WO2013056685A1

WO2013056685A1 - Sample slide preparation method and device

Info

Publication number: WO2013056685A1
Application number: PCT/CZ2012/000103
Authority: WO
Inventors: Martin Mistrik; Jiri Bartek; Jiri Lukas
Original assignee: Univerzita Palackeho; Cancer Research Technology Limited; Kraeftens Bekaempelse
Priority date: 2011-10-21
Filing date: 2012-10-17
Publication date: 2013-04-25
Also published as: GB201118282D0

Abstract

The present invention relates to a method for preparing a microscopy slide for analysis of a biological sample comprising i) placing the slide in a humid environment; ii) cooling the slide so that frost forms on its upper surface: and iii) applying the biological sample to the frost on the slide. It further relates to a microscopy slide preparation device for the preparation of a frosted microscopy slide, the device comprising: a humidity chamber for containing the slide; a support for holding the slide within the chamber; cooling means for cooling the slide; and humidifying means for humidifying the chamber. The invention further includes a kit comprising said microscopy slide preparation device and a fixative for adding to the biological sample.

Description

Sample slide preparation method and device

Field of the Invention

The present invention relates to methods for preparing sample slides and microscopy slide preparation devices for the

preparation of frosted microscopy slides.

Background to the invention

The preparation and analysis of sample slides for the analysis and observation of research samples is an operation routinely

performed by researchers in many scientific fields. The quality and reproducibility of the samples mounting is often critical to the quality of the data that can be collected from the sample, so there is an ongoing requirement for reliable methods for producing high quality sample slides.

The preparation of sample slides is particularly important in the field of cytogenetics in methods such as the metaphase spreading technique, an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH) , are greatly influenced by the quality of chromosome spreading. Obtaining compact and constant high-quality metaphase spreads is a difficult procedure which is affected by many difficult-to-control environmental conditions.

At present the successful execution of this technique requires highly trained people with the appropriate technical skills. The current standard methodology involves manual dropping of cells onto a glass slide with cells spreading on a layer of water, a process that is highly user-dependent, often difficult to control, and time consuming in achieving satisfactory quality results. Typically the cells to be spread are suspended in a fixative solution comprising methanol and acetic acid. This fixative maintains the cells in a state that is compatible with the current metaphase spreading technique. However, the use of acetic acid in the fixative often means that the spread cells cannot subsequently be used in imunohistochemistry (IHC) and immunofluorescence (IF) studies due to acetylation of antibody epitopes and/or protein wash-off. The difficulty of the currently available protocols for metaphase spreading is a major technical shortcoming in fields that require the quantitative analysis of mitotic BGC (breaks, gaps and constrictions) based on morphological evaluation of chromosomes. This analysis places a high demand on the quality of mitotic spreads and is extremely time-consuming. Moreover, subsets of the potentially small lesions may remain undetected in low-quality metaphase spreads.

Metaphase spreading technique has been recently advanced by the discovery that heating of mitotic spreads on a heating block immediately after the spreading procedure improves the quality of the resulting spreads (Henegariu et al . (2001) Cytometry, 43:101- 9) . In an attempt to make the technique simpler and easier to control, a number of commercially available microscopy slide preparation devices have been developed for use in spreading metaphase cells. For example, Thermotron's Cytogenetic Drying Chamber CDS-5 is a microscopy slide preparation device designed specifically for conducting cytogenetic slide drying tests during the harvest of in situ and non-in situ grown cultures; this device provides for control of the environmental temperature and humidity. Another microscopy slide preparation device is ADSTEC s HANABI metaphase spreader; this device is a bench-top device that provides an incubator with adjustable humidity and temperature conditions. US 2005/0042767 Al describes a method for preparing a slide. The authors describe a method in which the 'dryness' of the slide is controlled by monitoring and controlling the humidity and

temperature of the slide's environment. This document also describes a microscopy slide preparation device for carrying out this method. In one embodiment the device comprises a humidity chamber for containing the slide, the chamber having a vent for allowing the exchange of air with the external environment. To date, no microscope slide preparation devices or methods are described wherein the microscopy slide is actively cooled.

Disclosure of the Invention

It has been observed that covering the surface of a microscopy slide with a layer of frost prior to applying the sample reliably allows slow and gentle spreading of the sample. Furthermore, it has been observed that the introduction of a step in which the sample is frozen into the preparation protocol permits the use of fixatives that preserve antibody binding epitopes in the

biological sample.

It was also observed that a sample of cells applied to the surface of a frost-covered microscope slide, spread over a larger surface area and were significantly larger in size than those observed using previously-known cell spreading techniques. This increased the resolution of the sample allowing for easier visualisation of cell structures during microscopy analysis, without increasing the magnificatio .

Accordingly, the present invention provides a method for preparing a microscopy slide for analysis of a biological sample comprising: i) placing the slide in a humid environment; ii) cooling the slide so that frost forms on its upper surface; and iii) applying the biological sample to the frost on the slide. The invention also provides a method for preparing a microscopy slide for analysis of a biological sample comprising: i) placing the slide in a humid environment; ii) cooling the slide so that frost forms on its upper surface; iii) applying the biological sample to the frost on the slide; and iv) continuing to cool the slide after the sample has been applied until the sample is frozen.

In another aspect, the invention provides a microscopy slide preparation device for the preparation of a frosted microscopy slide, the device comprising: a humidity chamber for containing the slide; a support for holding the slide within the chamber; cooling means for cooling the slide; and humidifying means for humidifying the chamber.

The method and device of the invention may be used in particular for preparation of metaphase spreads.

These and further aspects of the invention are described in more detail herein below. Description of the drawings

Figure 1 shows chromosomes from fibroblasts after metaphase spreading. The chromosomes are stained for (A) ploidy and cohesion analysis and (B) sister chromatid exchange (SCE)

analysis .

Figure 2 shows metaphase spreads of U-2-OS cells for morphological analysis, stained with DAPI .

Figure 3 shows metaphase spreads stained for γ-Η2ΑΧ. U-2-OS cells were treated with Aphidicolin (APH) (0.1 μΜ) for 24 hours, enriched for mitotic cells for 2 hours of Colcemide treatment before the harvest. Mitotic spreads were prepared and stained for γ-Η2ΑΧ. γ-Η2ΑΧ co-localize with breaks in metaphase chromosomes.

Figure 4 shows a diagrammatic representation of the method used for automated analysis of immunostained metaphase spreads. Pictures with γ-Η2ΑΧ immunoflourescence signal taken by digital camera are processed by automatic routine in Adobe Photoshop 7.0 for background subtraction and then analysed in ImageJ 1.37v.

Data sets associated with every scored focus are further processed by statistical software. All parameters are fixed and routine is automated using macro program Mouse Recorder.

Figure 5. shows metaphase spreads stained for γ-Η2ΑΧ.

(A) Chart summarising average number of γ-Η2ΑΧ foci in DOX-induced and non-induced sh-ATR U-2-OS cell treated by different doses of APH. Cells were treated with APH for 24 hours and were then spread and stained with γ-Η2ΑΧ. Foci were scored as an average per cell in 100 spreads for every concentration in two independent experiments. (B) Examples of spreads scored for γ-Η2ΑΧ foci.

Figure 6 shows a schematic of a microscopy slide preparation device according to the present invention. (A) top view. (B) cross-sectional view taken along line 6B in Fig. 6A.

Figure 7 shows a schematic of another embodiment of a microscopy slide preparation device according to the present invention. (A) top view. (B) cross-sectional view taken along line 7B in Fig. 7A.

Figure 8 shows an example of metaphase spreads and interphase nuclei stained for γ-Η2ΑΧ (immunoflourescence) and fragile site FRA3B (FISH) . (A) Expression of FRA3B within the cell cycle measured as incidence of overlays of γ-Η2ΑΧ foci and FRA3B probe signals (200 cells were scored for each column) . (B) Examples of scored samples.

Figure 9 shows examples of U-2-OS cells stained for DAPI, images (A) -(C), or stained for both DAPI (blue) and γ-Η2ΑΧ (green), images (D)-(F) . Cells were either grown directly onto a microscope slide (images (A) and (D) ) ; dropped onto a microscope slide using the standard method (images (B) and (E) ) or; applied to a microscope slide using the frost spreading method described herein (images (C) and (F) ) . Cell nuclei are outlined in white.

Detailed description of the invention The finding that covering the surface of a slide with a layer of frost prior to applying the sample reliably allows for slow and gentle spreading of the sample cells and provides a novel means for the preparation of microscopy slides. Thus in one aspect, the invention provides a method of preparing a slide that comprises the steps of placing a slide in a humid environment, cooling the slide, until frost forms on the upper surface of the slide, and applying the biological sample to the frost on the slide and allowing the sample to spread on the slide.

The methods of the invention may further comprise the steps of continuing to cool the slide after the sample has been applied until the sample on the slide freezes. Subsequent to the

spreading and/or freezing of the sample, the methods of the invention may also comprise the steps of drying the sample on the slide, immunostaining the sample, and analyzing the sample with a microscope .

Other types of analysis may be used subsequent to the spreading and/or freezing of the sample, for example, classical banding techniques; standard morphological and ploidy analysis of

chromosmes (see examples below and Figures 1 and 2); quantitative analysis of mitotic BGC, and other types of metaphase spread analysis known in the art. The methods of the invention may be particularly useful for complex analysis applications which are greatly influenced by the quality of chromosome spreading, such as FISH, muliplex FISH and comparative genomic hybridization (CGH) .

The methods of the invention are also useful for analysis of cells which are not currently undergoing metaphase, for example

interphase cells (see Figure 9) . For the sake of brevity, a sample of interphase cells applied to a microscope slide is referred to herein as an interphase spread' .

Examples of immunostaining methods which can be used on interphase cells include the scoring of DNA breaks to estimate the degree of DNA damage after ionizing radiation and/or other genotoxic factors. Spreading of interphase nuclei can simplify these routines by, for example, reducing the depth of the sample (Z- coordinate) which can speed up the analysis because it is no longer necessary to take images at different depths of the sample (Z-stack) . Furthermore lower magnification can be used which has the advantage of avoiding problems associated with immersion objectives and all the spreaded interphase nuclei can be observed in much greater detail during the microscopic analysis due to the increased resolution of cells in interphase spreads.

Samples applied to a layer of frost spread more slowly than the equivalent sample would spread if applied to a layer of water, as in conventional spreading techniques. This enables the spreading process to be more easily manipulated.

Without being bound by theory, the slowing of the spreading process appears to occur because in order to spread the sample has to melt the frost from slide.

Consequently, by controlling the thickness of the layer of frost that accumulates on the slide prior to the application of the sample the rate and extent to which the sample spreads can also be controlled .

The thickness of the frost layer on the slide can be controlled by adjusting the temperature at which the cooled slide is maintained and the humidity of the environment surrounding the slide. In general, the lower the temperature of the slide and the more humid the environment, the greater the rate and degree of frost

formation on the slide. The thickness of the frost layer can also be controlled by altering the amount of time that frost is allowed to accumulate on the cooled slide. Therefore by controlling these variables the properties of the frost on the slide can be

consistently and reliably controlled. By continuing to cool the slide after the sample has been applied, the sample can be frozen. Typically, the freezing point of the applied sample is below the temperature at which the cooled slide is maintained. However, as the sample melts the frost from the slide, the resulting liquid water mixes with the sample and increases its water content. As the water content of the sample increases, so does its freezing point. When the freezing point of the sample exceeds the temperature of the slide, the sample will freeze . The freezing process induces a change in the sample which results in improved quality of the resultant spread. Without being bound by theory, it appears that the freezing of the sample induces a ^' mechanical force within the sample that acts on elements present in the sample. For example, the freezing of a sample containing eukaryotic nuclei results in very large and flattened interphase nuclei along with high quality mitotic spreads. Thus, in one aspect of the present invention, cooling of the slide is continued until the sample is frozen. The biological sample may be cooled prior to applying the sample to the frost on the slide.

It has been observed that cooling the sample can improve the quality of the sample spreads, for example the sample may be spread more homogeneously. Without being bound by theory it appears that such pre-cooling of the sample slows down the rate at which the frost layer dissolves, particularly in the early time points after the sample is placed on the frost layer. However, cooling the sampleis not an essential prerequisite for the spreading process. Therefore in some aspects the sample may be applied to the slide at the ambient temperature (for example 18°C- 25°C) . Also provided by the present invention is a microscopy slide preparation device for the preparation of a frosted microscopy slide, the device comprising: a humidity chamber for containing the slide; a support for holding the slide within the chamber; cooling means for cooling the slide; and humidifying means for humidifying the chamber.

In one aspect the device also comprises heating means for drying the sample on the slide once the spreading is complete. This defined drying performed immediately after the spreading improves the quality of the spread. In one aspect of the present

invention, the heating means is a thermoelectric heat-pump (also known as a Peltier heat pump) that may also be used to Cool the slide. In this aspect the polarity of the Peltier pump is reversed once the spreading is complete and the pump is used to heat the slide, to dry the sample. As such the slide can remain in situ on the Peltier heat pump throughout the spreading and drying protocol with no need to transfer the slide to a separate drying device. This increases the efficiency and reliability of the protocol .

The device of the invention may also comprise a microscopy slide preparation device comprising a temperature sensor for measuring the temperature of the slide. The device may also comprise a humidity sensor for measuring the humidity of the slide's

environment. The device may be automated. For example, the device may comprise feedback circuits for maintaining the

temperature and humidity within a pre-set range. The device may comprise an automated dropper, often called a liquid handling device, for applying a biological sample to the slide. Suitable devices are commercially available and known to the skilled person. The dropper may be of a 'fixed' type, so that the dropper remains stationary while a sample is moved underneath, or

'robotized' so that the dropper can move in relation to a

stationary sample. The device may also comprise slide processing systems for allowing the device to prepare multiple slides simultaneously .

Also described herein are kits comprising a microscopy slide preparation device as described herein, and a fixative.

Definitions

Microscopy slide

As used herein, a 'microscopy slide' refers to a slide suitable for analysis with a microscope, for example, an optical

microscope. Slides may be made from a range of light-transmitting materials such as glass, quartz crystal or Perspex, with thermally conductive materials, such as glass being preferred for use in the present invention.

Typically, microscopy slides are rectangular with dimensions of approximately 50 - 100 x 15 - 50 x O.5 - 2.0 mm, for example 75 mm x 25 mm x 1 mm, although slides of different dimensions may be utilized if required. Accordingly, each slide has two broad and approximately flat surfaces; in normal use one of these broad faces is laid on a surface with the other, upper surface being for receiving the sample to be mounted on the slide.

Frosted Slide

As used herein, a 'frosted microscopy slide' refers to a slide having frost on its upper surface. For the avoidance of doubt, this is distinct from a slide manufactured from 'frosted glass' .

Biological sample

As used herein, a 'biological sample' refers to a sample of analytical interest that is obtained from a biological source. In one aspect, the sample may comprise cells, preferably eukaryotic cells, such as mammalian cells. The invention is of particular use with human cells, such as foetal cells, tumour cells or cells comprised within a biopsy sample. Alternatively, the sample may comprise isolated cell components such as nuclei or chromosomes. The sample may comprise cells which have been synchronized and arrested at a particular stage of the cell cycle, such as

interphase or a stage of mitosis, for example prophase, anaphase, metaphase or telophase. Alternatively, the sample may comprise cells at different stages of the cell cycle, for example a mixture of interphase cells and mitotic cells.

In a preferred embodiment, the sample comprises metaphase cells. Accordingly, the method and device of the invention may be used for preparing metaphase spreads.

The sample may be formulated with diluents or carriers - for example buffers or fixatives may be added to the sample. Such buffers typically include salts, pH buffers and polymers such as polyethylene glycol.

Various fixatives are known to those skilled in the art and may be used in the present invention. For example, fixatives may comprise an alcohol, such as methanol, ethanol, propanol or isopropanol, and/or another organic solvent such as acetone or formaldehyde. Fixatives may also comprise acetic acid and/or water .

Fixatives may be combined in various ratios to produce a mixture. Typical fixatives may comprise a mixture of acetic acid and methanol. The mixture may be in a volume ratio of 3 methanol: 1 acetic acid (i.e. approximately 75% methanol by volume) . In one aspect it was found that the method of the invention may be improved by reducing the amount of acetic acid in the methanol- acetic acid fixative. For example, this fixative may contain no more than 20%, preferably no more than 15%, no more than 10% or no more than 5% acetic acid by volume. In one aspect the fixative may contain only trace amounts of acetic acid, by which it is meant that the fixative is substantially free of acetic acid e.g. less than 1% acetic acid by volume. Such fixatives having reduced, ^{^} or being substantially free of acetic acid may be used to preserve antibody binding epitopes in the biological sample. The use of fixatives having reduced, or being substantially free of acetic acid may facilitate the preparation of immunostaining-compatible cell spreads. Effects of different ratios of acetic acid, methanol and water are discussed in (Claussen et al . , 2002) . In other aspects, a sample may be fixed in a mixture of acetone and methanol, such as a 1 acetone: 1 methanol mixture by volume.

Cooling the biological sample

In one aspect it was found that the method of the invention may be improved by cooling the biological sample prior to applying the sample to the slide. This may also be referred to a pre-cooling the sample prior to applying the sample onto the slide. The biological sample may be cooled by storing the sample at a temperature cooler than the ambient temperature of the room for suitable length of time for the temperature of the sample to decreas below the ambient temperature. Techniques and equipment for cooling the biological sample are known to those skilled in the art. For example, in one aspect the biological sample may be cooled by placing the sample in a freezer. The biological sample may be cooled to a temperature of 0°C of lower, more preferably -5 °C or lower, -10°C or lower or -15°C or lower prior to applying the sample on to the slide. Preferably the sample is cooled to -20°C prior to applying the sample on to the slide. Humid environment , Humid, Humidity

As used herein, a ^Ahumid environment' is one in which water vapour is present. Typically the water vapour will be present in a mixture with normal air. The degree of humidity in an environment can be defined in terms of either the relative humidity or the absolute humidity.

Relative humidity is defined as the ratio of the partial pressure of water vapour in a gaseous mixture of air and water vapour to the saturated vapour pressure of water at a given temperature, i.e. the amount of water vapour in a sample of air compared to the maximum amount of water vapour the air can hold at any specific temperature. Accordingly, relative humidity is expressed as a percentage of the maximum amount of water the air can hold at that specific temperature.

In the present invention, the humidity of the environment affects the extent to and rate at which moisture is deposited on the microscopy slide. A range of different environmental humidities may be used with the present invention. The relative humidity in the chamber will be dependant upon temperature of the environment and the precise conditions of frost deposition may be varied by those of skill in the art taking into account factors such as the dimensions of the chamber, the temperature of the slide and the ease of interaction between the humid environment and the slide. Preferably the humidity of the chamber is maintained at a constant level so that the slide is kept in a controlled humidity

environment. Generally, the relative humidity may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 100% relative humidity. Preferably the relative humidity is at least 50% relative humidity.

The relative humidity of the environment may be measured at any temperature within a range such as 5 - 100°C, preferably 10 - 60°C, such as 15 - 50°C or 20 - 40°C. For example, the relative humidity of the environment may be measured at 10, 20, 30, 40 or 50°C, preferably at about 20°C.

The absolute humidity is the quantity of water in a particular volume of air, and is typically measured in grams of water per kilogram of air (g (H₂0) /kg (air ) ) .

In one aspect of the present invention, the absolute humidity of the environment may be within the range 5 - 200g (H₂0) /kg (air ) , such as 10 - lOOg (H₂0) /kg (air) , for example 10 - 60g (H₂0) /kg (air ) or 20 - 50g (H₂0) /kg (air) . Cooling

As used herein, 'cooling' refers to the active process by which the temperature of the microscopy slide is maintained at a temperature below that of the ambient environment. For the purposes of the invention, the cooling of the slide is sufficient to maintain the slide at a temperature where frost forms on the upper surface of the slide. Typically this temperature is 0°C or below; such as in the range of 0 to -40°C, for example, in the range of -20°C to -40°C; such as in the range of -0°C to -30°C, for example from -10°C to

-30°C. In the accompanying examples, a temperature of -30°C is used. This temperature ±5°C is generally suitable, particularly when a thermoelectric heat-pump is used. The temperature of the slide can be reduced by a number of different cooling means. For example, the slide may be placed in thermal contact with a pre-cooled heat sink, or placed in contact with a cooling medium such as liquid nitrogen, or a refrigerant, for example a flow of cooled refrigerant. Alternatively, the slide may be cooled by a Peltier solid state heat pump. Of these, cooling by a Peltier solid state heat pump is preferred because of the precision with which the degree of cooling can be controlled.

Continuing to cool

As used herein, 'continuing to cool' the slide refers to the ongoing maintenance of the slide at a temperature below that of the ambient environment. In one aspect, the temperature of the slide is not maintained below ambient once the biological sample has been applied to the slide. In another aspect, the temperature of the slide is maintained below the ambient after application of the biological sample to the slide until the applied sample has frozen. Maintaining the temperature of the slide below the ambient temperature may be preferred when the sample is applied without being pre-cooled. The slide is typically cooled for at least about 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes or at least 6 minutes after the sample has been applied in order to freeze the sample. For example, the slide may be cooled for 0.5 - 5 minutes after the sample has been applied, preferably 1 - 4 minutes, such as 2.5 - 3.5 minutes, for example 2 - 3 minutes, or preferably 3-5 minutes, such as 4-5 minutes. An appropriate cooling time can be selected by those skilled in the art according to the 'fixative used.

Frost

As used herein 'frost' is used to refer to a deposition of ice crystals on a surface. Typically the ice crystals, form when the surface is cooled below the temperature at which water vapour condenses from the surrounding environment and the temperature of the surface itself is below the freezing point of water. This results in the condensation of water vapour from the environment onto the slide, where the vapour freezes.

In one aspect the ice crystals comprise frozen water and are deposited on the upper surface of the slide. Preferably the frost will substantially cover the upper surface of the slide in a layer of approximately even thickness. The layer of frost may be approximately 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 5.0 mm thick, for example 0.1 - 5 mm thick, preferably 1 - 3 mm thick, such as 1.5 - 2.5 mm thick. In the accompanying examples, we have used a frost layer that is approximately 2 mm thick. The rate at which the ice crystals are deposited on the surface, the size and structure of the individual ice crystals and the thickness of the layer of frost that is deposited affect the spreading of the biological sample when it is applied to the slide. Preferably, the ice crystals form a layer of branched tree-like structures on the slide. These variables in turn depend upon the humidity of the slide's environment and the temperature of the slide; both of these factors may be controlled in the present invention. Controlled cooling

As used herein, 'controlled cooling' and 'controlling the amount of cooling' refer to the ability of a person carrying out a method according to the present invention to readily select the

temperature at which the cooled slide is maintained. Typically this temperature is 0°C or below; such as in the range of 0 to - 40°C, for example, in the range of -20°C to -40°C; such as in the range of -0°C to

-30°C, for example from -10°C to -30°C. In the accompanying examples, a temperature of -30°C is used. This temperature ±5°C is generally suitable, particularly when a thermoelectric heat- pump is used.

The person may also be able to control the rate at which the slide is cooled. For example, the person may be able to control the rate at which the slide is brought from the ambient temperature to the selected temperature.

In one aspect the cooling of the slide is controlled using a Peltier solid state heat pump; by varying the voltage across this device the temperature difference across the pump can be precisely and reliably controlled. In one aspect this device is used in conjunction with a temperature sensor that measures the

temperature of the slide. This allows the temperature of the slide to be monitored and subsequently adjusted by modulating the voltage across the pump. Using this method a user can keep the temperature of the slide within a narrow pre-determined range such as within 0.1, 0.2, 0.5, 1, 2 or 5°C of a selected temperature.

Alternatively, the temperature regulation may be automated using a thermostatic control program.

In another aspect the slide may be cooled by being placed in thermal contact with a cooling medium such as liquid nitrogen, or a refrigerant, for example a flow of refrigerant. The temperature of the slide may be adjusted by altering the temperature or flow rate of the refrigerant. Frozen

As used herein, ^frozen' and ^freezing' refer to the formation of ice crystals throughout the sample until the sample is substantially solidified. In one aspect the solidification of the sample occurs when the sample is cooled below its freezing point as a result of thermal contact with the cooled slide. Typically, the presence of fixatives such as an acetic

acid/methanol mixture means the freezing point of the sample is initially lower than the temperature at which the cooled slide is maintained. As a result the sample does not freeze. However, contact with frost on the slide and exposure to the humid

environment increases the water content of the sample which raises its freezing point. Once the freezing point of the sample exceeds the temperature at which the slide is maintained the sample freezes. The time taken for the sample to freeze can be decreased by cooling the sample prior to applying the sample to the slide. As stated above, a further advantage of cooling the sample is that the time taken for the frost to dissolve is increased giving rise to better homogeneity in the quality of the sample spreads.

Heating the slide

As used herein, heating the slide refers to the application of thermal energy to the slide in order to increase the slide's temperature. Typically the slide is heated in order to dry the sample on the slide. In one aspect the slide is maintained at a temperature of up to 40°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100°C.

The slide may be heated by a number of different heating means, such as an induction heater or a Peltier solid state heat pump or resistance-based heater. In one aspect heating is with a Peltier solid state heat pump; this allows the degree of heating to be controlled.

A Peltier solid state heat pump may be used to both heat and/or cool the slide, since the polarity of the Peltier heat-pump can be inverted instantaneously by reversing the polarity of the applied voltage. Accordingly, in one aspect the Peltier heat pump functions sequentially as both cooling and heating means; once cooling of the slide is no longer required, the Peltier heat pump is used as a heating means to dry the sample. In this aspect, the slide can remain in situ between the cooling and drying steps, increasing the efficiency of the method and reducing the

likelihood of damage or disruption to the mounted sample.

Controlled heating

As used herein, 'controlled heating' and 'controlling the amount of heating' refer to the ability of a person carrying out a method according to the present invention to readily select the

temperature at which the heated slide is maintained. Preferably the person will also be able to control the rate at which the slide is heated. For example, the person may be able to control the rate at which the slide is brought from a selected temperature below ambient at which the slide is maintained to a selected temperature above ambient.

In one aspect, the heating of the slide is controlled by using a Peltier solid state heat pump. By varying the voltage across this device the temperature difference across the pump can be precisely and reliably controlled. In one aspect this device is used in conjunction with a temperature sensor that measures the

temperature of the slide. This allows the temperature of the slide to be monitored and subsequently adjusted by modulating the voltage across the pump. Using this method a user may keep the temperature of the slide within a narrow pre-determined range such as within 0.1, 0.2, 0.5, 1, 2 or 5°C of a selected temperature. Alternatively, temperature regulation may be automated using a thermostatic control program. In another aspect the slide may be heated using an induction heater or a resistance-based heater. The temperature of the slide may be adjusted by altering the voltage across the heater. For example, a substantially flat resistance-based heater may be placed between a Peltier heat pump and the sample which may be used in the heating process. Alternatively, a substantially flat piece of magnetic metal may be placed between the Peltier heat pump and the slide which may be heated using electromagnetic induction .

Drying the sample

As used herein, 'drying the sample' refers to the reduction in the amount of liquid present on the slide. The sample may be

partially dried so that some liquid remains on the sample.

Typically the sample is dried until it is substantially free of liquid or, preferably until no liquid remains on the sample.

The sample may be dried by heating the slide, causing the liquid on the slide to evaporate.

Furthermore, as an additional drying force, a source of air flow may be directed at the center of the spreaded area. Air flow may be generated by an air pump, for example, a membrane pump. In one embodiment, the air pump is placed outside the chamber and the power supplied to the pump is regulated to control the rate of air flow. The air may be carried via a tube from the air pump to an outlet at the end of the tube, inside the chamber. Preferably, the outlet may be positioned to direct air flow at the slide for drying the slide. For example, the outlet may be adjustably mounted inside the chamber such that the direction of air output may be adjusted. The outlet may be adapted with a jet-like ending. Furthermore, the air pump system may be equipped with a dust and/or moisture filter.

Peltier solid state heat pump

As used herein, 'Peltier pump', 'Peltier unit', 'Peltier heat pump' or 'Peltier solid-state heat-pump' refer to a solid-state thermoelectric heat pump as is known in the art.

Briefly, the pump is typically an approximately planar element comprising P-doped and N-doped Bismuth Telluride. By applying a voltage across the pump thermal energy is transferred from one face to the other, resulting in a 'hot side' and a 'cold side' . The temperature difference between the two sides may be altered by changing the voltage passed across the pump. In addition, reversing the polarity of the applied voltage also reverses the identity of the ^¾hot' and ^cold' sides. As such, a slide in contact with one face of the pump can be either cooled or heated to a greater or lesser extent depending on the polarity and magnitude of the applied voltage.

One face of the heat pump may be placed in thermal contact with a heat sink. This enables thermal energy to be efficiently

dissipated from or supplied to the face of the pump in contact with the heat sink, increasing the efficiency of the heat pump. In one aspect the heat sink may be thermally insulated from the environment apart from the region of thermal contact with the heat pump. In a further aspect the heat sink may itself be cooled; for example, the heat sink's temperature may be maintained at 0°C by placing it in contact with water ice. Alternatively, the heat sink may be cooled by placing it in thermal contact with a refrigerant or cooling liquid. The refrigerant or cooling liquid may be circulated between a heat exchanger and thermal contact with the heat sink, for example through tubing.

Immunostaining

As used herein, immunostaining' refers to the labeling of antibodies or antigens with reporter molecules. Once labeled, the position of the antigen of interest may be identified through the activity of the reporter molecule.

In one aspect, the immunostaining uses two sets of antibodies: i) a primary antibody against the antigen of interest, and ii) a secondary antibody that recognizes the primary · antibody and is also coupled to a reporter molecule.

A reporter molecule can be any molecule that produces or can be induced to produce a signal, including but not limited to

fluorescers, radiolabels, enzymes, chemiluminescers or

photosensitizers . Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

Suitable reporter molecules include radiolabels such as ¹³¹I or ⁹⁹Tc, which may be attached to antibody molecules using

conventional chemistry known in the art of antibody imaging.

Reporter molecules also include enzyme reporter molecules such as horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase ( "G6PDH" ) , alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase and acetylcholinesterase.

Reporter molecules include fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochrome , rhodamine compounds and derivatives and GFP (GFP for "Green Fluorescent Protein") . Reporter molecules further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge

Immunostaining can be used to report the position of any molecule to which a primary antibody can be raised. Typically, the molecules to be visualised are proteins, but may alternatively be DNA, RNA, lipids, carbohydrates or any complex thereof.

Molecules to be visualised by immunostaining may be bound by any binding member or substance having an antibody antigen-binding site with the required specificity and/or binding to the molecule to be visualised. For example, the molecule to be visualised may be bound by any antibody, such as a polyclonal antibody or a monoclonal antibody; any antibody fragment, such as Fab, Fab' , Fab'-SH, scFv, Fv, dAb and Fd; engineered antibody molecules, such as Fab₂, Fab₃, diabodies, triabodies, tetrabodies and minibodies; and any other polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Accordingly, immunostaining can be used to analyse any manner of biological sample that may be spread on a slide using the methods of the present invention.

In one aspect the primary antibodies bind proteins associated with chromosomes, for example histones, such as H2AX. The antibodies may be a mouse monoclonal anti-y-H2AX such as 05-636 (Upstate) , or a rabbit polyclonal anti-phospho-Histone H2AX (serl39) antibody (Cell signaling) . In another aspect, primary antibodies may bind antigens associated with DNA breakages, such as MDC1, 53BP1 and BRCAl polypeptides.

In some aspects, primary antibodies directly labeled with a reporter molecule are used. Direct labeling decreases the number of steps in the staining protocol and may reduce any antibody cross-reactivity. For example, primary antibodies directly conjugated to a reporter molecule such as a fluorescent dye.

Analysing

As used herein, 'analysing' the sample refers to study of and/or extraction of data from a sample mounted on a slide. In one aspect the analysis is a quantitative analysis of mitotic breaks, gaps and constrictions based on a morphological evaluation of chromosomes spread on the slide.

The analysis may be performed manually or, alternatively, may be scored using an automated procedure. In one aspect, the

automated procedure may involve the collection of images from slides using a microscope followed by computer analysis of immunostained foci from the images. This data may then undergo statistical analysis in order to identify patterns or

correlations .

Microscope

As used herein, 'microscope' refers to an instrument used for the magnification and analysis of a sample mounted on a slide. The microscope may be an optical microscope, preferably a microscope for analysing electromagnetic radiation with wavelengths between 100 - 1000 nm, preferably 300 - 800 nm. In one aspect the microscope has a magnification range of about lOx or more (e.g. 10 - lOOx) to about lOOOx (e.g. 500 - lOOOx) . For example, the microscope may be a Zeiss Axioplan II.

The microscope may be coupled to a camera for capturing the images of the sample produced by the microscope.^' In a further aspect the microscope may also be coupled to a computer for analysis of the images captured by the camera.

Metaphase

As used herein, 'metaphase' is used to refer to the stage of the eukaryotic cell cycle following prophase and preceding anaphase in which the condensed chromosomes are paired and preparing to separate. Typically during metaphase the paired chromosomes align along an axis toward the centre of the cell. For some eukaryotic cells, prometaphase' is used to refer to the stage of the cell cycle immediately following prophase and before metaphase.

Prometaphase is characterised by nuclear envelope breakdown, microtubule organisation and condensed chromosomes.

Metaphase spread

As used herein, 'metaphase spread' refers to a preparation of a metaphase or prometaphase cell in which the condensed chromosomes are substantially spread out and may be analysed, for example by immunostaining and microscopy. 'Metaphase spreads' may comprise metaphase cells, prometaphase cells, or a mixture of both

metaphase cells and prometaphase cells. For example, metaphase spreads of predominantly metaphase cells may be obtained by methods known to persons skilled in the art by treating cells with inhibitors of mitosis.

Interphase

As used herein, 'interphase' is used to refer to the stage of the eukaryotic cell cycle in which the cell is not undergoing mitosis or cytokinesis. Interphase is the stage in which the cell spends the majority of its time and during which protein synthesis and cell growth occurs. Typically, during interphase the nuclear membrane is intact, chromatin has not yet condensed and

chromosomes are not visible. In the cell cycle, interphase is generally preceded by telophase and cytokinesis of the M phase and followed by prophase of the M phase. Cells which divide rarely or do not divide may spend the majority of their time in the G₀ phase of interphase, a resting phase in which the cell is neither dividing nor preparing to divide.

Adjustable

As used herein, Adjustable' refers to a device where the

parameter referred to can be readily controlled (as defined above for cooling/heating) .

The humidity of the environment within the humidity chamber may be adjusted by altering the power supply to the heating element heating the aqueous solution. This will alter the temperature of the solution and, consequently, the rate at which water vapour is generated. In one aspect, the humidity of the environment within the humidity chamber may be reduced by the opening of vents in the walls of the humidity chamber to allow exchange of air from outside the chamber with that inside the chamber. Preferably, the rate of exchange of air may be adjusted using a device for controlling air flow, such as a fan or an air pump.

The present invention provides a microscopy slide preparation device for the preparation of a frosted microscopy slide.

Described below are elements which may be comprised in a

microscopy slide preparation device according to the present invention.

Figure 6 shows detailed top (A) and cross-sectional (B) views of a preferred embodiment of the microscopy slide preparation device of the present invention. According to the present embodiment, the device 1 comprises a humidity chamber 2, a lower portion of which may be filled with an aqueous liquid 17, which contains a heater 13 for heating the liquid 17 and humidifying the chamber, and a Peltier unit 6, for cooling a microscopy slide 16.

The heater 13 is submerged in the aqueous liquid 17 and is connected to a power source 14. The chamber has a heated lid 2a, having a spiral heating element 19 embedded therein and an input passage 3 arranged above the slide position 16 for applying a biological sample 18 to the microscopy slide 16. The heating element 19 of the lid 2a is connected to a power source 4.

In the present embodiment the Peltier unit 6 acts as a cooling means for cooling the slide, a support to hold the slide and a heater for drying a sample on the slide. The Peltier unit 6 is connected to a power source 15 which may be used to control the current and voltage supplied to the Peltier unit 6 and to control the polarity of the Peltier unit for switching between cooling and heating functions.

The Peltier unit 6 is in thermal contact with and is mounted on a heat sink 7. The heat sink 7 is mounted in a thermally insulating platform 5 above the level of the aqueous liquid 17. The heat sink 7 is in thermal contact with a cooling liquid (not shown) which circulates through tubing 8 from a cooler unit 9 outside the chamber, comprising a heat exchanger and a cooling liquid pump (not shown) .

An air outlet 10 is pivotally mounted on the platform 5 such that air flow may be directed at the slide 16 for additional drying force. The outlet 10 is connected to an air pump 12 via a pipe 11. The air pump 12 comprises a membrane-based air pump, an air filter and a moisture absorber (not shown) . Humidity Chamber

As used herein, a ^xhumidity chamber' refers to a chamber or compartment comprised with a device that may contain a humid environment and is suitable for containing a microscopy slide. In one aspect the chamber may be sealed so that the environment within the chamber is substantially isolated from the external environment. For example, the chamber may be arranged such that the exchange of gases between the internal and external

environments is restricted or prevented.

In another aspect the chamber walls comprise vents for permitting exchange of gases between the internal and external environments of the chamber. This allows water vapour to leave the humidity chamber, reducing the humidity of the environment inside the chamber. Air from outside the chamber may also be allowed to flow into the chamber through the vents. Furthermore, the vents may be connected to a device for controlling airflow such as a fan or an air pump. In some aspects, the chamber may have a heated lid. This prevents condensation from collecting on the inside of the chamber lid, which could result in water dripping on the sample. When the lid is transparent, this may also ensure that the user can see inside the chamber so that he may have visual control over the spreading process. In preferred embodiments, the lid comprises a lid heating element, such as an electrical element heater.

Accordingly, the lid may be connected to a power source for powering the lid heating element. The power source may be adjustable for controlling the heating of the lid. In some embodiments, the lid is glass and comprises a spiral electrical heating element embedded therein.

The lid may comprise an input passage through which the biological sample is applied to the slide. For example, the input passage may be a hole in the lid through which a sample application device, such as a pipette, is insertable. Preferably, the sample may be applied to the slide while keeping the environment within the chamber substantially isolated from the external environment.

The chamber may contain devices such as a heater and/or a Peltier heat pump. These devices may be connected to a power source external to the chamber via electrical wires that pass through channels in the chamber wall. In a further aspect, the chamber wall may contain an inlet aperture through which water vapour may enter .

Preferably, the chamber is watertight such that the lower portion of the chamber may be filled with an aqueous medium for

humidifying the chamber. US 2005/0042767 Al describes a microscopy slide preparation device for carrying out a method of preparing a sample slide. Fig 8 of US 2005/0042767 Al shows a device comprising a humidity chamber. The aspects of this device used to control the humidity of the slide's environment are suitable for use in the present invention. This microscopy slide preparation device could be adapted to become a microscopy slide preparation device according to the present invention by addition of means to cool the slide

sufficiently to form frost on the slide's surface. Humidifying the Chamber, Humidifying means

As used herein, 'humidifying the chamber' refers to increasing the amount of water vapour within the humidity chamber.

In one aspect, the humidifying means comprise a port in the wall of the humidity chamber through which water vapour can enter the chamber. Alternatively, the humidifying means may comprise a heater for heating an aqueous medium. The heater may be an electrical element heater.

In one aspect the heater is contained within the humidity chamber and may be integral to the chamber. In a further aspect the heater is located toward the bottom of the chamber so that, in use, the lower portion of the chamber may be filled with an aqueous medium that may be heated by the heater, so humidifying the chamber. Heater

As used herein, 'heater' refers to a device for heating an aqueous medium such that water vapour is generated. The generated water vapour may then be used to humidify the humidity chamber. The heater may be any device capable of heating the aqueous medium, for example, an electrical element. The aqueous medium may be any medium that produces water vapour when heated,

preferably pure, or substantially pure water. In one aspect the heater is contained in the chamber.

Alternatively, the heating element may be outside the chamber, with the water vapour generated by the heating of the aqueous solution conveyed into the chamber via an inlet aperture in the chamber wall.

Support

As used herein, 'support' refers to an element for supporting a slide. The support may be a platform on which the slide rests. Alternatively the slide may only contact the support at either end, leaving the centre of the slide unsupported. In another aspect the support may be a clip or grip that grips the slide at only at one end. In one aspect the support is adapted to hold a microscopy slide of dimensions 50 - 100 x 15 - 50 x O.5 - 2.0 mm. The means for cooling and/or heating the slide may be comprised within the support. Accordingly, the slide may be cooled or heated via conduction from the support. In one aspect the support is a platform comprising a Peltier solid state heat pump on which the slide rests; in this aspect the support is in thermal contact with the slide across substantially the whole of the slide's lower surface, enabling efficient heat exchange between the heat pump and the slide. In a further aspect, the support is also in thermal contact with a heat sink to enable the efficient

dissipation of thermal energy.

Heat sink

As used herein, ^Aheat sink' is used to refer to a passive element for dissipating thermal energy absorbed from a second body.

Typically the heat sink will be made from a material with high thermal conductivity and will have heat capacity sufficient to dissipate thermal energy from the second body without the

temperature of the heat sink rising significantly.

In one aspect of the present invention the heat sink is a block of metal or metal alloy, such as aluminum (Al) , copper (Cu) or steel, and the second body is a Peltier solid state heat pump.

In a further aspect, the heat sink may be in thermal contact with a coolant, such as ice, a refrigerant, or a cooling liquid, in addition to being in thermal contact with the second body. For example, the heat sink may be in thermal contact with an ice bath. Figure 7 shows detailed top (A) and cross-sectional (B) views of an embodiment of the microscopy slide preparation device of the present invention wherein the coolant is ice.

In other aspects, the refrigerant or cooling liquid may be circulated between a heat exchanger and thermal contact with the heat sink, for example through tubing.

The heat sink may be thermally insulated from the environment apart from the thermal contact with the second body and/or coolant, refrigerant or cooling liquid.

Kits

The present invention also provides a kit comprising a microscopy slide preparation device as described herein and a fixative for adding to the biological sample. Various fixatives are known to those skilled in the art and may be used in kits of the present invention. Specific examples of fixatives are described above. A kit may comprise one or more fixatives and/or mixtures of fixatives. In some preferred embodiments, a kit may comprise one or more separate fixatives selected from: a fixative comprising methanol; a fixative comprising ethanol; a mixture of methanol and acetic acid; and a mixture of methanol and acetone. For example, a mixture of 3 methanol: 1 acetic acid, and/or a mixture of 1 methanol: 1 acetone. In some embodiments, a kit may comprise a fixative comprising less than 1% acetic acid.

Examples Cell Culture

Human U-2-OS osteosarcoma cells and primary BJ fibroblasts were grown in DMEM containing 10% fetal bovine serum (GIBCO) . A U-2-OS derivative cell line capable of down-regulating endogenous ATR in an inducible fashion was generated by cotransfecting U-2-0S cells with pcDNA6/TR and pSUPERIOR. puro-shATR and selecting cells with stably integrated plasmids in medium containing 5 μg/ml

Blasticidin S and 1 ug/ml Puromycin (Sigma) . For ATR knock-down, doxycycline (1 μg ml; Calbiochem) was users added to the culture medium 48 hours before analysis.

Cells synchronization and drugs treatment

Cells were synchronized by incubating them in medium containing 3 mM thymidine (Sigma T-9250) for 24 hours. Afterwards, cells were washed by PBS three times and released into medium containing 24 μΜ 2-deoxycytidine ( Sigma-Aldrich D-0776) . For CDK1 inhibition 25 μΜ Roscovitin (Calbiochem #557360) or 1 μΜ CGP74514A (Calbiochem # 217696) were applied to the cells synchronized as above in late G2. To release from the cell cycle block, the drugs were washed away from the cells by three washes of PBS after 4-5 hours. For induction of CFS expression we used 0.1 - 0.4 μ Aphidicolin (Biochemika 10797) for 24 hours in unsynchroni zed cells. In synchronized cells 0.4 μΜ Aphidicolin was added immediately after the release from the thymidine block for the time as specified in the figure legends. For arresting the cells in mitosis for metaphase spreads analysis we added 1 μg/ml of Colcemide (Karyo MAX, Gibco) two hours before analysis.

Preparation of the Frost Spreading device

The results of experiments (l)-(4) below were obtained using 100% humidity in the spreading device as follows. Before the spreading procedure the water bath inside the spreading device was filled with water and preheated to 45°C. This ensures that the internal environment will hold 100% humidity during the operation. Later experiments have demonstrated that the results of cell spreading experiments are equally good or better if the humidity in the spreading device is set to 50% and the temperature is 20°C. The results of example 5 were obtained under these conditions. For all experiments, the glass lid of the device was opened and directly on the ceramic surface of the Peltier unit was placed a standard microscopy glass slide. To ensure better heat contact a drop of pure ethanol may be placed on the ceramics before laying the slide. After the slide was in position and the glass lid closed, the frost layer forming program could be initiated.

Frost spreading of cells with intact γ-Η2ΑΧ epitope

U-2-OS and BJ-primary fibroblasts cells were trypsinized and resuspended in pre-cooled (4°C) PBS containing 5% of FBS,

immediately centrifuged in 15 ml tubes 500G at 4°C and resuspended in 8 ml of 60 mmol KC1/RT (at room temperature) . Cells suspension was incubated for 5 min in 37 °C water bath and for another 25 min on ice, centrifuged again 500G/ 4°C , resuspended in 10 ml of pre- chilled (-20°C) acetone/methanol 1:1 and incubated for 10 min/RT. The suspension was centrifuged again 500G/RT and drop-wise resuspended in 5 ml pre-chilled (-20°C) methanol for long term storage in freezer. Before the spreading procedure, the sample was centrifuged for 2 min at 700G/RT and most of the methanol supernatant was sucked away. Depending on the amount of cells 0.5-1 ml of methanol was left for resuspending of the pellet. 15 μΐ drop of the cell suspension was placed in the middle of the microscopy glass slide covered by approximately 2 mm thick frost layer, induced by incubation of the microscopy glass slide inside a wet chamber on a cold surface of a running Peltier heat pump at a power setting of a constant current of 3.5A. The power supply of the Peltier unit was programmed to allow setting of a constant power for defined time with the time of the process being the only factor which was modulated in this step. The polarity of the power supply was set to cool down the slide and the heat from the Peltier unit was transferred into the heat sink and subsequently into the external heat exchanger. The microscopy glass slide was further incubated inside the wet chamber while the Peltier unit was on for another 2-3 minutes to allow the methanol to be enriched by water via condensation to the degree when the mix freezes forming crystals. Crystallisation introduced a physical force contributing to disruption of the compact chromatin

structure making the cells more flattened during the subsequent drying process. After this moment the Peltier unit polarity was reversed, which led to immediate heating of the microscopy glass slide and the sample was dried quickly, with the Peltier unit set to 2.5A, and immediately processed for immunofluorescence (IF) staining. Timing and power setting for the Peltier unit and humidity and temperature of the humid chamber was kept constant for all the samples. As an additional drying force, a source of air flow was directed at the center of the spreaded area. Air flow was generated by a membrane pump which was placed outside the chamber and the power of the pump was regulated. The air was carried via a tube ended by a jet-like ending. The pump system was equipped with a dust and moisture filter.

Immunofluorescence and Scoring of γ-Η2ΑΧ foci by automatic software routine

Frost spreaded samples were washed for 10 minutes in a PBS solution containing 0.5% Tween detergent at room temperature (RT) and then incubated in PBS solution with mouse (or rabbit) primary antibody against γ-Η2ΑΧ for 1 hour/RT, washed in PBS/Tween solution and then incubated in PBS solution with anti-mouse (or anti-rabbit) secondary Alexa-flour dye conjugated antibody for 1 hour/RT. Slides were dried by ethanol series and stored at 20°C in 100% ethanol. Before analysis the dried sample was mounted in Vectashield mounting medium with DAPI (Vector Laboratories) .

Examination was done under fluorescent microscope (Zeiss Axioplan II) and photographed by Cool snap camera (Photometries) . The exposure time, binning, setting of the microscope and the UV light source was constant for all the samples. Each picture was saved as a TIFF and further examined by combining Adobe Photoshop, Image J and Excel software in a macro routine with fixed parameters. Foci were scored, analyzed for signal intensity and summarized automatically (Figures 4 & 5) . Data sets were graphically processed and statistically tested (T-test) in NCSS software (www . ncss . com) .

FISH

The DNA genomic vector (BACe3.6) containing FRA3B was obtained from RZPD, Germany (clone ID: RZPDB737B092164D6) . Clone was amplified in bacteria and isolated using the Large Construct DNA purification kit (Roche) . The DNA was labelled with digoxigenin (DIG) by the DIG-Nick translation Mix kit (Roche Cat.

No.11745816910) . DIG-labeled probes were detected with rhodamine- conjugated sheep anti-DIG specific antibodies (Roche Cat. No.

11207750910) . FISH on metaphase chromosomes was performed as previously described (Margaret A. Leversha, 1998).

Combined IF and FISH

For combined IF and FISH the sample was frost spreaded and immunostained for γ-Η2ΑΧ as described above. Slides were dried using ethanol series and stored in -20°C in 100% ethanol at least for two days. Before the hybridization, slides were air dried and than immersed in 50 mmol ethylene glycol-bis (succinic acid N- hydroxy-succinimide ester; E-3257, Sigma-Aldrich, St. Louis, MO) dissolved in mix of DMSO and 50% Acetic acid 3:1 ratio for two minutes. Afterwards samples were washed in PBS and dehydrated by ethanol series. Hybridization was performed as described above but no protease pretreatment was used.

Antibodies For IF γ-Η2ΑΧ detection mouse monoclonal anti-H2AX antibody

(Upstate 05-636) or rabbit polyclonal phospho-Histone H2A.X (Ser 139) (Cell signaling Lot : 2 #2577L) were used. Frost spreading of cells for morphological, FISH and ploidy analysis of mitotic figures

Cells were trypsinized and resuspended in pre-cooled (4°C) PBS containing 5% of FBS, immediately centrifuged in 15 ml tubes 500G/ 4°C and resuspended in 8 ml of 60 mmol KC1/RT. Cells suspension was incubated for 20-25 min at 37°C. Next, cells were pre-fixed by adding 2 ml of freshly prepared fixative (Methanol/Acetic acid 3:1,-20°C) into the hypotonic solution and incubated for another 10 min at RT . Next, cell suspension was centrifuged 500G/ 4°C and resuspended in 10 ml of pre-chilled fixative followed by 10 min/RT incubation. Last three steps were repeated three times. After the last washing step the cells were left in the fixative for long term storage in freezer at -20°C. Before the spreading procedure, the sample was centrifuged for 2 min at 700G/RT and most of the fixative supernatant was sucked away. The pellet was resuspended in 0.5-1 ml of supernatant depending on the amount of cells. 15 μΐ drop of the cell suspension was placed in the middle of microscopic glass covered by approximately 1 mm thick frost layer, induced by incubation of the glass inside a wet chamber on a cold surface of a running Peltier heat pump at a constant current of 3.5A for a defined time with the polarity of the power supply set to cool down the microscopy glass slide, as described above.

Peltier pump was lying on an aluminum heat sink dipped in ice. The Peltier pump was kept running in constant power mode after the drop of suspension of cells in fixative was placed on the slide. This was followed by slow gentle spreading in which the fixative dissolved the frost layer and became enriched by water. More water was also recruited via condensation. Enrichment by water contributed to shaping the mitotic figures, because acetylated proteins are hygroscopic (for example, see Figure 2). Once the drop was evenly spread on the surface of the glass, the Peltier unit polarity was reversed (leading to immediate heating of the microscopic glass) and the sample was dried quickly at 12V with unlimited amps. Timing and power setting for the Peltier unit, humidity and temperature of the wet chamber was kept constant for all the samples. As described above, as an additional drying force, a source of air flow is directed to the center of the spreaded area.

In general no further processing of the sample is necessary.

Further staining, banding and FISH procedures of these samples are fully compatible with standard protocols. Only for phase-contrast based microscopy, the sample could be incubated for 5-10 seconds in 50% acetic acid at 60°C and then washed in distilled water to increase the contrast of the chromosomes.

Sister chromatid exchange assay

BJ primary fibroblasts were incubated in the presence of 2.5nM Camptothecin (Sigma) and 10μΜ BrdU (Sigma) for 46 h, after which Colcemide (1 μg/ml, Gibco) was added to the medium for^" an additional 2 hours. Cells were harvested by trypsinization, resuspended in hypotonic buffer (75 mM KC1) , and incubated at 37°C for 25 min. Following centrifugation, cell pellets were incubated with fixative (75% Methanol; 25% Acetic Acid) for 10 min, washed twice, and mitotic spreads were prepared using the Frost method for spreading of cells for morphological, FISH and ploidy analysis of mitotic figures as described above. For chromatid staining slides were immersed in Acridine Orange (0.1 mg/ml in water, Molecular Probes) for 3 min, washed thoroughly in water and incubated in Sorenson Buffer, . pH 6.8 (0.1 M Na2HP04; 0.1 M NaH2P04) for 3 min. Finally, slides were dried and analyzed by fluorescence microscopy.

Comparison of cell nuclei sizes in cells grown directly onto microscope slides, dropped onto microscope slides or applied using the frost spreading technique.

U-2-OS cells were applied to glass microscope slides using three different methods: (1) Cells were grown directly on a glass microscopic cover slide using (METHOD) . (2) Cells were transferred into suspension, fixed by methanol /acetone fixative and then dropped on the microscopic slide and air dried (the 'classic' method for cell dropping. (3) Cells were transferred into suspension, fixed by methanol/acetone fixative and applied to the microscope slide by the frost spreading method described herein. All samples were stained for DAPI and γ-Η2ΑΧ and images were taken at 100X or 400X magnification as indicated.

Results

(1) Morphological and ploidy analysis

A sample was prepared using human lymphocytes and spread onto slides using the Frost method for spreading of cells for

morphological, FISH and ploidy analysis of mitotic figures as described above. The slides were subsequently prepared and their morphology and ploidy analysed. Results are shown below.

Therefore, of the total number of spreads scored, 100/129 = 78% were of sufficient quality to permit ploidy analysis. Using classical methods of manual dropping of cells onto a glass slide with cells spreading on a layer of water, as little as 10% or fewer cells may routinely be of sufficient quality to permit analysis, although results are highly dependant on the skill of the person carrying out cell spreading. , As shown herein, the Frost method provides a high percentage of spreads of sufficient quality, combined with ease of use for non-specialists.

A sample was prepared using fibroblasts using the Frost method as described above for morphological, FISH and ploidy analysis of mitotic figures. The slides were subsequently prepared and samples stained for ploidy and cohesion analysis. Results are shown in Figure 1A.

A sample was prepared using BJ primary fibroblasts using the Frost method as described above. The slides were subsequently prepared and samples stained for SCE analysis. Results are shown in Figure IB.

A sample was prepared using U-2-OS cells using the Frost method as described above. The slides were subsequently prepared and their morphology analysed following staining with DAPI . Results are shown in Figure 2.

Both Figures 1 and 2 show slow and gentle spreading leads to (i) a minimum of overlapping cells and (ii) a minimum of migrating chromosomes (ploidy analysis) .

(2) Immunofluorescence analysis

U-2-OS cells were treated with APH (0.1 μΜ) for 24 hours, enriched for mitotic cells for 2 hours of Colcemide treatment before the harvest. Mitotic spreads were prepared using the Frost method for spreading of cells with intact γ-Η2ΑΧ epitope as described above, and stained for γ-Η2ΑΧ. γ-Η2ΑΧ is a marker of DNA lesions. γ- H2AX co-localizes with breaks in metaphase chromosomes. Results are shown in Figure 3.

Cells lacking functional ATR are deficient in checkpoint response to agents that cause replication stress (e.g. Aphidicolin (APH)) and are known to respond to APH by a strong BGC expression in mitosis (Casper et al., 2002). Consistent with this, doxycyclin- induced sh-ATR-U-2-OS cells exposed to low doses of APH (0.1 μΜ) revealed massive BGC generation manifested by >100 γ-Η2ΑΧ foci per cell line in the majority of the mitotic spreads. Figure 5 (A) shows a chart summarising average number of γ-Η2ΑΧ foci in DOX- induced and non-induced sh-ATR U-2-OS cells treated by different doses of Aphidicolin (APH) . Figure 5 (B) shows examples of spreads scored for γ-Η2ΑΧ foci. (3) Automated Picture Analysis following Immunoflourescence

Analysis

Following γ-Η2ΑΧ immunoflourescence analysis (as described above) , the DNA lesions in metaphase spreads can be scored as a number of γ-Η2ΑΧ foci (green staining of breaks on foci). The immunostained metaphase spreads are amenable to automated picture analysis.

Figure 4 shows a diagrammatic representation of such a method. Pictures with γ-Η2ΑΧ immunoflourescence signal taken by digital camera are processed by automatic routine in Adobe Photoshop 7.0 for background subtraction and then analysed in ImageJ 1.37v. Data sets associated with every scored focus are further processed by statistical software. All parameters are fixed and routine is automated using macro program Mouse Recorder. Recognition and scoring of the foci is automatic, unbiased and sensitive and additional information is added in the form of the fluorescence signal intensity associated with the individual foci. The data obtained by this method is amenable to robust statistical analysis and is compatible with scoring spreads with large numbers of DNA breaks .

(4) Combined FISH and Immunofluourescence

Immunofluorescence detection of γ-Η2ΑΧ on metaphase spreads has been shown to be compatible with FISH (using FRA3B probe) which allows direct assessment of the integrity and expression of specific fragile sites independently on the cell cycle stage and degree of chromomsomal condensation. Figure 8 shows an example of metaphase spreads stained for γ-Η2ΑΧ ( immunoflourescence ) and fragile site FRA3B (FISH) . Figure 8 (A) shows expression of FRA3B within the cell cycle measured as incidence of overlays of γ-Η2ΑΧ foci and FRA3B probe signals (200 cells were scored for each column) . Figure 8 (B) shows examples of scored samples.

(5) Comparison of U-2-OS cell nuclei sizes

The results of this experiment are shown in Figure 9. Applying a culture of cells to a microscope slide using the frost spreading method described herein significantly increases the size of cells allowing easier visualisation of the cell nucleus.

Figures 9(A) and 9(D) (left column) shows cells which are grown directly on a glass microscopic cover slide; there are no mitotic cells in this sample. Figures 9(B) and 9(E) (middle column) show cells which were dropped on the microscopic slide and air dried (the Classic' method for cell spreading) . Mitotic cells are marked by ^λΜ' and arrows .

Figures 9(C) -(E) (right column) shows cells which were applied to the microscope slide by the frost spreading method described herein. The cells were fixed in the same methanol/acetone fixative as Figures 9(B) and 9(E) and mitotic cells are marked by ^λΜ' and arrows. Figures 9 (A) -(C) (top row) show DAPI stained nuclei at 100X magnification. Figures 9(D) -(F) (bottom row) show nuclei stained for DAPI (blue) and γ-Η2ΑΧ (green) at 400X magnification. The size of the cell nuclei is indicated with a white line. The cell nuclei shown in Figures 9 (C) and (F) which have been prepared using the frost spreading method are around 300% larger than the cells shown in Figures 9(A) (B) (D) and (E) .

These results demonstrate that applying cells to a microscope slide using the frost spreading method described herein significantly increases the size of cells undergoing both interphase and mitosis i.e cells undergoing all stages of the cell cycle. The size increase has the advantage of allowing the sample to be observed in much greater detail during microscopy analysis than a sample prepared using an alternative method and viewed using the same magnification. Furthermore, the size increase is accompanied by a significant flattening of the sample. The flattening of the cell samples has the advantage of allowing microscopy in minimum optical layers, which also allows the sample to be observed in greater detail. This also has the advantage of making the microscopy analysis faster, because no or minimum images from different depths within the sample (z-stacks) are necessary for obtaining the complete signal information. These results demonstrate that the method described herein is useful for preparing cells for all types of immunostaining analysis . References

Octavian Henegariu, Nyla A. Heerema, Lisa Lowe Wright, Patricia Bray-Ward, David C. Ward, Gail H. Vance. Improvements in

cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry 43 Issue

2, plOl - 109 (2001)

Margaret A. Leversha, in Cell Biology: A Laboratory Handbook, Julio E.Celis, Ed., Academic Press, San Diego, ch . 11, pp. 428-436 (1998) .

Casper AM, Nghiem P, Arlt MF, Glover TW. (2002) . ATR regulates fragile site stability. Cell. 111(6), 779-89.

Claussen,U., Michel, S., Muhlig,P., Westermann, . , Grummt,U.W., Kromeyer-Hauschild, K. , and Liehr,T. (2002) . Demystifying

chromosome preparation and the implications for the concept of chromosome condensation during mitosis. Cytogenet. Genome Res. 98, 136-146. Margaret A. Leversha (1998) . Mapping Cloned DNA on Metaphase

Chromsomes Using Fluorescence in Situ Hybridization. In Cell Biology: A Laboratory Handbook, Julio E.Celis, ed. (San Diego: ACADEMIC PRESS), pp. 428-436.

Abbreviations

APH Aphidicolin

BGC breaks, gaps and constrictions

°C Centigrade

CGH comparative genomic hybridization

DIG digoxigenin

DNA Deoxyribonucleic acid FBS foetal bovine serum

FISH fluorescent in situ hybridization g Grams

hr Hour

IF immunofluorescencelg Immunoglobulin min Minute

nm Nanometre

PBS Phosphate buffered saline

PCR Polymerase chain reaction

SCE sister chromatid exchange

Claims

1. A method for preparing a microscopy slide for analysis of a biological sample comprising:

i) placing the slide in a humid environment;

ii) cooling the slide so that frost forms on its upper surface; and

iii) applying the biological sample to the frost on the slide .

2. The method according to claim 1 wherein the biological sample is cooled prior to applying the sample to the frost on the slide.

3. The method according to claim 1 or claim 2 further

comprising the step of continuing to cool the slide after the sample has been applied until the sample is frozen.

4. The method according to any one of the preceding claims wherein the humidity of the environment is controlled.

5. The method according to claim 4 wherein the humidity is kept at approximately 50% relative humidity

6. The method according to any one of the preceding claims wherein the amount of cooling of the slide is controlled.

7. A method according to claim 6 wherein the slide is cooled to a temperature of approximately -30°C.

8. The method according to any one of the preceding claims further comprising the step of heating the slide to dry the sample.

9. The method according to claim 8 comprising the step of immunostaining the sample.

10. The method according to claim 8 or claim 9 comprising the step of analysing the prepared slide with a microscope.

11. The method according to claim 10 wherein the microscope is an optical microscope.

12. The method according to any one of the preceding claims wherein the biological sample comprises cells.

13. The method according to claim 12 wherein the cells are eukaryotic .

14. The method according to claim 13 wherein the cells are mammalian.

15. The method according to claim 13 or claim 14 wherein the cells are in metaphase.

16. The method according to claim 15 for preparing a metaphase spread.

17. The method according to claim 13 or claim 14, wherein the cells are in interphase

18. A microscopy slide preparation device for the preparation of a frosted microscopy slide, the device comprising:

a humidity chamber for containing the slide;

a support for holding the slide within the chamber;

cooling means for cooling the slide; and

humidifying means for humidifying the chamber.

19. A microscopy slide preparation device according to claim 18 wherein the humidifying means comprise a heater for heating aqueous liquid.

20. A microscopy slide preparation device according to claim 19 wherein the heater is contained within the chamber.

21. A microscopy slide preparation device according claim 19 or claim 20 wherein the heater is an electrical element.

22. A microscopy slide preparation device according to any one of claims 19 to 21 wherein the aqueous liquid is water.

23. A microscopy slide preparation device according to any one of claims 18 to 22 wherein the support is adapted to hold a microscopy slide of dimensions 50 - 100 x 15 - 50 x O.5 - 2.0 mm.

24. A microscopy slide preparation device according to any one of claims 18 to 23 comprising heating means for drying a

biological sample on the slide.

25. A microscopy slide preparation device according to any one of claims 18 to 24 wherein the cooling means comprises a Peltier solid state heat pump and optionally comprising heating means for drying a biological sample on the slide which comprises a Peltier solid state heat pump.

26. A microscopy slide preparation device according to any one of claims 18 to 25 wherein the cooling means are comprised within the support and optionally comprising heating means for drying a biological sample wherein the cooling and/or heating means are comprised within the support.

27. A microscopy slide preparation device according to any one of claims 18 to 26 wherein the amount of cooling or heating is adjustable .

28. A microscopy slide preparation device according to any one of claims 18 to 27 comprising a temperature sensor for measuring the temperature of the slide.

29. A microscopy slide preparation device according to any one of claims 18 to 28 comprising a humidity sensor for measuring the humidity inside the humidity chamber.

30. A microscopy slide preparation device according to any one of claims 18 to 29 comprising an automated dropper for applying a biological sample to the slide.

31. A microscopy slide preparation device according to any one of claims 18 to 30 wherein the support and/or the cooling means is in thermal contact with a heat sink.

32. A microscopy slide preparation device according to claim 31 wherein the heat sink is thermally insulated from the ambient environment of the chamber.

33. A microscopy slide preparation device according to any one of claims 18 to 32 wherein a metaphase spread is present on the microscopy slide.

34. A kit comprising the microscopy slide preparation device of any one of claims 18 to 33 and a fixative for adding to the biological sample.

35. A kit according to claim 34 wherein the fixative comprises methanol .

36. A kit according to claim 34 wherein the fixative comprises a mixture of methanol and acetic acid.

37. A kit according to any one of claims 34 to 36 wherein the fixative comprises less than 1% acetic acid by volume.

38. A kit according to claim 34 wherein the fixative comprises a mixture of methanol and acetone.

39. The method of any one of claims 1 to 17 wherein the method is performed in a microscopy slide preparation device according to any one of claims 18 to 33 or using a kit according to any one of claims 34 to 38.