Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/14—Condensers affording illumination for phase-contrast observation

Definitions

• the present invention relates to a detection system and a microscopy method for the rapid and sensitive detection and/or identification of biological materials on surfaces, in particular the detection or identification of bio-hazardous materials and infectious diseases including amyloid plaque diseases.
• the invention also relates to a microscopy apparatus, reagents and diagnostic aids for use in the method.
• TSE Transmissible spongiform encephalopathies
• prion diseases are fatal degenerative brain diseases involving conversion from the normal alpha helical PrP protein to the beta-pleated sheet PrP sc isoform.
• Current epidemiological and research evidence suggests that the risk of PrP sc transmission from TSE patients to other humans may be very low; nevertheless, TSE agents constitute a serious bio-medical hazard.
• a proven route of infection involves contaminated surgical instruments, particularly those in contact with neural tissues. Although government agencies, World Health Organization and other institutions have distributed some guidance for safe working and prevention of infection, these guidelines are not compulsory and are not harmonised globally.
• PrP S0 destruction requires prolonged autoclaving, which is only possible at the present time with the most recent rigid endoscopes. Indeed, there are general concerns about the efficacy of autoclaving, chemical disinfectants and the temperatures and contact times required.
• the present invention provides a microscopy method for use in the detection or identification of biological material on surfaces by implementing episcopic differential contrast (EDIC) microscopy plus epifluorescence (EF) microscopy wherein the microscope incorporates a DIC prism in the nosepiece , infinity corrected optics and and long distance objectives so that the materials can be visualised without requirement for a coverslip or oil or water immersion.
• EDIC episcopic differential contrast
• EF epifluorescence
• Long distance objectives are high power objectives that have a working distance of greater than 1 or 2 millimeters.
• Infinity Correction is composed of parallel optical paths, which enable the system to utilise a variety of different disciplines, to produce high resolution images within a range without the additional requirement for extra optics.
• the surfaces which can be examined include curved, ridged, smooth opaque or semi -opaque, transparent, fibrous, rough or corroded surfaces.
• the method is particularly useful for examination of stainless steel instruments, surgical instruments, work surfaces, plastic surfaces, pipes or pipe biofilms, clothes, fabrics, food, grains, indwelling devices, biological samples, biopsy materials biofilms, membranes, interior of cells or exterior of cells.
• the ability to examine instruments including forceps, surgical knives or scalpels, rigid or flexible endoscopes, cytoscopes, applanation tonometer tips is a particularly useful development .
• In dwelling devises which can usefully be examined include contact lenses and catheters.
• the invention enables the examination of a variety of biological material such as protein contamination and is particularly useful in the detection and identification of biohazardous materials, such as diseases including diseases caused by bacteria or viruses and amyloidogenic proteins.
• Disease materials able to be visualised according to the invention include helicobacter, campylobacteria, CMV, MRSA, TB, smallpox, and anthrax.
• the diseases can be diseases which affect humans or non-humans. Non human diseases include BSE, Scrapie or deer/elk Chronic Wasting Disease.
• a useful aspect of the ability of the biological materials to bind fluorophores is that the viability/vitality of the disease materials can also be determined using suitable staining techniques.
• suitable staining techniques include CTC or DAPI and/or PL This approach is particularly useful where the disease is cryptosporidium.
• Fluorophore agents useful in the invention method are fluorescent thiazole derivatives, such as Thioflavine T or S.
• general protein contamination is detected using Sypro Ruby fluorescent stain.
• the detection level on stainless steel is less than 1 picogram of protein.
• the present invention provides a microscope apparatus for use in the method.
• the microscope comprises an EDIC microscope with a high powered light system and a filter arranged in the light path so that light differences on the sample can be visualised.
• a suitable high powered mercury lighting system is for example 100 watt.
• an immunogold staining block in included.
• the microscope of the invention can be readily adapted to provide a handheld or portable device or for use in a conveyor belt or adapted for use in a modified containment cabinet.
• Devices according to the invention are useful for screening in the water industry for the examination of biofilms, within medical establishments, contamination within the food industry, on food surfaces (eg. salads), abbatoirs, veterinary practices, and dentistry practices.
• the invention also provides kits for use in diagnostic screening for prion disease in patients after tissue biopsy, or for quantitative assessment of the extent of contamination bound to surfaces comprising associated packs of reagents specifically designed to be used in conjunction with the method to enable visualisation of target material.
• the kits suitably comprise probes for the relevant biological materials and /or any necessary stains.
• the invention further provides a system for the diagnosis of disease including prion disease or any other amyloidogenic disease within bodily fluid of the human or animal subject, blood, urine, cerebral-spinal fluid , non-neuronal tissues (including spleen, lymph node), in cells, including living cells.
• Other uses include a system for rapidly screening biofilms and assessing their contents. Portable (handheld) or conveyor belt stage models can be used to enable the rapid scanning of large surface areas or numerous articles in very short periods of time. Another option is a quality control/safety scanner, able to rapidly visualise the structural integrity of opaque surfaces and tool/instruments there by quantifying the degree of pitting, scratching, etching or cracking that may have occurred.
• Other uses include a method of assessing or validation of the effects or effectivenesss of cleaning or disinfection methods on surfaces.
• Surfaces which can be examined according to the invention include curved, ridged, smooth opaque or semi -opaque, fibrous, rough or corroded surfaces.
• surfaces can be interior or exterior.
• Cells include all types of cells including, animals and plants as well as microrganisms such as bacteria.
• the present invention has applications in many environmental situations including environmental microbiology, entomology, biofilms, and in the investigation of disease.
• the present invention integrates the use of fluorescent marker techniques, monoclonal antibodies and advanced microscopy methods to produce a highly sensitive protocol for prion protein detection.
• This enables the identification of prion deposits on surgical instruments and work surfaces, as well as the possibility for its application on biopsy material.
• a useful aspect of the present invention is that it provides a pre-screen for use in selecting specimens for subsequent confirmation of the prion diagnosis using monoclonal antibodies.
• EDIC episcopic differential interference contrast
• EF epi-fluorescence
• the invention is useful for the screening of all surgical instruments for contamination in general, or more specifically for prion protein within medical establishments such as the UK National Health Service, Europe and world-wide. Other particular uses are for screening for contamination within the food processing industry, abattoirs, veterinary practices and dentistry practices. Also provided are quick diagnostic screening procedures for prion disease in patients after tissue biopsy.
• a particularly useful aspect of the present invention is in relation to providing early screening methods of a number of diseases. For example enabling detection of infected cells or infective prion aggregates in the peripheral blood, lymphoid tissues or other bodily fluids of species infected with TSE diseases. Such screening methods can be used to determine the incidence of infection in the general population of these species and accordingly the stage of disease progression in infected individuals. Other diseases which can be monitored is this way include CMN.
• the present studies demonstrate sub-micron amyloid plaques on stainless steel, corresponding to sub-picogram levels of contamination.
• This three-dimensional capability means that the technique is very good for z-direction detection and has been shown to be able to examine level differences of ⁇ 10 A; thus enabling detection of high extinction factor values, increasing image contrast and brightness.
• Devices according to the invention can also be used in the water industry for the examination of biofilms in addition to the screening of surgical instruments for contamination in general or more specifically for pathogens such as MRSA, TB, or prion protein within medical establishments or elsewhere and screening for contamination within the food industry, abattoirs, veterinary practices, dentistry practices.
• the apparatus and method also allows for the production of a quick diagnostic screening test for prion disease inpatients after tissue biopsy.
• Further uses include clinical pathology or any field requiring analysis for unwanted microbial contamination including pathogens or biohazardous materials. Campylobacter are readily visualised using this system. Also in the field of food microbiology the system has many uses eg, spot sampling for microbial contamination of eg salads). The system has uses in the area of plant pathology, for example the in-field diagnosis of plant diseases, especially those caused by various strains of micorganisms. The invention also offers uses in the area of forensics where rapid on site identification of deposits by non-destructive methods is important. Similarly the rapid identification of biohazardous materials for securing personal and public safety is particularly valuable.
• the present invention provides an improved microscope that implements Episcopic DIC illumination, hence EDIC, to visualise the sample thus providing many advantages over traditional techniques.
• the long distance lenses used are normally only applied in metallurgical applications and hitherto not biological applications.
• the present invention enables the apparatus to perform rapid, sensitive, non-contact screening of opaque and/or curved surfaces without the application of coverslips or immersion; thus making it ideal for inspection of clinical instruments.
• the apparatus can be adapted and adjusted to allow task specific operations. For example the rearrangement or removal of the stage allows the production of a handheld or portable device or a device suitable for isolation , for example in a containment cabinet.
• a microscope for EDIC microscopy has been described in Keevil, C.W. and Walker, J.T
• an improved episcopic DIC microscope has been constructed, as described in more detail below, which has successfully visualised low level contamination by infected brain homogenates and brain slices of stainless steel surfaces without the need for coverslips or oil immersion and enable the visualisation of surface contamination on curved, ridged or smooth opaque, or semi-opaque or transparent surfaces.
• the microscope technique implements episcopic differential contrast (EDIC) methodology in addition to episcopic fluorescent (EF) microscopy.
• EDIC episcopic differential contrast
• EF episcopic fluorescent
• the system adapts conventional Nomarski microscopy to incorporate episcopic imaging by redesign of the light path and microscope components.
• EDIC implements the destructive/constructive nature of light waves.
• the source light is split into two polarised parallel beams before it reached the specimen. Having trans versed the object the wave paths of the two beams have been altered in accordance with the specimen's thickness, slopes and refractive index. This variation causes interference between the light beams and allows detail to be visualised in a pseudo three-dimensional appearance. This enables the operator to not only visualise the object of interest but also gives an indication of the position of any such object.
• one new microscope (with reference to Figs. 1, 2 or Figures 40,41) includes a number of additional individual improvement features
• Low power objectives have the inherent ability to work at relatively long distances from samples. This is not true for most high power lens (>x20) and special long distance objectives, that have a working distance of greater than one or two millimetres, have been applied to the system.
• the present invention microscope optical configuration (see Figure 1 schematic ) is based upon infinity corrected optical system.
• the present system enables longer working distances with objective lenses to be achieved, a significant improvement from the mercury light source due to the infinity corrected light path and optical system , i.e. enabling better illumination.
• the device for EDIC is correctly positioned in the nosepiece just above the objective lenses enabling the quality EDIC to be achieved without the use of extra prisms for this technique to take place and extra optical glass wear to magnify the image to the next optical requirement.
• epi fluorescence In this case epi fluorescence.
• This infinity corrected EDIC system also enables you to use this on the current infinity corrected lenses of choice whether they are DIC specified or not which again is a improved technique enabling the user to use lenses that are specified DIC this means slightly better quality or those which are not specified the new improved EDIC enables you to work using any biological or material science objective.
• the present system can use new improved epi fluorescence filter combinations with 25mm filters and new improved filter combinations cubes i.e. new coatings, (eg Chroma filters , USA) enabling a wider saturation of required excitation of the particular wavelength and band pass required.
• new coatings eg Chroma filters , USA
• the earlier 1992 microscope was of a 160mm tube length optical configuration, with an extra 50mm insert required making 210mm for all episcopic material science lenses.
• the biological ones were 160mm. So when using material sciences lenses of long working distance these were the correct magnifications, when using biological these were giving more magnification due to the 50mm insert used to obtain the correct optical path for material science lenses and the epi fluorescence system.
• the new infinity corrected system results in a superior match of optical configurations.
• the material science long working distance objective had its own individual prism, the biological lenses had no prisms hence only used the IGS block and analyser to obtain polarisation and epi fluorescence imaging so limiting the 1992 EDIC system to the lenses that had the Nomarski prism in situ. In contrast, the present system allows any chosen objective to be used.
• the Nomarski interference imaging was obtained by the use of a prism directly behind the objective lens which could be engaged by a lever placing it in or out of the optical path.
• the rotating analyser also sat in the position above the fluorescent filter blocks and was rotated to obtain the Nomarski principal.
• This configuration of EDIC gave reasonable results but was not specified by the manufacturer as a current working system.
• the configuration according to the present invention using infinity corrected optics is capable of far superior optical quality.
• the original filters integrated into the filter blocks were only 18mm limiting their epi fluorescence function whereas it is now possible to use new filters integrated into new filter blocks which are 25mm.
• the extra available lighting in the new infinity corrected system coupled with the larger filter area provides excitation and illumination of specimens far beyond that possible with the 1992 EDIC.
• the 1992 EDIC included four filter blocks which were engaged into the optical pathway via a push pull rod, however the problem with this was that engaging the filter block correctly into the optical path could sometimes be a problem especially to a new user. This has been overcome and more versatility added by designing the new system so that four blocks easily click into place.
• the new system can also readily accommodate further e.g six filter blocks if required, also infinity corrected.
• the new system provides superior optical quality and versatility, in particular through the use of infinity corrected optics, fluorescent coatings, and configuration changes that compliment EDIC without extra glass wear and Nomarski prisms which limited the use of the earlier 160/210mm 1992 EDIC configuration.
• FIG. 1 Schematic of EDIC microscope.
• Figure 3 shows a murine section indicating PrP sc positive regions for ME7
• FIG. 4 shows PrP epitopes recognised by the SAF Mab antibodies in relation to the protein
• FIG. 5 shows Sypro Ruby excitation/emission spectra
• Figure 13 shows Sypro Ruby staining of brain contamination on stainless steel, showing proteinaceous material.
• Figure 14 EDIC/EF low magnification images showing detection, of protein deposits remaining after washing and stained with Sypro Ruby.
• Figure 15 shows high magnification EDIC/EF images showing detection of protein deposits remaining after washing and staining with Sypro Ruby.
• Figure 16 Thiofiavine-positive regions in 10 ⁇ m sections of infected dentate gyrus on stainless steel.
• Figure 17 Thiofiavine-positive area in 10 ⁇ m sections of infected CA3 region of hippocampus on stainless steel.
• Mab staining on stainless steel compares directly the immunohistological staining by 6H4 Mab (a) and the Thioflavines (b,c) within the thalamus of prion positive sections placed onto surgical stainless steel tokens.
• Figure 22 Spyro Ruby staining of contamination on lumenal surface.
• CMV Cytomegalovirus
• M83 Microgen Bioproducts CMV Antigenaemia kit
• FIG. 31 Peripheral blood mononuclear cells stained with monoclonal antibody against the early
• CMV Cytomegalovirus
• M83 Microgen Bioproducts CMV Antigenaemia kit
• FIG. 32 Fluorescent image (High Power) Peripheral blood mononuclear cells stained with monoclonal antibody against the early Pk65 protein of Cytomegalovirus (CMV), Microgen
• Bioproducts CMV Antigenaemia kit (M83).Hoffman configuration. (see Table 3 )
• Configuration 1 See Table 3
• Figure 35 Droplets excreted by fruit fly egg after laying (EDIC microscopy) x 1000 magnification.
• Configuration no 3 See Table 3)
• Figure 37 Drinking water biofilm on polyethylene pipe, room temperature (Composite of 10 stacked images). EDIC Confocal microscopy (Optigrid), Configuration no 6 (See Table 3)
• the configuration of the microscope of the present invention is based upon a mixture of biological and material science requirements with an emphasis on looking at contamination on opaque specimens.
• This microscope has the ability to mix fluorescence and DIC (Differential interference contract) in the incident mode (light from the lamphouse on the top of the microscope through the optical path down to the objective onto the sample then back up to the eyepieces to receive the image of choice).
• fluorescence and DIC Direct interference contract
• DIC microscopy implements the destructive/constructive nature of light waves from a mercury light source the light is split into two polarised parallel beams before it reaches the specimen, having transferred the object the wave paths of the two beams have been altered in accordance with the specimen's thickness slopes and refractive index.
• This variation causes interference patterns between the light beams and allows relief and light difference to be achieved, hence the user can see peaks and pits giving a 3D appearance.
• This three dimensional capability allows the excellent Z direction detection and has been shown to be able to examine level differences of 10 angstroms this enables detection of high extinction factor values, increasing contrast and depth of field in the specimen.
• a DIC prism incorporated into the nosepiece Two phase contrast position control enables the fine tuning of surface contamination and relief.
• EDIC EF cube slider Image superimposed blending two episcopic images for greater contrast and relief, coupled with the newly researched fluorochromes that also are used in this system.
• This epi fluorescence package provides a very powerful research and analytical system.
• EDIC is a two part process. Firstly, the use of an Immuno gold staining block IGS which is a polarizer and cross analyser. This gives direct polarisation to the sample, hence this will provide diagnostic biorefringence to any structure that its light path can be changed once it engages the specimen, this is a stand alone technique, epi polarization via a mercury HBO lamphouse. This IGS block is also required when engaging the DIC prism as the requirement for the EDIC is parallel light, the IGS block provides this, hence the two work together.
• IGS Immuno gold staining block
• An 8 slide stage with glass insert for reflective light microscopy is provided.
• a preferred design features the drive motor in the Y position moved to the opposite corners allowing for greater freedom of movement for the operator .
• the position of the stage is a problem due to protruding parts.
• a further advantage of the new configuration is that it deals with the protrusion of this part which then enables the microscope to be isolated, for example when placing the microscope in a ACDP (Advisory Committee on Dangerous Pathogens) category 1,2 or 3 containment cabinet.
• Other features include moving the control electronics input of this stage via computer from the right hand side to the left hand side. See figure 40 and 41
• the system uses fully automated computer scanning stage in XYZ axes or direction including software to focus on curved images, also dedicated software to establish a cell bar code reader, for counting cell's per square mm for analysis and research, and further software to perform two spatially identical scans that are designed to map surface properties under fluorescence and DIC.
• the images will be merged/overlaid with its composite partner image.
• Cool snap charge coupled device (CCD) low light camera system for improved 2D and 3D preparation, the interfacing optics from the microscope trinocular head to the CCD camera have been designed according to the invention to embellish the lighting and images contrast quality produced by the optics and imaging system thus maximizing surface quality.
• CCD Cool snap charge coupled device
• the present invention also relates to the introduction of a confocal adaptation without laser requirements.
• the introduction of the Optigrid real time digital optical sections for 3D mapping of textured specimens [Optigrid technology is described in GB 2,338,858 and US 6,376,818] as shown herein clearly provides increased and excellent resolution .
• Optigrid technology is described in GB 2,338,858 and US 6,376,818] as shown herein clearly provides increased and excellent resolution .
• customised diascopic differential interference contrast (DDIC) diascopic or transmitted light from below the specimen for contamination that required fluorescence and EDIC from light looking down onto the specimen to DDIC looking up through the specimen, the contrast technique Hoffman contrast is used for this. (US 4,200,353 Modulation contrast microscope).
• the IGS as a stand alone polarizing system incorporated into the EDIC in the episcopic format coupled with the Hoffmann polarized technique in the diascopic format adds considerable resolution and contrast to a translucent specimen. These can be used in combination or separately.
• By adding the Thales-Optem optigrid real time digital optical sectioning as referred to above enables resolution beyond the capability of the microscope and the lenses. Due to the combined techniques listed above the confocal is able to establish resolutions on samples now that a stack of images can be combined to give one sharp image, whilst taking light from diascopic and transmitted light simultaneously.
• the lens used for the Hoffman with its condenser is polarised to provided only the grey part of the spectrum whereby the light from the EDIC/fluorescence is providing the full spectrum of colours, hence the user can balance this system to work in such a way as to maximised the image texture on the specimen to define the contamination levels by altering the variation between the two relief techniques.
• the Hoffmann 40x lens is able to see high contrast and resolution on transparent, unstained and living cells, as this is the only Hoffmann lens on this microscope system when we move to another lens but without moving the polarized facility on the sub stage condenser we are still able to achieve with good resolution a "pseudo Hoffmann" image on biological and material science lenses in transmitted light format that do not have the Hoffmann facility. In essence this provides excellent relief on the specimen in transmitted light enabling height differential to be established picking out peaks and pits.
• the above features and adaptations provide a microscope to perform rapid sensitive, non contact screening of the opaque and curved surfaces without the application of the cover slips or oil. So making the microscope ideal for the inspection and analysis of opaque substrate and clinical surgical instruments.
• the microscope according to the invention represents a new revolutionary way in which contamination of surface structures Bio films etc. is viewed.
• the system has the ability to view any type of surface, either in 'white' light and under fluorescent excitation without the requirements for any major adjustments to the microscope set up.
• This allows both epi fluorescent (configuration 1) images and EDIC 'whitelight' (config. 3) or epi-polarised (config. 2) to be captured of the same area on interest.
• An example of the power of this is clearly shown in figure 13 where protein contamination of stainless steel surface is displayed under both fluorescence (fig 13 a) and EDIC (fig 13b) illumination.
• the subsequent application of the advanced software enables a composite image of the two configurations to be displayed and as such the accurate differentiation of contaminants to be achieved (fig 13c).
• the addition of the 'Thales-Optem optigrid' enables an extended field of depth and allows the system to scan multiple planes of the object in a similar way to laser confocal but without the requirement for expensive additional equipment.
• This system enables the visualisation of biofilms on polyethylene pipe sections either using 'whitelight' (config. 6/7, figure 37) or fluorescence (config 6).
• Transmitted illumination can only visualise samples in or on translucent media.
• the brightfield configuration (config 8) enables traditional microscopy to be performed such as traditional immunohistochemical staining (Figs 9 and 10).
• Hoffman modulation (config 9) enables relief to be visualised, it does not suffer the artefacts produced by plastic in other illumination methods and can be applied to such areas as the visualisation of cells (fig. 31)
• the ability of the system to combine light sources allows the flexibility to adjust the system in order to produce the best quality images for the media and subject involved in both fluorescent and whitelight modes.
• An example of this is the ability to visualise bacteria such as Campylobacter jejuni (config 13). Campylobacter is a bacterial pathogen that is the most important casue of gastroenteritis worldwide and transmitted through faecal contamination.
• the system is based around a Nikon Eclipse ME600, modified as required and fitted with a combination of fluorescent, metallic and Hoffman modulation contrast objectives as described below:
• the system is able to house 4 filter blocks, one Immuno-gold staining block for white light EDIC/epi-polarised illumination and 3 fluorescent filter blocks from Nikon and had the codes: UV- 2E/C, BV-2A, and G-2E/C.
• the samples were washed in water for 5 minutes at room temperature, they were then physically cleaned by rubbing the token surface with a surgical swab in the presence LabGuard scrub (Day-Implex Ltd, Essex, UK) for 2 minutes, and then washed in three changes of water for 5 minutes each.
• the tokens were then stained with Sypro Ruby and examined under the microscope.
• Formal saline-fixed tissue was used for immunocytochemistry.
• PrP sc detection the sections were pre-treated to destroy PrP c . This consisted of 15 minutes hydrated autoclaving, followed by 5 minutes with formic acid (>95%).
• the protocol then used the mouse on mouse kit 29 (Vector labs).
• the primary antibody 6H4 (Prionics), a monoclonal raised against the C-terminus region of the PrP protein, was left to incubate overnight at 4°C with a concentration of 1:4000. Positive staining was visualised using a diaminobenzidine (DAB) as the chromagen and counterstained with hematoxylin
• Thioflavine S (Sigma) - Sections of fresh tissue on glass slides or steel coupons were fixed in 4% paraformaldehyde (w/v) for 10 mins at 4°C. After washing with PBS the sections were incubated with Thioflavine S (0.01% w/v solution) for 10 minutes at room temperature. Subsequently the slides were washed in decreasing alcohol concentrations and the section covered with an aqueous fluorescent mounting medium (DakoCytomation).
• Thioflavine T (Sigma) - Sections of fresh tissue on glass slides or steel coupons were fixed in 100% ethanol at 4°C for 10 mins. After washing with PBS the sections were then incubated with Thioflavine T (0.03 % w/v solution) for 10 mins at room temperature. The samples were then washed with 1% acetic acid in de-ionised water for 40 minutes and the section covered with an aqueous fluorescent mounting medium (DakoCytomation).
• mice Female C57B1/6J mice were kept in-groups of five or six in plastic cages in a temperature-controlled room (21°C) with a twelve-hour day/night cycle. They had free access to water and food. Positive animals were those injected with 1 microlitre of 10% (w/v) ME7 brain homogenate, stereotaxically into the right dorsal hippocampal region of the brain (co-ordinates from bregma (the point on the top of the skull at which coronal and sagittal sutures meet): anterior - posterior -1.94mm, lateral -1.5mm, depth -1.5mm)
• NBH Normal Brain Homogenate
• ⁇ Defined areas of the mouse brain with known pathology, microglia activation and prion accumulation.
• the ME7 model has well defined PrP SQ characteristics' 8"15 -' with large concentrations of the abnormal protein forming within sections of the hippocampal region of the brain.
• dense areas of PrP sc deposition occur within the hilus of the dentate gyrus and following the mossy fibres of the CA3 region of the hippocampus.
• dense circular amyloid plaques found within the thalamus and occasionally above the corpus callosum (Fig. 3).
• a standard two step immunohistochemical technique was established on tissue sections cut onto stainless steel tokens.
• a microglial, CD68 marker, monoclonal antibody, FA11 (Serotec) was implemented as the primary antibody and the fluorescent signal produced by the linkage of the biotinylated secondary antibody and an FITC -avidin complex, Neutravidin (Molecular Probes Inc., Eugene, Oregon , USA).
• the tissue was formalin-fixated and underwent two pre-treatments: Porous autoclaving for 20 minutes at 121 °C, and formic acid (>95%) for 5 minutes, to destroy the normal form of the PrP protein (PrP c ) and reveal the epitopes of the aberrant PrP s0 .
• Table 1 SAF monoclonal antibodies and their epitopes on the Prp protein
• FIG. 4 shows PrP epitopes recognised by the SAF Mab antibodies in relation to the protein
• the sections were processed according to the Vector Labs, M.O.M. kit procedure with a SAF incubation time of 2 h; an avidin - biotin complex was then applied and followed by the standard diaminobenzidine (DAB) reaction.
• DAB diaminobenzidine
• Spyro Ruby Fluorescent dye [Molecular Probes, Inc. Eugene, Oregon 97402, USA] was developed for sensitive staining of low protein concentrations in gels only. General protein contamination was visualised by the modified application of Sypro Ruby stain. This stain has been show to possess a very high affinity for general proteins and be highly sensitive [27'32] on both gels and glass surfaces. The excitation and emission spectra for Sypro Ruby are given below (Fig.5). Its successful application to tissue on metal tokens indicated its suitability as a hygiene screen for medical instruments. Therefore, subsequent implementation of the stain was initiated on discarded medical devices.
• FIG. 5 shows Sypro Ruby excitation/emission spectra
• thiazole derivatives thioflavine T (ThT) and thioflavine S (ThS) (Fig. 6) have been shown to possess the ability to label amyloid deposits associated with amyloid plaques within histological sections from a number of neurodegenative diseases.
• Thiazoles's such as Thioflavine S and Thioflavine T have been used to detect amyloid deposits in fluids and post-mortem histological sections.
• ThT and ThS have emission spectra with maxima around 482 nm. 1491 ThS emission is stimulated by excitation at 385 nm, which is unchanged from that of the free dye in solution.
• ThT once bound, undergoes a change in excitation spectrum with a new peak appearing at 450 nm which does not exist for the free dye.
• a rigid cystoscope has a pencil thin extension and possesses both a light and lense at the tip to allow it to focus on the inner wall of the bladder or urethra. By this means the clinician is able to diagnose such conditions as: Persistant urinary tract infections
• the cystoscope set was scanned using the EDIC microscope and then cleaned.
• FIG. 8a and 8b The microscopy revealed that extensive activation and recruitment of microglia occurred within sections of the hippocampus, and that these were clearly visible on brain sections mounted on stainless steel. At greater magnification a single microglia and its processes can be visualised within the thalamus (Fig.8c) Figure 8 shows Activated microglia on stainless steel brain sections 1.13 Prion protein
• the dentate gyrus region of the hippocampus is a recognised area for PrP sc deposition as can be seen from Figure 9; the control tissue had no positive areas within the dentate gyrus (a).
• Figure 9 shows Positive SAF areas in dentate gyrus comparable to 6H4
• Figure 10 shows Positive SAF signal in the CA3 region of the hippocampus, similar to 6H4.
• Table 2 shows the suitability of the SAF Mab's for staining PrP sc in 10 ⁇ m brain sections.
• ME7-infected mouse brain was smeared on to a surgical stainless steel surface (Fig. 11) and visualised using EDIC microscopy at low power. The contrast between the neural deposition and the stainless steel can clearly be seen. The same brain smear at greater magnification is shown below (Fig.12)
• Figure 11 shows Low magnification EDIC image of brain contamination on stainless steel
• Figure 12 shows High magnification EDIC image of brain contamination on stainless steel 1.15 Sypro Ruby staining
• Figure 13 illustrates a brain-contaminated stainless steel token stained with a sensitive fluorescent protein marker (Sypro Ruby).
• Sypro Ruby a sensitive fluorescent protein marker
• Figure 13 shows Sypro Ruby staining of brain contamination on stainless steel, showing protenaceous material.
• Figure 14 shows EDIC/EF low magnification images showing detection, of protein deposits remaining after washing and stained with Sypro Ruby.
• Figure 15 shows high magnification EDIC EF images showing detection of protein deposits remaining after washing and staining with Sypro Ruby.
• PrP sc -positive sections of brain were placed on surgical stainless steel surfaces and studied with thiazoles.
• a positive signal within the dentate gyrus (Fig. 16) and CA3 (Fig.17) regions of the hippocampus can be seen.
• Thioflavine S (a) and Thioflavine T (b) staining are shown and a surface plot of the staining (c) can be produced to make the positive signal quantifiable.
• Figure 16 shows Thiofiavine-positive regions in 10 ⁇ m sections of infected dentate gyrus on stainless steel.
• Figure 17 shows Thiofiavine-positive area in 10 ⁇ m sections of infected CA3 region of hippocampus on stainless steel.
• Figure 17 shows Thiofiavine-positive regions in 10 ⁇ m sections of infected thalamus comparable to 6H4 Mab staining on stainless steel
• the minimum level of prion protein detection is less than l ⁇ m in diameter. This equates to less than lpg (Appendix 1) of prion protein and demonstrates the sensitivity, ease and speed of the EDIC/EF staining techniques.
• Figure 20 shows Contamination found on outside of the Spencer Wells forceps
• a Zoellner Sucker (Figs. 21 and 22) is used for the removal of debris from ears and brain.
• the sucker had part of its outer casing cut down to reveal its lumenal surface. With solely EDIC microscopy (Fig.22a), contamination is difficult to distinguish. However, after Sypro Ruby staining (Fig.22b), and subsequent picture combination (Fig.22c) the regions of proteinaceous deposition can be seen clearly.
• Figure 21 shows Zoellner Sucker with close up indicating exposed lumen
• Figure 22 shows Sypro Ruby staining of contamination on lumenal surface.
• a bladder cystoscope set (Fig. 23) is comprised of four pieces: 2 scopes with different lens angulations, and an obturator and sheath to provide clear passage for the scopes into the area of interest.
• Initial EDIC investigation began on the scopes: visible contamination was discovered and an indication of this is readily demonstrated in Figures 24 and 25.
• FIG. 23 shows Cystoscope set
• Figure 24 shows Contamination at tip of 70° cystoscope
• Figure 25 shows Contamination in the middle of 70° cystoscope
• the obturator sheath was looked at both on the inside on the visible lumenal surface (Fig.26) and the external surface (Fig. 27)
• the external scan picked up small areas of unknown deposits, whereas the extent of contamination on the lumen surface was such that 'clean' metal was difficult to visualise.
• Figure 26 shows Contamination on lumenal surface of cystoscope set obturator sheath
• Figure 27 shows Contamination on outside of cystoscope set obturator sheath
• the CMV antoigenaemia test is a rapid sensitive and quantifiable test useful in the early detection of CMV infections. Early detection allows clinicians to predict patients at risk and commence suitable treatment and monitoring.
• the appearance of the Pk65 protein is one of the first signs of CMV disease. If it is possible to detect whether someone has CMV early enough then it is possible to give suitable drugs.
• a method whereby transplant patients (or other patient populations typically at risk) can be routinely screened through eg, sampling of blood or other tissue, by looking for the variant protein can be a useful warning system.
• FIG 29 Shows the view of peripheral blood mononuclear cells stained with monoclonal antibody against the early Pk65 protein of cytomegalovirus (CMV).
• CMV cytomegalovirus
• M83 MicroGen Bioproduct CMV antigenaemia kit Obtainable from Microgen Bioproducts Limited, Camberley, surrey.
• Figure 32 Fluorescent image , High Power (Hoffman) Figure 33 Thioflavm T-stained dentate gyrus showing prion amyloid aggregates (FITC filter block) x 1000 magnification
• Figure 37 Drinking water biofilm on polyethylene pipe, room temperature , composite of 10 stacked images. EDIC/ Confocal microscopy (no 7 ) Optigrid
• ACDP/SEAC Transmissilbe Spongiform Encephalopathy Agents: Safe working and the prevention of infection. The stationary office, 1998.
• Vallet, P.G., et al. A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease. Acta Neuropathol (Berl), 1992. 83(2): p. 170-8.
• Brain Weight (whole brain): 482.3 mg (Av. gained from 129 mice within 23 litters 1 )
• Bio-Rad total protein assay was performed in order to assess the physical amount of protein within a section.
• a 10 ⁇ m section was taken from the late (coronal) hippocampus region of an ME7-infected mouse.
• the sections dimensions were obtained from the stereotaxic atlas which indicated that the section possessed dimensions of approximately 9(L) mm x 5.75(D) mm.
• the 10 ⁇ m section was homogenised in 50 ⁇ l of PBS and a 5 ⁇ l sample taken in accordance with the assay guidelines.

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