WO2002048692A1 - Apparatus and method for imaging a histological sample - Google Patents
Apparatus and method for imaging a histological sample Download PDFInfo
- Publication number
- WO2002048692A1 WO2002048692A1 PCT/GB2001/005432 GB0105432W WO0248692A1 WO 2002048692 A1 WO2002048692 A1 WO 2002048692A1 GB 0105432 W GB0105432 W GB 0105432W WO 0248692 A1 WO0248692 A1 WO 0248692A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sample
- autofluorescence
- imaging
- histological
- electromagnetic radiation
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Definitions
- This invention relates to a method for imaging a histological sample, associated apparatus and also an associated method of preparing histological samples.
- Imaging of histological samples is generally undertaken on sections or slices of sample that have been mounted on slides and then stained to reveal features within each sample slice.
- distortion of the slices occurs, for example due to shrinkage, causing a degree of misalignment between successive slices and complicating analysis of the features within the sample.
- Fiducial markers have been introduced into the sample prior to sectioning so that the distortion can be compensated for.
- sample preparation and subsequent image analysis is very time consuming and difficult to automate.
- An episcopic imaging method using bright field illumination and surface staining has been used to reveal tissue structure of a sample, with the uppermost surface of the sample being imaged as successive sections are in turn cut and removed.
- imaging the upper surface of the sample for around 1/100 th second before removing a thin slice to reveal the next surface of the sample distortion effects between the different images are avoided.
- the resolution of features within the sample tissue is limited due to penetration of the stain, as the depth of penetration of the stain restricts how thinly the sample can be sliced.
- a method of imaging a histological sample comprising stimulating autofluorescence in a sample and detecting autofluorescence in the sample, thereby to image features within the sample.
- Autofluorescence is the intrinsic fluorescence of a sample, and is an intrinsic property of the sample which under appropriate conditions can be stimulated to produce a weak intensity electromagnetic radiation.
- the stimulation of autofluorescence may be achieved by illuminating a sample surface with electromagnetic radiation in the range 400-750nm, preferably of wavelength 470nm. This may be achieved by using an excitation filter of 470nm wavelength in combination with a broadband electromagnetic radiation source.
- the method may further comprise detecting electromagnetic radiation produced by autofluorescence for a period of time.
- detection of the autofluorescence will generally be undertaken by a CCD camera, typically with an excitation filter of 510nm disposed between the camera and the sample, such that the camera records a large number of data sets over the period of time and obtains a large amount of data relating to autofmorescent radiation in the sample surface.
- the weak signal from autofluorescence can then be reinforced by manipulation of data acquired by the camera.
- the method may thus further comprise recording data relating to autofluorescence in a sample for subsequent analysis and manipulation to provide information on features within a sample.
- This data can be analysed and manipulated in a number of ways, for example by filtering, contrast enhancement, transforming to a grey-scale image. Movie generation of successive images is also possible by using simple programmes such as Quick Time, Movie Maker, Picts to Movie, to run a sequence of views one after the other. 3-D analysis and 3-D reconstruction may also be undertaken.
- the method may also comprise staining the sample, thereby to allow detection of specific staining patterns of particular cells or tissues within the sample in combination with obtaining an image of overall histological structure through detecting autofluorescence.
- the method may further comprise detecting autofluorescence from an upper face of the sample, removing an upper layer of the sample and imaging a next face of the sample, and repeating these steps thereby to obtain information on autofluorescence throughout the sample.
- the method enables detection of features for virtually any thickness of slice, and in particular slices can range from 50 ⁇ m to l ⁇ m, with the method generally being used to analyse slices of thickness 5-l ⁇ m.
- a method of preparing a histological sample for use in the above method comprising embedding the sample in a wax medium with stearic acid of around 3 weight per cent, the wax medium incorporating a dye to supress autofluorescence from below an upper surface of the sample.
- the chosen wax medium is more rigid than is usual for histological samples embedded in wax and as such is able to be sliced into thin sections of around l-2 ⁇ m.
- An example of a composition of wax medium in accordance with the invention is a wax medium comprising 73% Nybar, 24% paraffin wax, and 3% stearic acid containing 0.1% red aniline wax dye as supplied by Candlemakers Supplies London.
- the particular dye used allows for good surface autofluorescence at around 470/510 nm. However where other wavelengths are used to induce autofluorescence, an alternative dye may be chosen to supress autofluorescence.
- Another aspect of the invention lies in apparatus used for imaging autofluorescence in histological samples, the apparatus comprising a source of electromagnetic radiation adapted to stimulate autofluorescence in a sample, and a receiving means for receiving autofluorescent electromagnetic radiation emitted from a sample, thereby to stimulate and detect autofluorescence in a sample.
- the source of electromagnetic radiation produces radiation having a wavelength of around 470nm.
- the source may be provided by a pair of microscope lamps coupled to an input end of a fibre optic cable, an output end of the fibre optic cable having an excitation filter placed thereover so as to produce a wavelength of 470 nm.
- the receiver means may comprise a CCD camera in combination with an emission filter, the emission filter being placed over an input face to the CCD camera.
- the emission filter is chosen to filter out wavelengths other than 510nm.
- the CCD camera may be used with conventional software packages and data processing techniques to capture and analyse digital data relating to histological samples.
- the apparatus may further comprise a sample mounting means for mounting a sample thereon, and a cutting means for thinly slicing the sample. This allows an upper face of the sample to be examined, before slicing the upper face off to reveal to a next lower face for imaging. By slicing in this way throughout the sample so as to obtain a profile of the autofluorescence throughout the sample.
- the elements comprising the apparatus are in electrical communication with one another, so as to allow automation of the apparatus. This allows for automated examination of an entire sample.
- Figure 1 shows a schematic diagram of one embodiment of apparatus in accordance with the present invention
- Figure 2 shows a schematic diagram of a further embodiment comprising an automated system
- Figure 3 is a flow diagram of procedural steps undertaken when imaging histological samples, including sectioning, image capturing and image processing;
- Figure 4 is a flow diagram of steps undertaken during sectioning and image capturing
- Figure 5 is a flow diagram showing steps undertaken during image processing
- Figure 6 is a flow diagram showing steps undertaken during imagmg using the automated system.
- FIG. 1 illustrates apparatus 10 used for stimulating and analysing autofluorescence within a histological sample and which uses episcopic illumination and imaging by a CCD camera to view a surface of the sample.
- a mounting stage 12 of a sliding blade manual microtome 14 carries a sample 16 embedded in a wax mixture and an upper surface of the sample is illuminated by monochrome electromagnetic radiation of wavelength in the range 400- 750nm emitted by two microscope lamps 20, typically Scott KL2500 lamps.
- the radiation output of the lamps is directed along a wide fibre optic cable 22 with a focussing lens 24 at its output end and an excitation filter 26 of 470nm placed between the lens 24 and the sample 16.
- a bright field source is adapted to produce sufficient intensity of radiation to stimulate autofluorescence in the sample 16, and the radiation can be readily directed onto the sample using the adjustable fibre optic cable 22.
- the Monozoom microscope has a very long working distance and is adjusted so that the camera input views a clear focussed image of the sample face.
- An emission filter 36 of 510nm is placed between the camera input and the microscope, so that the camera 30 receives filtered radiation at the dominant wavelength of radiation emitted as a result of autofluorescence of the sample.
- a blade 40 mounted on the microtome is placed in proximity to the sample 16, so as to be able to slice away successive layers of the sample after imaging.
- the CCD camera 30 acquires a digital image of the sample surface by acquiring data in the usual way using a simple data capture software package (RS Image) run by a computer 42.
- the camera used is a relatively cheap, high resolution colour CCD device (1392 x 1040 pixels) and camera data is captured via a CoolSnap graphics PCI card as a 12 bit colour image.
- the resolution of the digital images is around 2 ⁇ m /pixel, although this can be increased.
- the sample is prepared in a manner already known for histological samples although the preparation process uses a novel embedding medium.
- embedding the sample one needs to replace water in the sample tissue with wax.
- the sample is exposed to increasing percentages of alcohol until gradually all water is replaced with 100% alcohol.
- the sample will be placed within 70%o ethanol, then 90% ethanol and lastly 100%) ethanol.
- the alcohol is then gradually replaced with an organic chemical, such as Toluol or Histoclear, which is miscible with alcohol and wax.
- the chemical is then replaced with molten wax to produce a sample where all the original water within the tissue is replaced with wax.
- the sample is placed in a mould and more wax added so as to provide a sample 16 where the sample tissue is infiltrated with wax and the sample is held within wax.
- the particular wax embedding medium chosen for use in the present invention consists of a mixture comprising Nybar (40-80%), paraffin wax (16-57%), and stearic acid (3-4%>) containing red aniline wax dye (0.1-0.35%>), as supplied from Candlemakers Supplies, London.
- This range of compositions produces a much stiffer embedding wax than is usual and allows the embedded sample to be thinly sliced at around a thickness of l-2 ⁇ m, as compared to a slicing thickness of 6-1 O ⁇ m for samples embedded in normal wax preparations.
- the red aniline wax dye is a liquid dye which supresses autofluorescence from below the top surface of the sample.
- the upper surface of the embedded sample is illuminated by radiation of wavelength 470nm which stimulates autofluorescence within a top layer of the sample of less than l ⁇ m thick.
- the sample surface emits radiation as a result of the autofluorescence, and this emitted radiation signal is detected by the CCD camera, which forms a digital image of the radiation over the sample surface.
- the radiation emitted by the sample varies over the surface depending on the tissue structure and thus by stimulating autofluorescence in the uppermost face 32 of the sample, an image of features within an upper thin layer of around 2 ⁇ m is obtained by the CCD camera 30. An intrinsic property of the sample is thus used to obtain information about the sample without the need for the sample to be stained.
- the autofluorescence is a very weak signal, and by carefully selecting the stimulating wavelength and the wavelength received by the CCD camera, a very clear image of features in the sample is obtained.
- a data set of 1000 images acquired over around three hours is sufficient to visualise an entire 16 day old embryo heart, when slicing in 2 ⁇ m sections.
- the data set can be as large as 6000 images.
- a complete data set for an embryonic heart is about 5 gigabytes in size.
- the degree of resolution obtained using detection of autofluorescence by the CCD camera is such that one can use the raw data from the camera as the sample image.
- enhancing and filtering steps can be carried out, and if appropriate 3-D models of the samples generated.
- the blade 40 is moved across to slice the sample at the chosen depth (usually 2 ⁇ m), and a slice of sample of the same thickness is removed. The camera then acquires image data for the next face of the sample. This process is repeated, slice by slice, until all the sample has been imaged as a series of layers. A three-dimensional reconstruction of the sample to a high degree of resolution is then possible. In previous episcopic imaging procedures, staining has been required to show features within the sample. However it is not possible to limit penetration of the stain to less than about 6 ⁇ m depth and thus structures within the sample can only be resolved to this limit.
- the detection of autofluorescence can also be combined with surface staining of the sample so as to obtain different types of information about the sample.
- specific regions of the sample can be stained to detect the presence of beta-galactosidase enzyme activity (a bacterial enzyme which has been introduced into the mouse genome by transgenesis and is expressed in only a subset of mouse tissues).
- beta-galactosidase enzyme activity a bacterial enzyme which has been introduced into the mouse genome by transgenesis and is expressed in only a subset of mouse tissues.
- FIG. 1 shows a further embodiment of the present invention in which the imaging process and analysis is automated.
- a sample block 50 is placed on a moveable mounting stage 52 of a rotary, fixed blade microtome 54 so that the sample can be moved to a rotary fixed blade 56 for sectioning.
- the sample 50 is illuminated by a light source 60 directed to the block surface through the optical path of a microscope 62 supported on a stand 64 and which has a filter wheel 66 containing filter sets of corresponding excitation and emission filters disposed between itself and the sample 50.
- a CCD camera 70 is placed at the uppermost end of the microscope 62 and computer connections 72, 74, 76 to the camera, filter wheel and microtome are provided.
- Figure 3 shows the various steps used to obtain images from a histological sample and Figure 4 shows a flow diagram illustrating how sectioning and imaging capturing is undertaken, Figure 5 shows a flow diagram illustrating how image processing is undertaken using Adobe Photoshop (batch function).
- Figure 6 shows a flow diagram illustrating control of the automated device of Figure 2 to obtain autofluorescence images of the sample.
- the method and apparatus disclosed herein is particularly suitable for accurate analysis of tissue, organ or embryo morphology and can be used for rapid routine screenings of the phenotype of genetically modified embryos, foetuses and neonates.
- the method also allows detection of spatially specific staining patterns within embryo or tissue sample, revealing for example gene expression or protein expression patterns by analysing whole mount stained samples. Routine pathological analysis of tissue samples is also possible.
Abstract
Description
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01270762A EP1350087A1 (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a histological sample |
AU2002256549A AU2002256549A1 (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a histological sample |
US10/433,870 US6992760B2 (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a histological sample |
JP2002549950A JP2004515780A (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a tissue specimen |
CA002427345A CA2427345A1 (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a histological sample |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0030310.7 | 2000-12-13 | ||
GBGB0030310.7A GB0030310D0 (en) | 2000-12-13 | 2000-12-13 | Apparatus and method for imaging a histological sample |
Publications (1)
Publication Number | Publication Date |
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WO2002048692A1 true WO2002048692A1 (en) | 2002-06-20 |
Family
ID=9904943
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Application Number | Title | Priority Date | Filing Date |
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PCT/GB2001/005432 WO2002048692A1 (en) | 2000-12-13 | 2001-12-11 | Apparatus and method for imaging a histological sample |
Country Status (7)
Country | Link |
---|---|
US (1) | US6992760B2 (en) |
EP (1) | EP1350087A1 (en) |
JP (1) | JP2004515780A (en) |
AU (1) | AU2002256549A1 (en) |
CA (1) | CA2427345A1 (en) |
GB (1) | GB0030310D0 (en) |
WO (1) | WO2002048692A1 (en) |
Cited By (3)
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WO2010138063A1 (en) * | 2009-05-29 | 2010-12-02 | General Electric Company | Method and apparatus for ultraviolet scan planning |
US11906723B2 (en) | 2018-11-01 | 2024-02-20 | Hust-Suzhou Institute For Brainsmatics | High-throughput optical sectioning three-dimensional imaging system |
US11940359B2 (en) | 2017-07-27 | 2024-03-26 | Agilent Technologies, Inc. | Preparation of tissue sections using fluorescence-based detection |
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DE102004022484B4 (en) * | 2004-05-07 | 2007-12-20 | P.A.L.M. Microlaser Technologies Ag | microscope stage |
DE102004023262B8 (en) * | 2004-05-11 | 2013-01-17 | Carl Zeiss Microimaging Gmbh | Method for processing a mass by means of laser irradiation and control system |
DE102005042367B4 (en) * | 2005-09-07 | 2009-11-12 | Leica Microsystems Cms Gmbh | Apparatus for generating 3-D images of a sample |
US7831075B2 (en) * | 2005-10-20 | 2010-11-09 | Case Western Reserve University | Imaging system |
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US7560689B2 (en) * | 2006-03-10 | 2009-07-14 | Canon Kabushiki Kaisha | High-sensitivity mass spectrometer and method |
US9008378B2 (en) | 2006-12-20 | 2015-04-14 | The Board Of Trustees Of The Leland Stanford Junior University | Arrangement and imaging of biological samples |
WO2010021744A1 (en) * | 2008-08-21 | 2010-02-25 | California Institute Of Technology | Microscope coupled tissue sectioning system |
JP5141816B2 (en) * | 2009-03-31 | 2013-02-13 | 株式会社島津製作所 | Mass spectrometer |
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JP7231345B2 (en) * | 2017-07-27 | 2023-03-01 | アジレント・テクノロジーズ・インク | Preparation of tissue sections using fluorescence-based detection |
DE102017128491B4 (en) * | 2017-11-30 | 2022-12-22 | Leica Biosystems Nussloch Gmbh | Microtome and method of positioning a microtome specimen head |
US20240102932A1 (en) * | 2021-02-01 | 2024-03-28 | Agilent Technologies, Inc. | Analysis of embedded tissue samples using fluorescence-based detection |
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- 2000-12-13 GB GBGB0030310.7A patent/GB0030310D0/en not_active Ceased
-
2001
- 2001-12-11 CA CA002427345A patent/CA2427345A1/en not_active Abandoned
- 2001-12-11 EP EP01270762A patent/EP1350087A1/en not_active Withdrawn
- 2001-12-11 US US10/433,870 patent/US6992760B2/en not_active Expired - Fee Related
- 2001-12-11 JP JP2002549950A patent/JP2004515780A/en not_active Withdrawn
- 2001-12-11 AU AU2002256549A patent/AU2002256549A1/en not_active Abandoned
- 2001-12-11 WO PCT/GB2001/005432 patent/WO2002048692A1/en not_active Application Discontinuation
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WO1998040007A1 (en) * | 1997-03-13 | 1998-09-17 | Biomax Technologies, Inc. | Methods and apparatus for detecting the rejection of transplanted tissue |
WO1999037204A1 (en) * | 1998-01-26 | 1999-07-29 | Massachusetts Institute Of Technology | Fluorescence imaging endoscope |
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WO2010138063A1 (en) * | 2009-05-29 | 2010-12-02 | General Electric Company | Method and apparatus for ultraviolet scan planning |
US8063385B2 (en) | 2009-05-29 | 2011-11-22 | General Electric Company | Method and apparatus for ultraviolet scan planning |
EP2435873A1 (en) * | 2009-05-29 | 2012-04-04 | General Electric Company | Method and apparatus for ultraviolet scan planning |
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CN102449529B (en) * | 2009-05-29 | 2014-08-20 | 通用电气公司 | Method and apparatus for ultraviolet scan planning |
US11940359B2 (en) | 2017-07-27 | 2024-03-26 | Agilent Technologies, Inc. | Preparation of tissue sections using fluorescence-based detection |
US11906723B2 (en) | 2018-11-01 | 2024-02-20 | Hust-Suzhou Institute For Brainsmatics | High-throughput optical sectioning three-dimensional imaging system |
Also Published As
Publication number | Publication date |
---|---|
US20040026630A1 (en) | 2004-02-12 |
JP2004515780A (en) | 2004-05-27 |
AU2002256549A1 (en) | 2002-06-24 |
GB0030310D0 (en) | 2001-01-24 |
CA2427345A1 (en) | 2002-06-20 |
US6992760B2 (en) | 2006-01-31 |
EP1350087A1 (en) | 2003-10-08 |
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