arrays (FPA) of longwave IR uncooled detectors can significantly improve sensor's parameters. Surface relief of the binary micro-lenses is built of annular stair step structures of heights and widths smaller than the radiation length. Scalar diffraction theory cannot correctly describe diffraction on these micro-structures and therefore the rigorous electromagnetic theory should be applied. In this aper, we have applied the electromagnetic eignemode method to study binary micro-optics for the longwave IR FPA of 50 micrometers pixel width. We have shown that binary refractive micro-lenses outperform their diffractive counterparts allowing for detectors of 10 micrometers width. The effective refractive micro-lenses require the 8-level surface relief. Geometrical optics predictions of the focal position agree quite well width electromagnetic calculations.
Kinoform is a phase modulating computer-generated hologram. Most often, algorithms for nonparaxial kinoform synthesis use discrete propagation operators and solution is given in a discrete form. Kinoform is obtained by interpolation from these discrete samples in its plane. This paper gives the relationship between the continuous reconstruction of that kinoform and the discrete synthesis solution in the image plane. The relationship allows us to describe complex amplitude distribution in diffraction orders, predicts the possibility of speckle-like reconstruction, and is a basis for an efficient method of zero-order image calculation. Spatial frequency filtering properties of discrete algorithms are analyzed and criteria for algorithm sampling parameters are derived.
Kinoform for irregular space-variant nonparaxial optical interconnection (01) was calculated by the generalized error-reduction algorithm (ERA). Diffraction limited spots easy to align for detectors of 10 im width and diffraction efficiency r 34 were obtained for the binary kmoform case. This approach is fast takes into account the incident beam intensity distribution and an equalization of the intensity between spots is easy. However the resulted surface relief is complicated. NONPARAXIAL KINOFORM SYNTHESIS Free-space Ols for clock distribution will require a low f-number (F/i) holograms due to expected detector sizes of 10 m x 10 im and typical laser diode (LD) light divergence angles of 30 and iS . Large field angles needed to obtain large fanouts and to deflect beams towards detectors located near the chip boundary also cause that paraxial conditions are not satisfied. Kinoforms which are computer generated phase holograms afford possibilities for Ols with complicated detector patterns and high . Free-space nonparaxial propagation can be described by the operator N FT QFI where Fr U f Uexp(-ikcxx1 ) dx1 U(x1) is the complex amplitude in the kinoform plane Q is the spectrum phase shift operator QFJ'' U exp ( ikz I 1 - 2 ) j''U k 2 vt/A X is the wavelength is the normalized spatial frequency and z is the separation between planes. N is an unitary transform because we may neglect evanescent waves in