WO1999035463A1 - Spectral imaging apparatus and methodology - Google Patents
Spectral imaging apparatus and methodology Download PDFInfo
- Publication number
- WO1999035463A1 WO1999035463A1 PCT/US1999/000319 US9900319W WO9935463A1 WO 1999035463 A1 WO1999035463 A1 WO 1999035463A1 US 9900319 W US9900319 W US 9900319W WO 9935463 A1 WO9935463 A1 WO 9935463A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sample
- interferometer
- interferogram
- detector
- detector array
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 32
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J3/2823—Imaging spectrometer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J3/45—Interferometric spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
- G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
- G01J3/28—Investigating the spectrum
- G01J2003/2866—Markers; Calibrating of scan
Definitions
- FISH fluorescence in situ hybridization
- a multi-colored electrophoresis pattern reading apparatus which irradiates a sample with one or more light sources.
- the light sources can either be used individually or combined into a single source.
- Optical filters are used to separate the fluorescence resulting from the irradiation of the sample into a plurality of fluorescence wavelengths.
- U.S. Patent No. 5,190,632 a multi-colored electrophoresis pattern reading apparatus is described in which one or more light sources are used to generate a mixture of light capable of exciting two or more fluorescent substances. Both optical filters and diffraction gratings are used to separate the fluorescence by wavelength.
- the plate may be illuminated with light of a second wavelength and the scattered light versus location monitored.
- the second wavelength is selected such that it does not exhibit increased scattering for the objects of interest and therefore represents background scatter.
- Fig. 7 illustrates one aspect of the invention that can be used to quickly obtain a fringe free image of the sample
- Fig. 8 illustrates the polarizing beam splitter used in a specific embodiment of the invention
- This type of probe is very useful for determining chromosome structure, as they more or less uniformly hybridize to the entire length of a given chromosome. Paints are used to determine chromosome complements of a cell, structural abnormalities such as translocations, and to identify the origin of marker chromosomes.
- an epifluorescence/phase microscope 201 creates an image of a sample (not shown) which is then relayed to a Sagnac interferometer 203 using relay optics 205.
- optics 205 form a collimated beam containing the sample image.
- Fig. 3 illustrates a monolithic form of interferometer 300 that can also be used with the present invention.
- the monolithic interferometer is more immune to vibration, misalignment, and thermal effects than other forms of interferometers.
- the monolithic interferometer also has a very large acceptance angle.
- Interferometer 300 is comprised of a first piece of glass 301 bonded to a second piece of glass 303 along the plane of a beam splitter coating 305. Light is incident on the interferometer along path 307. When this light ray hits beam splitter coating 305, the ray is split into two rays, one ray following path 309 and the other ray following path 31 1. After being reflected by interferometer mirrors 313, the rays exit the optic along paths 315 separated by a distance 317.
- the interferometer of the spectral discriminator must be properly aligned.
- the following procedure has been determined to be both rapid and accurate. Referring to Fig. 4, the alignment procedure assumes the use of a Sagnac interferometer 400 and collimating input and output relay lens 401 and 403, respectively.
- the laser can be removed, the 10X objective replaced, and the fringes due to source 427 of microscope 409 incident on array 421 may be observed via monitor 425.
- laser 407 and mirror 41 1 are removed, the 10X objective is re-inserted into the system, and darkfield light source 427 and detector array 421 are turned on.
- the output of microscope 409 in conjunction with interferometer 400 is then centered onto array 421 by adjusting mirrors 417 and 419. Tilting mirrors 417 and 419 accomplish two things. If the two mirrors are tilted in the same direction relative to the beam path, the fringes will be changed. If the two mirrors are tilted in opposite directions relative to the beam path, the image will be shifted. This is true along both the x- and y-axes of the image.
- Calibration slit 503 is illuminated by a light source 505.
- the light from source 505 is focussed onto slit 503 with one or more focussing optics 507.
- the image of slit 503 is collected by optics 401, passed through the spectral discriminator 400, and imaged onto detector 421 by output relay optics 403.
- the image of slit 503 can be recorded in an adjacent, unused portion of array 421.
- At least two, and preferably three, wavelengths must be used during the system calibration. This allows a range of wavelengths to be interpolated from the two or more known wavelengths.
- multiple laser diodes 505 are utilized, each operating at a different, known wavelength.
- source 505 emits multiple wavelengths and one or more bandpass filters 509 are used to select the desired wavelength.
- Fig. 7 illustrates one aspect of the invention that can be used to quickly obtain a fringe free image of a sample.
- the spectral discriminator 700 of Fig. 7 is suitable for inclusion within a spectral imaging system such as that illustrated in Figs. 2, 4, and 5.
- spectral discriminator 700 includes a beam splitter 701, a first turning mirror 703, and a second turning mirror 705.
- this system includes an optic 707 that can be inserted in front of one of the mirrors, in this case mirror 703, without altering the position of either turning mirror.
- the insertion of optic 707 produces an extremely large offset between the legs of the interferometer, thus causing the fringe density to become too large to resolve by the individual pixels of detector array 709. Therefore it is not necessary to computationally remove the fringe bars and a monochrome image of the sample is obtained with a single frame exposure.
- optic 707 is a window 8 millimeters thick formed of BK-7 with less than 3 arc minutes of wedge.
- optic 707 is coated with an anti-reflection coating.
- optic 707 may be manually inserted into the beam path, in the preferred embodiment optic 707 is attached to a translation stage 711 that is coupled to a motor 713. Stage 711 and motor 713 allow the user to easily insert optic 707 whenever a fringe free, monochrome image of the sample is desired.
- beam splitter 801 may be replaced by a polarizing beam splitter 801 as illustrated in Fig. 8.
- Polarizing beam splitter 801 offers several benefits including increased efficiency and decreased ghosting.
- beam splitter 801 for example a polarizing beam splitter cube, preferentially reflects one polarization (e.g., s-polarization) while preferentially transmitting a second polarization (e.g., p-polarization).
- spectral imaging system of the present invention When using the spectral imaging system of the present invention, it is often desirable to be able to locate a specific area of interest. For example, in systems designed to study and identify chromosomes, the ability to locate metaphase spreads is desirable. If such an area can be quickly and efficiently located, the user can avoid the time consuming task of processing the entire sample in detail. Rapid location techniques are especially important in conjunction with the present invention due to the time required to process an interferogram.
- the present invention takes advantage of the phenomena commonly known as Rayleigh scattering, referring to the scattering of light by objects that are small in comparison to the wavelength of the incident light.
- Fig. 9 illustrates the basic concept of the invention.
- One or more metaphase spreads are contained on a microscope slide 901 along with various other cell matter and debris.
- Light of a first wavelength from a source 903 irradiates slide 901 and a detector 905 monitors the intensity of scattered light versus location.
- the wavelength of source 903 is selected to achieve maximum scattering given the dimensions of the metaphase spreads (or other objects) in question.
- slide 901 is also irradiated by light of a second wavelength, either by altering the wavelength of source 903 or simply substituting a different source.
- the second wavelength is selected such that it does not exhibit increased scattering for the objects in question.
- a laser may be used as the light source and the relative positions of the laser and the detector may remain constant.
- a x-y translation stage e.g., numerically controlled stage
- the scattering from the entire sample may be mapped.
- the beam from the laser(s) may be raster scanned across the sample.
- this approach requires accurate knowledge of the beam location on the sample in order to be able to later return to the locations containing the objects of interest (e.g., metaphase spreads).
- the light scattered from the sample may be imaged onto a detector array, thus offering a one-to-one correspondence between locations on the sample and the detector array.
- the sample may either be raster scanned using a laser source, or the entire sample may be illuminated by a large area source in the wavelength or wavelengths of interest.
- an entire sample 1001 may be illuminated by a single source 1003.
- a detector array 1005 monitors the scattered radiation passing through sample 1001, thus providing both the intensity of the scattered radiation as well as the locations of maximum scatter.
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Physics & Mathematics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Spectrometry And Color Measurement (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000527803A JP3712937B2 (en) | 1998-01-07 | 1999-01-06 | Apparatus and method for calibration of a spectral imaging system |
DE69941565T DE69941565D1 (en) | 1998-01-07 | 1999-01-06 | DEVICE AND METHOD FOR SPECTRAL IMAGING |
EP99902098A EP1046016B1 (en) | 1998-01-07 | 1999-01-06 | Spectral imaging apparatus and methodology |
CA002316984A CA2316984C (en) | 1998-01-07 | 1999-01-06 | Spectral imaging apparatus and methodology |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/004,180 US6051835A (en) | 1998-01-07 | 1998-01-07 | Spectral imaging apparatus and methodology |
US09/004,180 | 1998-01-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999035463A1 true WO1999035463A1 (en) | 1999-07-15 |
Family
ID=21709565
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/000319 WO1999035463A1 (en) | 1998-01-07 | 1999-01-06 | Spectral imaging apparatus and methodology |
Country Status (6)
Country | Link |
---|---|
US (3) | US6051835A (en) |
EP (1) | EP1046016B1 (en) |
JP (2) | JP3712937B2 (en) |
CA (1) | CA2316984C (en) |
DE (1) | DE69941565D1 (en) |
WO (1) | WO1999035463A1 (en) |
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DE102010024266A1 (en) * | 2010-06-18 | 2011-12-22 | Karlsruher Institut für Technologie | Micro-optical interferometer i.e. optical delay interferometer, for e.g. refraction index measurement, has monolithic molded bodies with optical boundary surfaces having entry point, beam splitter, reflection point and interference point |
Also Published As
Publication number | Publication date |
---|---|
DE69941565D1 (en) | 2009-12-03 |
CA2316984C (en) | 2006-03-14 |
US20060250616A1 (en) | 2006-11-09 |
JP2005164612A (en) | 2005-06-23 |
EP1046016A4 (en) | 2004-05-26 |
US6108082A (en) | 2000-08-22 |
JP3712937B2 (en) | 2005-11-02 |
CA2316984A1 (en) | 1999-07-15 |
JP2002500375A (en) | 2002-01-08 |
EP1046016B1 (en) | 2009-10-21 |
US7330268B2 (en) | 2008-02-12 |
US6051835A (en) | 2000-04-18 |
EP1046016A1 (en) | 2000-10-25 |
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