Diffuse reflectance near infrared hyperspectral imaging is an important analytical tool for a wide variety of industries,
including agriculture, consumer products, chemical and pharmaceutical development and production. Using this
technique as a method for the standoff detection of explosive particles is presented and discussed. The detection of the
particles is based on the diffuse reflectance of light from the particle in the near infrared wavelength range where CH,
NH, OH vibrational overtones and combination bands are prominent.
The imaging system is a NIR focal plane array camera with a tunable OPO laser system as the illumination source. The
OPO is programmed to scan over a wide spectral range in the NIR and the camera is synchronized to record the light
reflected from the target for each wavelength. The spectral resolution of this system is significantly higher than that of
hyperspectral systems that incorporate filters or dispersive elements. The data acquisition is very fast and the entire
hyperspectral cube can be collected in seconds. A comparison of data collected with the OPO system to data obtained
with a broadband light source with LCTF filters is presented.
We present a novel hyperspectral imaging technique based on tunable laser technology. By replacing the broadband
source and tunable filters of a typical NIR imaging instrument, several advantages are realized, including: high spectral
resolution, highly variable field-of-views, fast scan-rates, high signal-to-noise ratio, and the ability to use optical fiber
for efficient and flexible sample illumination. With this technique, high-resolution, calibrated hyperspectral images over
the NIR range can be acquired in seconds. The performance of system features will be demonstrated on two example
applications: detecting melamine contamination in wheat gluten and separating bovine protein from wheat protein in
Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-µm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a “volume camera,” consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 µm of stage travel) to collect sharply focused images sampled at 0.3×0.3 µm2/pixel at a stage speed of 2.3 mm/s. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.
With recent advances in high-speed confocal imaging, data storage, and computational power, practical high-speed 3D cytometry instrumentation is on the horizon. For 3D cytometry to become practical for example from the perspective of a pathologist, speed attained in part by walk-away automation is fundamentally important. This level of automation can only be obtained with fully automated segmentation of image objects from background. Accuracy of this first image analysis task is crucial since it determines the results of all subsequent quantitative analyses. Confocal cell images often have low contrast due to both inherently low signal-to-noise ratios and high cell- cell contrast ratios that can occupy much of the available imaging dynamic range. A contrast-enhancing technique previously developed for 2D images of fluorescent cell nuclei was extended for 3D confocal images (stacks of 2D image slices). Edge sharpening and contrast-enhancement necessary for automatic thresholding are achieved by filtering with a finite impulse response (FIR) filter. These optimal FIR filters range in size from 3 X3X3X to 13X13X13 and were designed by utilizing the perceptron criterion and nonlinear least squares on confocal training datasets derived from fluorescent microspheres. By utilizing fluorescent beads of known shapes and sizes, the ideal (or standard) segmented image is known a priori. The contrast-enhancing performance of these filters on 3D confocal images of DAPI stained cell nuclei demonstrates that they should lead to accurate, fully automated 3D image segmentation.
Simultaneous multi planar microscope imaging enables parallel computation of autofocus for high-sped image cytometry. Although image cytometry exhibits many potential advantages over flow cytometry, substantially slower speed has limited use to fewer applications. In commercial image cytometry instruments, long scanning times have typically been circumvented by identification of small areas of interest during high speed, low resolution scans for subsequent analysis at high resolution. This two-pass strategy of analyzing only a few cells at high resolution is a disadvantage and often cannot be used at all where dim fluorescence demands higher numerical aperture (NA) objectives. Continuous stage motion synchronized with line array or time-delay-and-integrate (TDI) CCD image acquisition is capable of increasing scan speed by an order of magnitude or more, but until recently lacked the autofocus required for higher resolution objectives where depth of field is about the thickness of a cell monolayer. Here we describe an improved design for simultaneous multi planar acquisition and on-the-fly autofocus. This new system replaces more complicated and less light efficient fiberoptic imaging bundles with beamsplitters and mirrors. This new image-splitting design also enables addition of magnification correction optics not easily added to the earlier fiberoptic version. The result is a simplified, high sensitivity, magnification-matched, parallel multi planar acquisition module containing an array of CCD sensors for high-speed focus tracking and 3D imaging.