WO2002006880A2

WO2002006880A2 - Plastic substrates slide apparatus for infrared microscopy analysis

Info

Publication number: WO2002006880A2
Application number: PCT/CA2001/001031
Authority: WO
Inventors: Paul Anthony Presta; Michael Maszkiewicz
Original assignee: Veracel Inc.
Priority date: 2000-07-17
Filing date: 2001-07-16
Publication date: 2002-01-24
Also published as: AU2001275617A1; WO2002006880A3

Abstract

A slide apparatus with a plastic substrate for infrared microscopy analysis. The plastic substrate is formed from material with optical transmission characteristics in the mid-infrared region or 7 to 12 micron range. In one embodiment, the plastic substrate is formed from polyethylene (PE) and provides optical transmission in the mid-infrared bands 975 to 1000 cm-1 and 1155 to 1180 cm-1. In another embodiment, the plastic substrate is formed from polymethyl pentene (TPX) and also provides optical transmission in the mid-infrared bands 975 to 1000 cm-1 and 1155 to 1180 cm-1.

Description

TITLE: PLASTIC SUBSTRATE SLIDE APPARATUS FOR INFRARED

MICROSCOPY ANALYSIS

FIELD OF THE INVENTION

The present invention relates to medical and biological specimen analysis, and more particularly to a plastic substrate slide apparatus and technique for infrared microscopy analysis.

BACKGROUND OF THE INVENTION

Fourier Transform Infrared (FTIR) spectroscopy is an established technique for the extraction of quantitative measures in a wide variety of materials. Recently, FTIR spectroscopy has been applied to human tissue samples in order to discriminate cancerous or pre-cancerous tissue from normal tissue.

Many of the absorption spectra of organic compounds are generated by the vibrational overtones or the combination bands of the fundamentals of 0-H, C-H, N-H, and C-C transitions. These transitions in the near-infrared regions produce spectra in the easily accessible range between 0.7 microns and 2.5 microns. However, the strengths of these spectra are one to three orders of magnitude smaller than the associated fundamentals. As a result special care and steps must be taken to recover and analyze this information.

Research has revealed that the mid-Infrared (i.e. 2.5 - 25 microns) response of normal and pre-cancerous human tissue comprises a variety of structural and chemical changes which may be used to discriminate between the two. These changes include increases in glycogen content, extensive hydrogen bonding of phosphodiester groups in nucleic acids, tighter physical packing of nucleic acids, phosphorylation of C-OH groups in carbohydrates and proteins, increased disorder of methylene chains in membrane lipids, increased ratio of methyl to methylene, reduction in the hydrogen bond strength in the amide groups of α-helical segments and an increase in the hydrogen bond strength in the amide groups of the β-sheet segments.

Proposals have been made to utilize these mechanisms in cancer screening protocols, such as, the early detection of pre-cancerous lesions of the uterine cervix and carcinomatous breast tissue.

Automated systems for the rapid and accurate screening of cytological specimens are being developed to address cost, labour and liability issues. An example is the Pap test, a screening test that searches for evidence of precancerous lesions in exfoliated cervical cells. Involving the manual examination of tens of thousands of cervical epithelial cells, the Pap test is costly to apply and is subject to human error. Nevertheless, it has an enviable record of reducing cervical cancer mortality in the countries where it is applied. As a result considerable effort has been put into developing automated alternatives for the manual Pap test.

The most successful automated Pap test systems emulate cytotechnologists, the highly-trained professionals that screen this and other tests. As the cytotechnologist relies on the visual evaluation of cervical cells, so the automated systems depend upon some type of image analysis. For machines, image analysis may be thought of as a series of four steps. First, the microscopic image is digitized, putting it in a form that may be readily used by electronic hardware and the computer software. Second, the image must be segmented. Segmentation is generally the most difficult and crucial step for image analysis systems. Segmentation involves the separation of the relevant portions of the digitized image from everything else. Since this must be done well in advance of any pattern recognition operation, the segmentation procedure must be designed to use visual keys such as edges to find and separate the important image components. The third step is known as feature extraction. During feature extraction, each of the segmented regions or objects in the image is subjected to a range of mathematical measures that seek to encapsulate the visual appearance in numerical form. The fourth step and final step is classification. Classification comprises utilizing the numerical features to arrive at some type of conclusion about the object's identity.

As applied to cervical epithelial cells in a Pap test for example, the mid- infrared absorption properties of the cervical epithelial cells may be used to yield a new and useful channel of information in addition to the usual visible-light imaging measures. As described in U.S. Patent No. 6,181 ,414 owned by the common assignee of the subject application, these new mid-IR channels may be used to improve segmentation performance, to create a new set of features for classification, or as discrimination measures on their own. It has been found that restricting the use of the mid-IR channels to regions of the samples that contain nuclear material improves the effective signal-to-noise ratio of the measurement over bulk tissue measurements offering a greater power of discrimination to the technique. The use of a differential mid-IR measure (one channel acting as an interrogator, the other as a reference) allows a high-speed real-time scan of cellular material to quickly discriminate between normal and abnormal cells or tissue and may be extended to other important diagnostic properties of the cells.

The nature of the substrate or carrier holding the specimen poses an acute problem for practical applications of mid-infrared spectroscopy to anatomic pathology. The most common substrate for anatomic pathology evaluations is the glass microscope slide. Glass slides are typically manufactured from borosilicate glass to relatively relaxed tolerances. The problem with borosilicate glasses (and most other reasonable substitute glasses) is the lack of a suitable transmission window for mid-infrared radiation. As shown in Fig. 4, the optical transmission of borosilicate glass declines rapidly after 1.5 microns. This is well short of the mid-IR region of interest which falls between 7 and 12 microns. Thus, the interrogation signal required by an automated system is essentially absorbed indiscriminately by the glass substrate.

There are known glass compositions, such as BaF₂ and ZnS, which transmit infrared radiation. However, there are several drawbacks to these glass compositions. First, the known infrared transparent glass compositions are expensive and not well suited to manufacture in forms appropriate to standard cytology and involve difficult-to-handle compounds. Secondly, known infrared transparent glass compositions pose health risks through their use by and near human beings. Yet another problem arises from unwanted interactions between these materials and the various compounds used to stain and otherwise process cellular specimens in the cytology laboratory. Accordingly, there remains a need for apparatus which overcomes the shortcomings associated with glass slides in order to better exploit the advantages of infrared microscopy analysis.

BRIEF SUMMARY OF THE INVENTION The present invention provides an apparatus and technique for a microscope slide or carrier suitable for application of mid-IR spectroscopic techniques, such as, discrimination between normal and pre-cancerous cells prepared in the form cells or cell groupings in a specimen placed on the slide.

According to one aspect, the present invention provides a microscope slide comprising a plastic substrate having mid-infrared transmissive characteristics in the range of 7 to 12 microns.

In a first aspect, the present invention provides a slide apparatus for infrared microscopy analysis of a biological specimen, the slide apparatus comprises: (a) a plastic slide substrate for receiving the biological specimen; (b) the plastic slide substrate has optical transmission characteristics in the mid- infrared range.

In a second aspect, the present invention provides a slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 975 to 1000 cm^-1. ln another aspect, the present invention provides a slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 1155 to 1180 cm^"1.

In further aspect, the present invention provides slide a apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 975 to 1000 cm^"1.

In yet another aspect, the present invention provides slide a apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, the slide apparatus comprises: (a) a plastic slide substrate for receiving the cytological sample; (b) the plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 1155 to 1180 cm^"1.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

Reference will now be made to the accompanying drawings, which show, by way of example, a preferred embodiment of the present invention, and in which:

Fig. 1 shows in diagrammatic form a plastic slide apparatus suitable for infrared microscopy analysis according to the present invention;

Fig.2 shows in graphical form the optical transmittance characteristics for the plastic slide formed from polyethylene (PE) according to one embodiment of the invention;

Fig.3 shows in graphical form the optical transmittance characteristics for the plastic slide formed from polymethyl pentene (TPX); and

Fig.4 shows in graphical form the optical transmittance characteristics for a conventional borosilicate glass specimen slide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference is first made to Fig. 1 , which shows in diagrammatic form a plastic slide 10 suitable for infrared microscopy analysis in accordance with the present invention. The slide 10 comprises a substrate or carrier indicated by reference 12. Cells indicated by reference 20 are deposited on the slide substrate 12. The cells 20 may be covered by a plastic cover slip 14. The plastic cover slip 14 is formed from the same material as the substrate 12. As will be described in more detail below, the plastic slide 10 provides optical transmission in the mid-infrared region in the range of 7 to 12 microns.

Once deposited on the plastic slide 10, the cells 20 may be stained for the usual visual analysis (i.e. the Papanicolaou procedure for epithelial cell observations) and mounted in the usual mounting medium in an automated analysis system for example.

The cells in the specimen 20 that respond to the stain comprise naturally- derived, encapsulated protein and this is what the stains will generally bind to. For example, in the Pap stain, the hematoxylin will bind to DNA or RNA in the cell nucleus, and the Orange G stain will selectively bind to keratin in the cytoplasm, etc.

In the context of automated analysis of cervical cells for pre-cancerous and cancerous effects, the presence of an infrared absorbance signal at 975 cm^" ¹ to 1000 cm^"1 and again at 1155 cm^"1 to 1180 cm^"1 may be used to discriminate between normal and abnormal cells. The infrared band between 975 cm^"1 to 1000 cm^"1 corresponds to the symmetric stretching vibration of the dianionic phosphate monoesters of phosphorylated proteins and nucleic acids. The infrared band at 1155 cm^"1 to 1180 cm^"1 is associated with the stretching vibrational modes of the C-O groups serine, threonine, or tyrosine amino acid residues of cellular proteins. Techniques for infrared spectroscopy for medical imaging are described in U.S. Patent No. 6,181 ,414 which issued January 30, 2001 , and is owned by the common assignee of the subject application, and is herein incorporated by reference. According to one embodiment of the invention, the substrate 12 for the plastic slide 10 is manufactured from polyethylene (PE). The cellular sample 20 to be analyzed is fixed using known techniques and then deposited onto the surface of the plastic slide 10. Reference is made to Fig. 2, which shows in graphical form optimal transmittance characteristics for polyethylene. As shown, polyethylene has an optical transmission in the mid-IR 'windows' or bands of 975 - 1000 cm^"1 and 1155 - 1180 cm^"1 which is never less than 50%.

According to another embodiment of the invention, the substrate 12 for the plastic slide 10 is made from polymethyl pentene (TPX). As shown in Fig. 3, the substrate 12 of polymethyl pentene shows an optical transmission of more than 20% in the mid-IR bands of 975 - 1000 cm^"1 and 1155 - 1180 cm^"1.

According to another aspect of the invention, a cell or cells 20 deposited on the plastic slide 10 are analyzed according to the following method. In a first step, the cell or cells 20 of interest on the slide 10 are brought into focus using conventional light microscopy by either automatic or manual means. Once the focal position is set, the microscope stage is translated to a blank region that contains no cells at all. At this 'blank' position a background reading is obtained for the infrared absorption through the substrate 12 of the plastic slide 10 using an infrared microscopy apparatus (not shown). The background infrared bands of interest lie between 970 cm^"1 and 1000 cm^"1 and again between 1145 cm^"1 and 1190 cm^"1.

Next in data acquisition mode, the slide 10 carrying the sample or specimen is rapidly scanned with the infrared microscope (not shown) across all of the cells in the specimen. Scan data is collected in the infrared bands of 970 cm^"1 and 1000 cm^"1 and 1145 cm^"1 and 1190 cm^"1. The absorbence is corrected for the presence of the background, and the following ratios are calculated:

R₁ - Abs _{9 8 cm} / Abs ₁₀oo_cm -1

R₂ = Abs ιι_{7 cm} / Abs ₁₁₅₅ > cm

These ratios may then be correlated to the increase in the malignant potential of individual cells.

In summary, the plastic slide according to the invention provides apparatus which increases the signal-to-noise ratio of the relatively weak mid-IR absorbencies and makes possible the practical realization of mid-infrared microscopy analysis for cytological specimens.

The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Certain adaptations and modifications of the invention will be obvious to those skilled in the art. Therefore, the presently discussed embodiments are considered to be illustrative and not restrictive, the scope of the invention being indicated by the

appended claims rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A slide apparatus for infrared microscopy analysis of a biological specimen, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the biological specimen;

(b) said plastic slide substrate having optical transmission characteristics in the mid-infrared range.

2. The slide as claimed in claim 1 , wherein said plastic slide substrate comprises polyethylene.

3. The slide as claimed in claim 2, wherein said mid infrared range comprises wavelengths substantially in the range 975 to 1000 cm^"1.

4. The slide as claimed in claim 2, wherein said mid infrared range comprises wavelengths substantially in the range 1155 to 1180 cm^"1.

5. The slide as claimed in claim 1 , wherein said plastic slide substrate comprises polymethyl pentene.

6. The slide as claimed in claim 5, wherein said mid infrared range comprises wavelengths substantially in the range 975 to 1000 cm^"1.

7. The slide as claimed in claim 5, wherein said mid infrared range comprises wavelengths substantially in the range 1155 to 1180 cm^"1.

8. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 975 to 1000 cm^"1.

9. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the range 1155 to 1180 cm^"1.

10. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polyethylene having optical transmittance characteristics substantially in the ranges 975 to 1000 cm^"1 and 1155 to 1180 cm^"1.

11. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 975 to 1000 cm^"1.

12. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in the range 1155 to 1180 cm^"1.

13. A slide apparatus for infrared microscopy analysis of a cytological sample prepared according to the Pap staining protocol, said slide apparatus comprising:

(a) a plastic slide substrate for receiving the cytological sample;

(b) said plastic slide substrate comprises polymethyl pentene having optical transmittance characteristics substantially in ranges 975 to 1000 cm^"1 and 1155 to 1180 cm^"1.

14. An apparatus as substantially described herein.

15. An apparatus as substantially depicted herein.

16. A method as substantially described herein.

17. A method as substantially depicted herein.