Wide-field fluorescence microscopy is generally limited to either small volumes or low temporal resolution. We present a microscope add-on that provides fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror of update rate 20kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.
A hyperspectral Fourier transform spectrometer (HS-FTS) has been developed to study biological material binding to
surfaces through spatially resolved, spectral self-interference fluorescence microscopy and also label-free white light
reflectance spectroscopy. Spectral self-interference fluorescence microscopy yields the height of fluorescent tags bound
to a specific location on biomolecules tethered to a surface, and from this the biomolecule conformation can be
predicted; white light reflectance spectroscopy yields the average height of an ensemble of biomolecules relative to the
surface. The HS-FTS is composed of a small, step scanning Michelson interferometer made by Optra, Inc., a series of
commercial off the shelf imaging lenses, and a 12-bit thermoelectrically-cooled CCD camera. The system operates over
the 500 to 900 nm spectral range with user defined spectral resolution, thereby supporting use of a host of fluorescent
tags or white light spectral windows. The system also supports near real-time hyperspectral cube acquisition via
undersampling with the use of a spectral filter and user defined interferometer step increments. The overall approach
offers flexible yet sensitive measurement capability for a variety of biological studies. Preliminary results are presented
of both spectral self-interference fluorescence microscopy and white light reflectance spectroscopy measurements of
artificial, photographically etched surfaces with feature heights on the order of 10 nm. Planned future work includes
spectral self-interference fluorescence microscopy measurements of biomolecule conformation as manipulated by
external electrical and magnetic fields as well as label-free white light reflectance spectroscopy measurements of DNA
Light that would typically be discarded at a confocal microscope's detector pinhole will be collected and processed to allow a reduced spatial sampling rate and thus an improved image acquisition time. It is shown that collecting and appropriately processing the out-of-focus light will allow an axial sampling rate below that specified by the Nyquist criterion. To achieve this, a central detector pinhole and a number of out-of-focus regions are collected concurrently. This corresponds to imaging through several different channels, with differing point spread functions, in parallel. Since the spatial sampling rate is below the Nyquist frequency, aliasing occurs in the data from each of the channels. However, since the point spread functions are different, the aliasing effects are different in each channel. This allows the ensemble of aliased images to be processed into a single dealiased and deconvolved image. This potential utility of out-of-focus light is demonstrated through simulated examples for differing collection schemes and scanning rates. Results are shown for under-sampling by up to a factor of four. Collecting the out-of-focus light also improves instrument collection efficiency.
A reconstruction algorithm is developed that uses
specific a-priori knowledge to produce higher
resolution images than standard approaches.
Deconvolution is an important image
reconstruction tool in fluorescence microscopy.
This is especially true for modern interferometric
instruments (such as I5M and 4Pi systems), as
they may have complicated oscillatory point
spread functions. Current methods are designed
to work on an arbitrary object - i.e. it is assumed
that there is no available a-priori knowledge of
the object (with the possible exception of a non-
negative condition on the fluorophore-emission
intensities). In situations where there is a-priori
knowledge of the object, it may be possible to use
this information to produce a higher quality
reconstruction of the object. A useful a-priori
condition is investigated here.
It is assumed that the object can be represented
by the sum of not more than L basis functions. The
simplest example of this is when the basis
functions are impulses - this leads to an object of
L or less non-zero points on a background of
zeros. This a-priori condition can be applied
directly; applied to a limited region of the object;
applied in one dimension (for an object with a
layered structure such as lipid bilayers); or
applied in two dimensions (for an object with a
filamentary structure such as actin fibers.) A
reconstruction algorithm is described and applied
to some illustrative simulated examples. The
results are found for several fluorescence
microscopy methodologies and compared to the
results produced by standard deconvolution
An original technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), can determine the location of fluorescent markers above a reflecting surface with sub-nanometer precision. SSFM was used to resolve the position of a fluorescent marker bound to either the top or the bottom leaflet of a lipid bilayer -- the difference in distance is only 4 nm. SSFM is a valuable tool in studying the conformation of DNA molecules immobilized on surfaces. A fluorescent label attached to a DNA molecule tethered to the surface can help elucidate its spatial orientation. This method is based on the fact that spontaneous emission of fluorophores located near a mirror is modified by the interference between direct and reflected waves, which leads to an oscillatory pattern in the emission spectrum. Spectral patterns of emission near surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra yielding information about the location of the emitters with very high precision.
We present a new method of fluorescence imaging, which yields nm-scale axial height determination and ~15 nm axial resolution. The method uses the unique spectral signature of the fluorescent emission intensity well above a reflecting surface to determine vertical position unambiguously. We have demonstrated axial height determination with nm sensitivity by resolving the height difference of fluorescein directly on the surface or on top of streptavidin. While different positions of fluorophores of different color are determined independently with nm precision, resolving the position of two fluorophores of the same color is a more convoluted problem due to the finite spectral emission widow of the fluorophores. Hence, for physically close (<λ/2) fluorophores, it is necessary to collect multiple spectra by independently scanning an excitation standing wave in order to deconvolute the contribution to the spectral pattern from different heights. Moving the excitation standing wave successively enhances or suppresses excitation from different parts of the height distribution, changing the spectral content. This way two fluorophores of the same color can be resolved to better than 20 nm. Design aspects of the dielectric stack for independent excitation wave scanning and limits of deconvolution for an arbitrary height distribution will be discussed.
We demonstrate a through the substrate, numerical aperture increasing lens (NAIL) technique for high-resolution inspection of silicon devices. We experimentally demonstrate a resolution of 0.2 micrometers , with the ultimate diffraction limit of 0.14 micrometers . Absorption limits inspection in silicon to wavelengths greater than 1 micrometers , placing an ultimate limit of 0.5 micrometers resolution on standard subsurface microscopy techniques. Our numerical aperture increasing lens reduces this limit to 0.14 micrometers , a significant improvement for device visual inspection (patent pending). The NAIL technique yields a resolution improvement over standard optical microscopy of at least a factor of n, the refractive index of the substrate material, and up to a factor of n 2. In silicon, this constitutes a resolution improvement between 3.6 and 13. This is accomplished by increasing the numerical aperture of the imaging system, without introducing any spherical aberration to the collected light. A specialized lens made of the same material as the substrate is placed on the back surface of the substrate. The convex surface of this lens is spherical with a radius of curvature, R. The vertical thickness of the lens, D, should be selected according to D equals $ (1 + 1/n)-X and the substrate thickness X.
Proc. SPIE. 3626, Testing, Packaging, Reliability, and Applications of Semiconductor Lasers IV
KEYWORDS: Multimode fibers, Metals, Spectroscopy, Composites, Reliability, Near field scanning optical microscopy, Near field, Associative arrays, Vertical cavity surface emitting lasers, Near field optics
We have studied the spatial and spectral characteristics of vertical-cavity surface-emitting laser emission using near- field scanning optical microscopy. We report the multi- transverse-mode characteristics of 15 micrometers diameter proton- implanted 850 nm devices used in a 2 Gbit/s multimode fiber- optic links. Spectrally resolved and integrated intensity scans over a 20 X 20 area were performed. The intensity of each resolvable transverse mode was integrated and its wavelength range false-colored at each scan position. The resulting composite image displays relative intensity and spatial distribution information for each transverse mode. Correlation with the shear force data allows mapping of the optical distributions to topographical features. Lasing filaments were observed at high drive currents. Gain competition among spatially overlapping transverse modes was observed while spatially isolated modes coexisted without competition.
This is a one-day lecture in the theory, development, and applications of high-resolution optical microscopy with a concentration on solid immersion lens and near-field scanning optical microscopy techniques. The course covers the necessary general background to optics and microscopy near the diffraction limit. Subjects covered include the development of scanned probe microscopies; the development and theory of near-field scanning optical microscopy; the design, construction, and assessment of near-field microscopes; the application of near-field microscopy to biological systems, to waveguides and photonic devices, and to semiconductor characterization; the theory, development, and practical application of solid immersion microscopy, including semiconductor inspection and thermal imaging.