Hyperspectral Imaging Basics

"Analytical Hyperspectral Systems Convert Spectral
Signatures Into Targeted Images"

Advanced hyperspectral imaging systems acquire all wavelengths simultaneously by using an imaging spectrometer coupled to a CCD. All spectra that meet a user-determined threshold are pseudo-colored, and mapped onto a grayscale image of the sample.

Introduction

The earliest use of hyperspectral imaging occurred in the remote sensing community with Landsat. This instrument used just four filters yet even so was remarkably successful.

Successor instruments now use hundreds of wavelength data points that are acquired with imaging spectrometers rather than filters. Remote Earth sensors "fly-over" a field of view (FOV) collecting a continuous stream of data.

PARISS has its origins in the remote Earth sensing community and uses similar methods to characterize biological and other materials. Rather than moving PARISS, we translate the FOV with a computer controlled microscope stage. This technique is commonly known as "push-broom" spectral imaging.

How the PARISS Wavelength Dispersive Hyperspectral Imaging System Works

The following animation illustrates how this works with the PARISS system: The movie is broken up into four parts

1. An animation showing schematically how PARISS works
2. A spectral library of eight highly overlapping fluorophores
3. A hyperspectral image showing how the eight fluorophores were "unmixed" in objects in the FOV
4. How individual spectra can be identified for further study, or classification

Figure 1: Bioconjugated Au nanoparticles internalized within living cells observed in dark field	Figure 2: Movie illustrating how PARISS works: After the movie has finished downloading you, can "jump" between segments by clicking in the left side-column. You can also "stop," "pause" or "start" at any time	Figure 3: Tissue section stained with Alexa 555, 569 and 594. The distribution of each fluorophore is clearly segmented.

For simplicity, we chose to demonstrate the principle with one of Invitrogen Corp's FocalCheck (TM) Fluorescent Microsphere slides. Each microsphere presents one of eight fluorophores with different emission intensities, each of which was characterized by PARISS and inserted into a spectral library . All the fluorophores were excited at ~436 nm, and all wavelengths were acquired simultaneously.

Each Library spectrum was assigned a pseudo-color. If a spectrum from the FOV correlates at greater than a certain user-selected percentage (we used 98% in this experiment) then it was painted to an image-pixel in the FOV. All spectra that failed to correlate appeared in grayscale units.

Unlike the majority of algorithms presently used in competitors spectral imaging applications the PARISS "unmixing" software does not require that spectra mix linearly. The figures 1 and 3 show two examples of how PARISS was able to segment, and differentiate, highly complex spectral mixtures in "difficult" samples.

Security settings and your ability to play the animations
Your computer may have security settings that will not allow the flash application to run unless you have deliberately enabled it. You may have to keep your finger on the "CTRL" when you click the link or answer "yes" to a message at the top of the content window. If you have a problem getting the animation to run, or you would like to discuss it, call me!

Most if not all versions of Windows XP and Vista already have a Flash player installed. If you have Win 2000 or an older version of XP you can download a free version of Macromedia Flash here. Note you may have to reboot your computer before it takes effect.

Prism assembly is manufactured under a license to US patent 5,127,728 owned by The Aerospace Corporation.
_________________________