Robert T. Kester

Snapshot Image Mapping Spectrometer (IMS) with high sampling density for hyperspectral microscopy

A snapshot Image Mapping Spectrometer (IMS) with high sampling density is developed for hyperspectral microscopy, measuring a datacube of dimensions 285 × 285 × 60 (x, y, λ). The spatial resolution is ~0.45 µm with a FOV of 100 × 100 µm2. The measured spectrum is from 450 nm to 650 nm and is sampled by 60 spectral channels with average sampling interval ~3.3 nm. The channel’s spectral resolution is ~8nm. The spectral imaging results demonstrate the potential of the IMS for real-time cellular fluorescence imaging.

Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy

An image slicing spectrometer (ISS) for microscopy applications is presented. Its principle is based on the redirecting of image zones by specially organized thin mirrors within a custom fabricated component termed an image slicer. The demonstrated prototype can simultaneously acquire a 140 nm spectral range within its 2D field of view from a single image. The spectral resolution of the system is 5.6 nm. The FOV and spatial resolution of the ISS depend on the selected microscope objective and for the results presented is 45 x 45 microm(2) and 0.45 microm respectively. This proof-of-concept system can be easily improved in the future for higher (both spectral and spatial) resolution imaging. The system requires no scanning and minimal post data processing. In addition, the reflective nature of the image slicer and use of prisms for spectral dispersion make the system light efficient. Both of the above features are highly valuable for real time fluorescent-spectral imaging in biological and diagnostic applications.

Real-time snapshot hyperspectral imaging endoscope

Hyperspectral imaging has tremendous potential to detect important molecular biomarkers of early cancer based on their unique spectral signatures. Several drawbacks have limited its use for in vivo screening applications: most notably the poor temporal and spatial resolution, high expense, and low optical throughput of existing hyperspectral imagers. We present the development of a new real-time hyperspectral endoscope (called the image mapping spectroscopy endoscope) based on an image mapping technique capable of addressing these challenges. The parallel high throughput nature of this technique enables the device to operate at frame rates of 5.2 frames per second while collecting a (x, y, λ) datacube of 350 × 350 × 48. We have successfully imaged tissue in vivo, resolving a vasculature pattern of the lower lip while simultaneously detecting oxy-hemoglobin.

Snapshot advantage: a review of the light collection improvement for parallel high-dimensional measurement systems.

Abstract. The snapshot advantage is a large increase in light collection efficiency available to high-dimensional measurement systems that avoid filtering and scanning. After discussing this advantage in the context of imaging spectrometry, where the greatest effort towards developing snapshot systems has been made, we describe the types of measurements where it is applicable. We then generalize it to the larger context of high-dimensional measurements, where the advantage increases geometrically with measurement dimensionality.

High numerical aperture microendoscope objective for a fiber confocal reflectance microscope

A disposable high numerical aperture microendoscope objective has been designed, fabricated, and tested for use with a fiber confocal reflectance microscope. The objective uses high precision LIGA fabricated components to integrate imaging components and hydraulic suction lines into a housing that measures only 3.85 mm in outer diameter and 14.65 mm in length. The hydraulics are used to translate tissue through the focal plane for three dimensional imaging. This device is diffraction limited for lambda = 850 nm, has a numerical aperture of 1.0, a field of view of 250 microm, and a working distance of 450 microm. The objective is intended for in vivo imaging of precancerous cells.

Development of image mappers for hyperspectral biomedical imaging applications.

A new design and fabrication method is presented for creating large-format (>100 mirror facets) image mappers for a snapshot hyperspectral biomedical imaging system called an image mapping spectrometer (IMS). To verify this approach a 250 facet image mapper with 25 multiple-tilt angles is designed for a compact IMS that groups the 25 subpupils in a 5 x 5 matrix residing within a single collecting objectives pupil. The image mapper is fabricated by precision diamond raster fly cutting using surface-shaped tools. The individual mirror facets have minimal edge eating, tilt errors of <1 mrad, and an average roughness of 5.4 nm.

Depth-resolved image mapping spectrometer (IMS) with structured illumination

We present a depth-resolved Image Mapping Spectrometer (IMS) which is capable of acquiring 4D (x, y, z, λ) datacubes. Optical sectioning is implemented by structured illumination. The device’s spectral imaging performance is demonstrated in a multispectral microsphere and mouse kidney tissue fluorescence imaging experiment. We also compare quantitatively the depth-resolved IMS with a hyperspectral confocal microscope (HCM) in a standard fluorescent bead imaging experiment. The comparison results show that despite the use of a light source with four orders of magnitude lower intensity in the IMS than that in the HCM, the image signal-to-noise ratio acquired by the IMS is 2.6 times higher than that achieved by the equivalent confocal approach.

Low cost, high performance, self-aligning miniature optical systems

The most expensive aspects in producing high quality miniature optical systems are the component costs and long assembly process. A new approach for fabricating these systems that reduces both aspects through the implementation of self-aligning LIGA (German acronym for lithographie, galvanoformung, abformung, or x-ray lithography, electroplating, and molding) optomechanics with high volume plastic injection molded and off-the-shelf glass optics is presented. This zero alignment strategy has been incorporated into a miniature high numerical aperture (NA = 1.0 W) microscope objective for a fiber confocal reflectance microscope. Tight alignment tolerances of less than 10 microm are maintained for all components that reside inside of a small 9 gauge diameter hypodermic tubing. A prototype system has been tested using the slanted edge modulation transfer function technique and demonstrated to have a Strehl ratio of 0.71. This universal technology is now being developed for smaller, needle-sized imaging systems and other portable point-of-care diagnostic instruments.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer.

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic &bgr;-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope.

Design and evaluation of an ultra-slim objective for in-vivo deep optical biopsy

An estimated 1.6 million breast biopsies are performed in the US each year. In order to provide real-time, in-vivo imaging with sub-cellular resolution for optical biopsies, we have designed an ultra-slim objective to fit inside the 1-mm-diameter hypodermic needles currently used for breast biopsies to image tissue stained by the fluorescent probe proflavine. To ensure high-quality imaging performance, experimental tests were performed to characterize fiber bundle’s light-coupling efficiency and simulations were performed to evaluate the impact of candidate lens materials’ autofluorescence. A prototype of NA = 0.4, 250-µm field of view, ultra-slim objective optics was built and tested, yielding diffraction-limited performance and estimated resolution of 0.9 µm. When used in conjunction with a commercial coherent fiber bundle to relay the image formed by the objective, the measured resolution was 2.5 µm.