Expand this Topic clickable element to expand a topic
Skip to content
Optica Publishing Group
Journal Home About Issues in Progress Current Issue All Issues Feature Issues

Large-image-format computed tomography imaging spectrometer for fluorescence microscopy

Bridget K. Ford, Michael R. Descour, and Ronald M. Lynch

Open Access Open Access

Abstract

Multispectral imaging has significantly enhanced the analysis of fixed specimens in pathology and cytogenetics. However, application of this technology to in vivo studies has been limited. This is due in part to the increased temporal resolution required to analyze changes in cellular function. Here we present a non-scanning instrument that simultaneously acquires full spectral information (460 nm to 740 nm) from every pixel within its 2-D field of view (200 µm×200 µm) during a single integration time (typically, 2 seconds). The current spatial and spectral sampling intervals of the spectrometer are 0.985 µm and 5 nm, respectively. These properties allow for the analysis of physiological responses within living biological specimens.

©2001 Optical Society of America

Full Article  |  PDF Article
More Like This
Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy

Liang Gao, Robert T. Kester, and Tomasz S. Tkaczyk
Opt. Express 17(15) 12293-12308 (2009)

Assessment of a computed tomography imaging spectrometer using an optimized expectation-maximization algorithm

F. Narea-Jiménez, J. Castro-Ramos, J. J. Sánchez-Escobar, and A. Muñoz-Morales
Appl. Opt. 61(20) 6076-6085 (2022)

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

Liang Gao, Robert T. Kester, Nathan Hagen, and Tomasz S. Tkaczyk
Opt. Express 18(14) 14330-14344 (2010)

View More...

Supplementary Material (1)

Media 1: MOV (820 KB)     

Cited By

Optica participates in Crossref's Cited-By Linking service. Citing articles from Optica Publishing Group journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (9)
Fig. 1.
Fig. 1. Digital image of the large-format CTIS aligned to the side photo port of an Olympus IX70 inverted fluorescence microscope.

Download Full Size | PDF

Fig. 2.
Fig. 2. Optical microscope image of a segment of the CGH disperser (Courtesy of D. W. Wilson and P. D. Maker, Jet Propulsion Laboratory).

Download Full Size | PDF

Fig. 3.
Fig. 3. Mapping of the signal from individual voxels to the imaging array. The signal on the imaging array is distributed in a diffraction pattern that depends on a voxel’s (x, y, λ) coordinates in the object cube. Calibration images are shown for two different voxels that occupy the same spatial position but different spectral bands. Part (a) corresponds to a voxel with a center wavelength of 470 nm while Part (b) corresponds to a center wavelength of 740 nm.

Download Full Size | PDF

Fig. 4.
Fig. 4. (821 KB) Movie demonstrating how the CTIS maps a 3-D (x,y,λ) object cube onto a 2-D image plane. The sequence corresponds to simulated raw images of the same specimen viewed through a filter of increasing spectral bandwidth. The movie begins with a narrow-band image (500 nm–510 nm). The bandwidth increases in steps of 10 nm to a maximum of 240 nm (500 nm–740 nm). The simulated object cube consists of eight spheres with four different emission maxima (530 nm, 515 nm, 560 nm, 615 nm).

Download Full Size | PDF

Fig. 5.
Fig. 5. Representative raw image of embryonic rat cerebellum glia. The cells were probed with primary antibodies to glial fibrillary acidic proteins (GFAP) and subsequently labeled with Alexa Fluor 488 goat anti-rabbit IgG (H+L) conjugate secondary antibody and the DNA/RNA probe, Ethidium Bromide. The image was taken using a 40x, NA=1.35 microscope objective. Object cubes were reconstructed with 55 spectral bands (470nm–740 nm, spectral sampling distance of 5 nm) and 203×203 spatial resolution elements. The spatial sampling distance between adjacent pixels is 0.985 µm for this application

Download Full Size | PDF

Fig. 6.
Fig. 6. Reconstructed image of a mixture of microspheres with different emission spectra. Color image is generated by converting the reconstructed spectra into sets of RGB values.

Download Full Size | PDF

Fig. 7.
Fig. 7. Reconstructed spectra corresponding to three different locations within the microsphere sample. See Fig. 6 for locations (A), (B) and (C). Crosses denote comparison spectra measured with a radiometrically calibrated non-imaging spectrometer.

Download Full Size | PDF

Fig. 8.
Fig. 8. Color image reconstructed from the raw image shown in Figure 5. Green fluorescence indicates the distribution of GFAP while orange-red indicates nucleic acid distribution.

Download Full Size | PDF

Fig. 9.
Fig. 9. Reconstructed spectra of two pixel locations within the reconstructed image of Figure 8. The locations (A) and (B) are indicated in Figure 8.

Download Full Size | PDF

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

f ̂ k + 1 = f ̂ k H T g H T H f ̂ k ,
Select as filters
Select Topics Cancel
Top
       
© Copyright 2024 | Optica Publishing Group. All rights reserved, including rights for text and data mining and training of artificial technologies or similar technologies.
Privacy | Terms of Use