Approaches to Spectral Imaging Hardware
Instruments used for spectral, multispectral, and hyperspectral imaging in the biosciences have evolved significantly over the last 15 years. However, very few are calibrated and have had their performance validated. Now that spectral imaging systems are appearing in clinics and pathology laboratories, there is a growing need for calibration and validation according to universal standards. In addition, some systems produce spectral artifacts that, at the very least, challenge data integrity if left unrecognized. This unit includes a comparison of the band-pass and light-transmission characteristics of electronic tunable filters, interferometers, and wavelength-dispersive spectral imaging instruments, as well as a description of how they work. Methods are described to test wavelength accuracy and perform radiometric calibration. A real-life example of spectral artifacts is dissected in detail in order to show how to detect, diagnose, verify, and work around their presence when they cannot be eliminated. Curr. Protoc. Cytom. 53:12.20.1-12.20.40. © 2009 by John Wiley & Sons, Inc.
Keywords: acousto optic tunable filter; liquid crystal tunable filter; imaging spectrometer; prism spectrometer; diffraction grating ghosts; diffraction grating spectrometer; holographic diffraction grating; ruled diffraction grating
Table of Contents
- Spectral Imaging Basics
- Wavelength-Dispersive Spectral Imaging (WDSI)
- Need to Know Diffraction Grating Characteristics
- Electronic Tunable Filters (ETF)
- System-Based Light Throughput Considerations
- Wavelength Detectors
- Calibration, Validation, and Radiometry
- Comparison of Spectral Imaging Systems
- Contact Information
- Literature Cited
Figure 12.20.1 (A) A nanowire internalized within a living cell as seen in polarized light, in transmission. (B) A spectral image of the nanowire in panel A; the spectrum confirms the presence of gold and nickel.
Figure 12.20.2 The spectral lines from a MIDL wavelength calibration lamp are shown in the top panel. The bottom panel shows the spectrum; each line is an image of the entrance slit of the spectrometer.
Figure 12.20.5 Prism-based imaging spectrograph employs a prism with curved sides to correct astigmatism and other aberrations over a wide FOV.
Figure 12.20.6 Refractive index and wavelength dispersion curves for a flint prism. Band-pass decreases nonlinearly with wavelength.
Figure 12.20.7 Spectral resolution is measured at the full width at half maximum of an infinitely narrow emission line. In this case, the 405-nm Hg line is resolved with an optimum of three camera pixels.
Figure 12.20.8 The diffraction efficiency profiles of a ruled and a holographic grating present discontinuities in their efficiency profiles. The internal transmission curve of a flint glass prism offers a flat response from 365 nm to 950 nm.
Figure 12.20.9 (A) A 600 g/mm diffraction grating distributes light at 532 nm into 3 positive and 3 negative orders. (B) A 150 g/mm diffraction grating distributes light at 532 nm into 12 positive and 12 negative orders.
Figure 12.20.16 (A) LCTF transmission is the product of the transmission of each cell in the assembly. (B) LCTF percent transmission bands in randomly polarized light; spectral overlap increases with wavelength.
Figure 12.20.17 Layout of a Sagnac interferometer. An image of a fixed FOV is acquired at each wavelength sequentially when the Optical Path Difference is changed by rotating mirrors M1 and M2.
Figure 12.20.18 (A) QE curves of a PMT and a scientific CCD camera. Quantum curves vary widely from PMT to PMT and camera to camera. (B) Relative intensity curves for halogen and xenon lamps.
Figure 12.20.19 (A) The spectrum of an MIDL wavelength calibration lamp presenting both Hg and Ar lines. The Ar lines fade rapidly after the lamp is turned on, and the Hg lines increase in intensity. (B) Chart of wavelengths present in a MIDL lamp.
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