WO2009040627A1

WO2009040627A1 - Counting chamber for analysis of samples

Info

Publication number: WO2009040627A1
Application number: PCT/IB2008/002470
Authority: WO
Inventors: Nicolò Manaresi; Gianni Medoro
Original assignee: Silicon Biosystems SpA
Current assignee: Silicon Biosystems SpA
Priority date: 2007-09-24
Filing date: 2008-09-23
Publication date: 2009-04-02
Anticipated expiration: 2010-03-24
Also published as: ITBO20070646A1; WO2009040627A8

Abstract

Counting chamber (1) for analysis of samples; the counting chamber (1) comprises a base wall (2), an upper wall (3), which faces the base wall (2), a spacer element (4), positioned between the base wall (2) and the upper wall (3), and an inner chamber (5), which is suitable for housing the sample and is delimited by the base wall (2) and upper wall (3) and by the spacer element (4); the base wall (2) comprises an inner reflecting surface (12) facing towards the upper wall (3).

Description

"COUNTING CHAMBER FOR ANALYSIS OF SAMPLES"

TECHNICAL FIELD

The present invention relates to a counting chamber, a method for the production thereof and a use of said counting chamber.

BACKGROUND ART

To count the cells (or corpuscles of other types such as dust or microspheres) in a sample, a glass chamber is currently used which is substantially transparent (often with references that highlight regions with a well-defined area, for example Burker's chamber) containing the sample and inverted microscope systems, in which the light comes from above while the objective is positioned below to collect the light that crosses the glass chamber.

Nevertheless, due to the fact that the majority of cellular components are transparent to visible light, it is very difficult to identify the position of the cells on the slide unless a phase contrast microscope is used. In this regard, it should be noted that, once a cell has been crossed, the light rays undergo a phase change; the phase contrast microscope converts these phase changes into differences in light density, improving the contrast between cell and background.

The known techniques described above have various drawbacks including:

. weakness of the signal

• need for use of a phase contrast microscope • need for use of an inverted microscope

. difficulty with manual counting of fluorescent cells

(or other corpuscles) .

In relation to the latter point, it is important to stress that in the case of some cells being highlighted by means of fluorescence entailing absorption with limited wavelength range, the above-mentioned traditional method does not normally permit the "manual" counting of cells - i.e. with inspection by the operator via the eyepieces - since it is not possible to display the references (for example a grid) currently used.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a counting chamber, a method for the production thereof and a use of said counting chamber which allow the drawbacks of the known art to be overcome, at least partially, and are at the same time easy and inexpensive to produce.

The present invention provides for a counting chamber, a method for the production thereof and a use of said counting chamber as claimed in the following independent claims and, preferably, in any one of the claims depending directly or indirectly on the independent claims.

Unless explicitly specified to the contrary, in this text the following terms have the meaning indicated below.

Substantially reflecting surface is a surface suitable for reflecting the majority of incident light rays. According to some embodiments, substantially reflecting surface is a surface having a reflectance greater than 0.50, in particular greater than 0.60, advantageously greater than 0.70, advantageously greater than 0.85. Preferably, substantially reflecting surface is a surface with a specular reflectance greater than 0.50, in particular greater than 0.60, advantageously greater than 0.70, advantageously greater than 0.85.

Reflectance is the ratio between the intensity of the reflected light flux and the intensity of the incident light flux. According to .some embodiments, reflectance is measured with respect to non-polarised electromagnetic radiation which covers in a substantially uniform manner the field of the visible light, ultraviolet (UV) and near infrared (NIR) (1400nm-lnm, in particular 780nm-200nm) . Advantageously, the reflectance is measured with respect to a ray of non-polarised white light which has electromagnetic radiation that covers in a substantially uniform manner the visible light field (780nm- 400nm) .

Specular reflectance is the ratio between the intensity of the non-polarised light reflected in a specular manner (i.e. with an angle of reflection substantially equal to an angle of incidence) and the intensity of the incident non-polarised light (Fresnel equation) . According to some embodiments, specular reflectance is measured with respect to a ray of nonpolarised electromagnetic radiations which cover in a substantially uniform manner the field of the visible light, the UV and the NIR (1400nm-lnm, in particular 780nm-200nm) . Advantageously, specular reflectance is measured with respect to a ray of non-polarised electromagnetic radiations which cover in a substantially uniform manner the visible light field (780nm-400nm) .

Advantageously specular reflectance is measured at an angle of incidence of 60°. Advantageously, specular reflectance is measured in accordance with the DIN EN ISO 2813 standard.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described below with reference to the accompanying drawings, which illustrate some non- limiting embodiment examples, in which: figure 1 is a schematic overhead view of a counting chamber produced in accordance with the present invention; figure 2 illustrates a section according to the line II-II of the counting chamber of figure 1 ; figures 3-5 are 4x photographs at different focuses of a portion of a counting chamber produced in accordance with the present invention during its use; figures 6-8 are 10x photographs at different focuses of a portion of the counting chamber of figures 3-5 during its use ; figures 9-11 are 4x photographs at different focuses of a portion of a Burker chamber of known type during its use; figures 12-14 are 10x photographs at different focuses of a portion of the Burker chamber of figures 9-11 during its use; figures 15 and 16 are 4x and 10x photographs respectively of a portion of a counting chamber produced in accordance with the present invention during its use with fluorescent cells; - figures 17 and 18 are 10x photographs of a portion of a Burker chamber of known type during its use with fluorescent cells; figure 19 is a perspective and schematic view of a further embodiment of a counting chamber produced in accordance with the present invention; figure 20 is a lateral view of the counting chamber of figure 19; figure 21 shows a possible 10x photograph of a further embodiment of a counting chamber produced in accordance with the present invention; figure 22 is a schematic overhead view of a further embodiment of a counting chamber produced in accordance with the present invention; figure 23 is a schematic overhead view of a further embodiment of a counting chamber produced in accordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

With reference to figures 1 and 2, 1 indicates overall a counting chamber, which comprises a base wall 2, an upper wall

3 which is substantially parallel to the base wall 2, and a spacer element 4, which is positioned between the base wall 2 and upper wall 3 to maintain the base wall 2 and upper wall 3 at a given distance equivalent to its height.

The counting chamber 1 has an inner chamber 5 which is delimited at the top and bottom by the upper wall 3 and the base wall 2 respectively and laterally by the spacer element 4. The inner chamber 5 has a substantially parallelepiped form with square base.

The upper wall 3 has a substantially parallelepiped form with square base, is transparent and, in particular, is made of glass. The upper wall 3 has two apertures 6, which are arranged each corresponding to a respective corner of the upper wall 3 and are suitable for connecting the inner chamber 5 with the external environment .

In use, when one of the apertures 6 is used by an operator to insert a sample (in a substantially liquid phase) in the inner chamber 5, the other aperture 6 acts as a vent.

The upper wall 3 has a substantially square shaped inner surface 7 facing towards the base wall 2 and the apertures 6 are transverse to the inner surface 7.

The base wall 2 has a substantially parallelepiped form with square base and comprises a supporting layer 8 and a covering layer 9, which comprises a metallic material and has a substantially reflecting face 10 which faces towards the upper wall 3.

According to some embodiments, the inner chamber 5 has a width from 6 to 3 mm, in particular approximately 5 mm, and a depth from 6 to 3 mm, in particular approximately 5 mm.

Advantageously, the layer 8 in turn has a first semi -layer (not illustrated) , which comprises (in particular, consists of) silicon, and a second semi -layer (not illustrated) which comprises (in particular consists of) SiO₂ and is positioned between the first semi-layer and the covering layer 9. The layer 9 has interruptions 11, corresponding to which the layer 8 is exposed towards the inside of the inner chamber 5. The interruptions 11 define on an inner surface 12 of the base wall 2 a grid 13, which comprises a plurality of substantially parallel lines 14 and a plurality of substantially parallel lines 15 which are transverse, in particular perpendicular, to the lines 14. It should be noted that, despite the interruptions 11, the layer 9 covers the majority of the layer 8 towards the inside of the inner chamber 5.

The spacer element 4 has a substantially annular form and is connected in a fluid-tight manner to the base wall 2 and upper wall 3. In particular, the spacer element 4 comprises two lateral walls 4', which are substantially parallel, and two lateral walls 4'', which are substantially parallel and transverse, in particular perpendicular, to the lateral walls 4'; the lateral walls 4' and 4'' have substantially identical lengths and are connected so as to form one single piece.

According to some embodiments, the spacer element 4 keeps the surfaces 7 and 12 at a distance from 10 to 600 μm, advantageously from 50 to lOOμra, advantageously from 10 to 90μm.

It should be observed that the spacer element 4 can be obtained by appropriately shaping the base wall 2 or alternatively the upper wall 3, or can consist of a layer of different material which comprises (in particular consists of) a photo-resistant polymer.

The counting chamber 1 furthermore comprises two further reference signs or crosses 16, which are arranged in different positions on a plane (i.e. on the base wall 2 and/or on the upper wall 3) parallel to the surface 7. In particular, the crosses 16 are positioned outside the grid 13 on opposite sides of said grid 13. A further simplified embodiment of the counting chamber 1 (not illustrated) comprises two reference crosses (advantageously three), without the grid 13.

The crosses 16 allow, in use, the counting chamber 1 to be used with an automatic detector (not illustrated) . An automatic detector can for example comprise a personal computer, connected to a camera for acquisition of the images of the counting chamber, in which an image recognition software program is installed.

The automatic detector identifies (in use) the position of the inner chamber 5 by means of the crosses 16 and this permits automatic counting of the cells in the presence or absence of the grid 13.

According to a further embodiment, the surface 12 is treated so that the grid 13 and/or the interruptions 11 can be detected at a wavelength different from the wavelength of the radiations striking it.

Advantageously, the surface 12 is treated so that the grid 13 and/or the interruptions 11 are suitable for receiving radiations at a first wavelength, in particular in the UV outside the visible light field or also in the visible light field, and emitting at a second wavelength, the second wavelength being longer than the first (grid 13 and/or interruptions 11 substantially fluorescent) .

Figures 19 and 20 illustrate a further embodiment of a counting chamber 1. The counting chamber 1 of the figures 19 and 20 has a base wall 2 substantially identical to the base wall 2 described above, differing in that it is part of a substrate 17. The substrate 17 has four transverse depressions 18 and two transverse supporting elements 4 positioned on opposite sides of the base wall 2. It should be observed that, in this embodiment, the two supporting elements 4 are obtained in the appropriately shaped substrate 17. Contrary to what is described for the embodiment of figures 2 and 3, in this case the inner chamber 5 is open laterally and the upper wall 3 is not provided with apertures 6. In use, the sample spreads in the inner chamber 5 by capillarity, once it has been placed inside laterally in position P by means of a pipette.

Alternatively, in a further embodiment not illustrated, the two supporting elements 4 can be obtained in the upper wall 3 appropriately shaped.

The counting chamber 1 of figures 19 and 20 has a configuration analogous to that of a standard haemocytometer (Burker chamber) and differs from it mainly due to the structure of the base wall 2 (in particular the structure of the inner surface 12 at least partially substantially reflecting) .

Figure 22 schematically illustrates, with details removed for the sake of clarity, a further embodiment of a counting chamber 1. The counting chamber 1 of figure 22 is substantially identical to the counting chamber 1, from which it differs only in that the upper wall 3 comprises one single aperture 6 and the spacer element 4 has a capillary hole 19, which connects the inner chamber 5 to the external environment. In use, the sample is placed inside by means of a pipette via the aperture 6 and the hole 19 acts as a vent. Due to the small diameter of the hole 19, the sample cannot leak out of the inner chamber 5.

The main advantage of this embodiment is that evaporation of - S -

the sample is minimised, since the vent hole has a minimal surface in contact with the air. The device is generally more difficult to clean since, even when strong pressures are applied (up to the point where any further pressure would damage the chip) , it is difficult to create a high flow of cleaning liquid due to the hydraulic resistance of the outlet capillary. It is suitable for one single use and re-use should therefore be avoided.

Figure 23 schematically illustrates, with details removed for the sake of clarity, a further embodiment of a counting chamber 1. The counting chamber 1 of figure 23 is substantially identical to the counting chamber 1, from which it differs only due to the fact that the upper wall 3 does not comprise any aperture and the spacer element 4 has two capillary holes 19, which connect the inner chamber 5 with the external environment; each hole 19 is positioned on a respective wall 4' ' . In use, the counting chamber 1 is gripped by the user via a pair of tweezers and placed in contact with a solution of the sample to be examined in the area of one of the holes 19. The solution enters the counting chamber 1 by capillarity through the hole 19 immersed in the solution, while the other hole 19 acts as a vent. The solution substantially has no possibility of leaking out of the inner chamber 5.

The advantage of this embodiment consists in the fact that no preliminary machining of the upper wall 3 is required to obtain the apertures 6, and the holes 19 are obtained directly during production of the chamber 1.

According to embodiments not shown, the inner surface 12 is not smooth but has a plurality of depressions and/or reliefs. In particular, the inner surface 12 has a plurality of first depressions and/or reliefs which are substantially parallel and a plurality of second depressions and/or reliefs which are substantially parallel and transverse, in particular perpendicular, to the first depressions and/or reliefs. Advantageously, the depressions and/or reliefs define the grid 13. The covering layer 9 is substantially continuous (i.e. does not have the interruptions 11) and has a substantially uniform thickness. The grid 13 and the various inner areas of the chamber 5 can also be discerned in this case due to the fact that the face 10 is undulating with portions at different levels .

The counting chamber 1 as defined above can be produced by means of a method which comprises an affixing phase, during which a spacer layer is positioned on the base wall 2 and an inner part of the spacer layer is removed in order to obtain the spacer element 4; a covering phase, during which the upper wall 3 is placed over the spacer element 4, so that the spacer element 4 is positioned in contact between the base wall 2 and upper wall 3 and so that the upper wall 3 and base wall 2 delimit at top and bottom the inner chamber 5. The method furthermore comprises a phase of preparation of the base wall 2, which is previous to the affixing phase and during which the covering layer 9 is deposited on the supporting layer 8. The affixing and covering phases are performed so that the covering layer 9 has its face 10 towards the upper wall 3.

During the phase of preparation of the base wall, the interruptions 11 are created, by removal, so that the supporting layer 8 is exposed towards the inside of the inner chamber 5 in the area of the interruptions 11.

Advantageously, after the covering phase, the spacer element 4 is heated so that the spacer element 4 connects in a fluid- tight manner to the base wall 2 and the upper wall 3.

The counting chamber 1 as defined above can be used for counting corpuscles, in particular cells, of a sample with the help of an upright microscope (known per se and not illustrated) .

In this regard, it is important to stress that the particular structure of the counting chamber 1 (in particular the presence of one or more substantially reflecting areas on the inner surface 12) allows the microscope to receive a sufficiently strong signal such as not to require a phase contrast .

The use of a counting chamber 1 as defined above is particularly advantageous if the corpuscles have a fluorescent pigment which absorbs at a given wavelength (in particular in the blue light) and emits at a longer wavelength (in particular in the green light) .

In this case the presence of indicators (in particular the grid 13) comprising one or more fluorescent substances is particularly useful when manual counting is desired. Advantageously, the fluorescent substances absorb (and preferably emit) at wavelengths substantially coinciding with those of the fluorescent pigment .

Further characteristics of the present invention will be evident from the following description of a merely illustrative non-limiting example.

Example 1

This example describes the production of a plurality of counting chambers starting from one single support (slice or wafer) of silicon of a substantially circular shape with diameter of approximately 15.24 cm and a thickness of approximately 600 μm.

A layer of SiO₂ was deposited for growth in a hydrogen and oxygen environment at a temperature of 1100⁰C on the silicon support .

A layer of aluminium (copper 4%) with thickness of approximately 0.53 μm was deposited by sputtering on the layer of SiO₂. The layer of aluminium was deposited on one side of the slice of silicon.

At this point, the layer of aluminium was incised by means of photolithography. In order to do this, the support covered by the layer of aluminium was covered by a layer of photosensitive material (fotoresist) by means of spin coating.

After deposition of the fotoresist, the support was heated to evaporate the residual solvent and relieve stress caused by the previous spinning in order to improve uniformity of the fotoresist on the support.

The fotoresist surface was then exposed to a beam of ultraviolet light via a mask-aligner able to project the design of a first photomask on the surface of the fotoresist.

The parts of the fotoresist that were radiated underwent a process of photopolymerisation while those protected by the photomask were subsequently removed via a development bath in which the non-radiated fotoresist is soluble.

At this point, the substrate was immersed in a solution of acid etching (HCl) to remove the non- protected parts of the layer of aluminium.

Due to the shape of the mask-aligner a plurality of areas are obtained (over 300) substantially covered in aluminium; the areas substantially covered in aluminium had dimensions of 6mm x 6mm and were distributed substantially over the whole area of the silicon support. For each area, incisions (interruptions) were obtained on the layer of aluminium; said incisions defined a respective grid comprising a plurality of first parallel lines and a plurality of second parallel lines perpendicular to the first lines. Each line had a width of approximately 5μm. Each grid delimited portions of a substantially square shape (approximately 195μm x 195μm) without interruptions of the layer of aluminium (said area is indicated in figures 1 and 2 by a Z) .

After photolithographing the layer of aluminium, the fotoresist was removed. Subsequently, the substrate partially covered with aluminium was covered by a further photoresistant polymer (Ordyl 550 by Elga Europe - this polymer consists of a layer of resist protected on one side by a layer of polyester

(PET) and on the other by a layer of polyethylene (PE) - the polymer is applied by removing the layer of PE) with thickness of approximately 60μm by means of lamination, a second mask is applied, the exposed material is radiated (UV light in the range 250nm-345nm) , developed and rinsed to remove the excess material by means of a rinsing solution (the procedure followed is analogous to the one described in P. VuIto et al , Microfluidic channel fabrication in dry film resist for production and prototyping of hybrid chips, Lab Chip, 2005, 5, 158-162, the contents of which are referred to in their entirety for the sake of completeness of description) . The mask was chosen so as to obtain a spacer lattice which extended around each grid. In practice, the spacer lattice consisted of a plurality of spacer elements, each of which were substantially ring-shaped and laterally delimited a respective inner chamber; inside each chamber a respective grid was positioned.

A Pyrex glass having a substantially circular shape (diameter approximately 15.24 cm thickness 500μm) was bored with abrasive jets (a process known as powder-blasting) to produce a plurality of apertures. The Pyrex glass thus treated was laid over the spacer element.

The apertures were made in order to provide two apertures in the area of each inner chamber; in particular the apertures were positioned near the respective spacer elements.

At this point, the silicon-polymer-glass sandwich was treated at a temperature and pressure such that the polymer of the spacer element acted as an adhesive and the spacer element was connected in a fluid-tight manner to the support and to the Pyrex glass (wafer-bonding,) .

Having completed this phase, the slice of silicon was cut along the spacer lattice so as to obtain a plurality of counting chambers each of which included a respective inner chamber.

The counting chamber can be easily produced in series at a relatively low cost. For these reasons the counting chamber thus obtained can be advantageously used as disposable, facilitating and considerably accelerating the work of the operators using the counting chamber.

Although it is more advantageous to produce a multiplicity of chambers starting from one single slice, it is nevertheless possible to produce a single counting chamber using a support previously cut to the final required dimensions and a Pyrex glass of corresponding dimensions. In these cases, the support and the Pyrex glass are normally cut previously.

Example 2

This example compares the uses of a counting chamber produced according to example 1 and a known Burker chamber of traditional type.

The photographs were taken with an epifluorescence upright microscope with broad spectrum white light source, a first filter block (FITC block) with an excitation filter 460-495 nm

(blue) , emission filter 515-550 nm . (green) and dichroic mirror at 505 nm, and a second filter block for bright field illumination.

Cells K- 562 were loaded with calcein and washed twice with a buffer solution.

Figures 3-5 and 6-8 show photographs (4X and 10X) of the counting chamber of example 1, using the filter block for bright field. Three different focuses were used (Fl, F2 , F3) for each enlargement. In figures 3 and 6 (Fl) the cells tend to be luminous, while in figures 4, 7, (F2) 5 and 8 (F3) the cells become progressively darker. This phenomenon is important for having different colour gradients during identification of the cells. In the counting chamber the grid is well-defined and visible and this permits easy counting of the cells by the operator.

Figures 9-11 and 12-14 show photographs (4X and 10X) of the

Burker chamber taken using the above-mentioned upright microscope, again in bright field. In all the figures 9, 12

(Fl), 10, 13 (F2) , 11 and 14 (F3) the cells are less defined than those detected in the counting chamber and consequently identification of the cells is more difficult. Furthermore, the grid has almost disappeared; counting of the number of cells per area is, therefore, impossible or at least very difficult and subject to errors.

Since the Burker chamber is made of transparent glass, in order to increase the contrast, a dark background was positioned below the Burker chamber. Even using this technique, however, the photographs were poorly defined as in the figures 9-14. To conclude, using an upright microscope, the Burker chamber does not permit accurate counting of the cells. On the other hand, the counting chamber provides a well-defined background on which the cells and the different areas can be easily identified.

It should be noted that the ease of identification of the cells due to use of the counting chamber according to the present invention can be advantageously exploited when either manual or automatic counting is required.

Example 3

This example compares the uses of a counting chamber produced according to example 1 and a known Burker chamber of traditional type for counting fluorescent cells.

The photographs were taken with the upright microscope described above, using the filter block for the fluorescence (FITC block with excitation 460-495 nm (blue) , emission filter 515-550 nm (green) ) .

Figures 15 and 16 (4x and 10x respectively) are photographs taken by manually controlling the exposure to avoid saturation of the sample. The gain of the digital camera was set to 1.0 (minimum value) while the exposure time was 1/30 of a second. The low gain is important for preventing electronic noise in the photographs; there was no need to increase the sensitivity of the camera since the sample was clearly visible already at 1/30 of a second. The good quality of the photographs of figures 15 and 16 permits both manual and automated analysis of the cells.

Figure 17 (1Ox) is a photograph of a Burker chamber in the same conditions as above, but it is completely dark and therefore unusable. Figure 18 (1Ox) is a photograph of a Burker chamber in almost the same conditions as above. In this case the photograph was taken with a gain of 11.3 (more than ten times the one used with the counting chamber) and an exposure time of 0.5 seconds (15 times superior to the value used for the counting chamber) . Due to the electronic noise amplified by the high gain in acquisition, the photograph has a poor contrast and the sample is not well defined; the quality is too poor to identify the cells (in either manual or automated mode) . Furthermore the time required to acquire one or more images by scanning the surface of the subminiature camera is necessarily longer due to the longer exposure times.

To conclude, the Burker chamber has a very low possibility of being used for identification (and if necessary counting) of fluorescent cells, whereas said identification is possible with the counting chamber according to the present invention.

Example 4 In this example it is shown that a counting chamber provided with a fluorescent grid can permit manual counting also at wavelengths determined in fluorescence.

Figure 21 shows how a sample would appear positioned inside a counting chamber produced as in example 1 in which the grid is treated in order to be fluorescent.

The fluorescence of the grid can be obtained in different ways, for example due to the presence of SiO₂ and/or rare earth ions (for example Er, Eu, Gd, Tb, Ce, Tm and Nd) .

Claims

1.- Counting chamber for analysis of samples,- the counting chamber (1) comprises a base wall (2) , which has a first inner surface (12); an upper wall (3), which is substantially transparent and has a second inner surface (7) facing the first inner surface (12); at least one spacer element (4), which connects the base wall (2) and upper wall (3) so as to maintain the first and second inner surfaces (12, 7) spaced from each other; an inner chamber (5) , which is suitable for housing the sample and is delimited at the top and bottom by the base wall (2) and the upper wall (3); the counting chamber

(1) comprises indicators (11; 13; 16) which are arranged in a given position in the counting chamber (5) and can be detected; the counting chamber (5) is characterised in that the first inner surface (12) has at least one substantially reflecting area.

2.- Counting chamber as claimed in claim 1, wherein the distance between the first and the second inner surface (12, 7) is between 10 and 600 μm.

3.- Counting chamber as claimed in claim 1 or 2 , wherein the first inner surface (12) is for the most part substantially reflecting.

4. - Counting chamber as claimed in one of the preceding claims, wherein the substantially reflecting area has a reflectance greater than 0.50.

5.- Counting chamber as claimed in one of the preceding claims, wherein the substantially reflecting area has a specular reflectance greater than 0.50.

6.- Counting chamber as claimed in one of the preceding claims, wherein the indicators (11; 13; 16) comprise at least one first optically detectable reference sign (11; 13).

7.- Counting chamber as claimed in claim 6, wherein the first reference sign (11; 13) is positioned in the area of the base wall (2) .

8.- Counting chamber as claimed in claim 7, wherein the first reference sign (11; 13) is positioned on the first inner surface (12) and can be optically discerned from the substantially reflecting area.

9.- Counting chamber as claimed in claim 8, wherein the first inner surface (12) has a plurality of substantially reflecting areas; the first reference sign (11; 13) separates the substantially reflecting areas from one another.

10.- Counting chamber as claimed in claim 9, wherein the first reference sign (11; 13) comprises a plurality of substantially parallel first lines (14) and a plurality of substantially parallel second lines (15) transverse to the first lines (14) .

11.- Counting chamber as claimed in one of the preceding claims, wherein the base wall (2) comprises a supporting layer (8) and a covering layer (9), which is positioned towards the inside of the inner chamber (5) ; the material of the supporting layer (8) and of the covering layer (9) are different from each other; the covering layer (9) has a face

(10) that faces towards the second surface which defines the substantially reflecting area.

12.- Counting chamber as claimed in claim 11, wherein the covering layer (9) comprises a metallic material.

13.- Counting chamber as claimed in claim 11 or 12, wherein the supporting layer (8) comprises silicon.

14.- Counting chamber as claimed in one of the claims from 11 to 13, wherein the covering layer (9) has interruptions (11) on the first inner surface ; the indicators (11; 13; 16) comprise the interruptions (11) ; the supporting layer (8) is exposed towards the inside of the inner chamber (5) in the area of the interruptions (11) .

15.- Counting chamber as claimed in claim 14, wherein the interruptions (11) comprise a plurality of substantially parallel first lines (14) and a plurality of substantially parallel second lines (15) transverse to the first lines (14).

16.- Counting chamber as claimed in one of the preceding claims, wherein the indicators (11; 13; 16) comprise at least one second and one third reference sign (16) , which can be optically detected, and are arranged in different positions with reference to a plane substantially parallel to the base wall (2); the second and third reference sign (16) are suitable for indicating the position of the inner chamber (5) with respect to a detector.

17.- Counting chamber as claimed in one of the preceding claims, wherein the inner chamber (5) is laterally delimited by the spacer element (4), which is connected in a fluid-tight manner with the upper wall (3) and the base wall (2) ; the counting chamber has at least one aperture (6) via which, in use, the sample is placed inside; the counting chamber (1) has at least one further vent aperture .

18.- Counting chamber as claimed in claim 17, wherein the aperture (6) is positioned on the base wall (2) or on the upper wall (3 ) .

19.- Counting chamber as claimed in one of the preceding claims, wherein the indicators (11; 13; 16) comprise a first substantially fluorescent reference sign (11; 13) .

20.- Counting chamber as claimed in claim 19, wherein the first reference sign (11; 13) is suitable for absorbing at a first wavelength and emitting at a second wavelength longer than the first wavelength.

21.- Counting chamber as claimed in claim 19, wherein the first reference sign (11; 13) comprises SiO₂.

22.- Counting chamber for analysis of samples; the counting chamber comprises a base wall (2), which has a first inner surface (12); an upper wall (3), which is substantially transparent and has a second inner surface (7) facing the first inner surface (12) ; at least one spacer element (4) , which connects the base wall (2) and upper wall (3) so as to keep the first and the second inner surface (12, 7) spaced from each other; an inner chamber (5) , which is suitable for housing the sample and is delimited at the bottom and top by the base wall (2) and upper wall (3) ; the counting chamber (1) comprises indicators (11; 13; 16), which are arranged in a given position of the counting chamber (1) and can be detected; the counting chamber (1) is characterised in that the indicators (11; 13; 16) comprise at least one first substantially fluorescent reference sign (11; 13) .

23.- Counting chamber as claimed in claim 22, wherein the first reference sign (11; 13) is suitable for absorbing at a first wavelength and emitting at a second wavelength longer than the first wavelength.

24.- Counting chamber as claimed in claim 22, wherein the first reference sign (11; 13) comprises SiO₂.

25.- Counting chamber as claimed in one of the claims from 22 to 24, and in accordance with one of the claims from 1 to 18.

26.- Method for the production of counting chambers as claimed in one of the claims from 1 to 25, and comprising a covering phase, during which an upper wall (3) is overlaid on a spacer element (4) , so that the spacer element (4) is positioned in contact between the upper wall (3) and a base wall (2) so that the upper wall (3) and base wall (2) delimit at the top and bottom an inner chamber (5) of the counting chamber (1) .

27.- Method as claimed in claim 26, and comprising an affixing phase, during which a spacer layer is positioned on the base wall (2) and an inner part of the spacer layer is removed so as to obtain the spacer element (4) .

28.- Method as claimed in claim 27, comprising a phase of preparation of the base wall (2) , which precedes the affixing phase and during which a covering layer (9) is deposited on a supporting layer (8) ; the material of the supporting layer (8) and of the covering layer (9) are different from each other; the affixing and covering phases are performed so that the covering layer (9) has a face (10) facing towards the substantially reflecting upper wall (3) .

29.- Method as claimed in claim 28 wherein, during the phase of preparation of the base wall (2), interruptions (11) of the covering layer (9) are produced by removal so that the supporting layer (8) is exposed towards the inside of the inner chamber (5) in the area of the interruptions (11) .

30.- Method as claimed in one of the claims from 26 to 29, and comprising a heating phase which is subsequent to the covering phase and during which the spacer element (4) is heated so that said spacer element (4) connects in a fluid-tight manner to the base wall (2) and upper wall (3) .

31.- Method as claimed in one of the claims from 26 to 30, wherein during the covering phase several inner chambers (5), one single upper element defining a plurality of upper walls (3) and one single base element defining a plurality of base walls (2) are simultaneously delimited; the method furthermore comprising a cutting phase, which is subsequent to the covering phase and during which the upper element and base element are cut so as to separate the counting chambers (1) from one another.

32.- Use of a counting chamber (1) defined as claimed in one of the claims from 1 to 25 for counting corpuscles in a sample; said use entails counting of the corpuscles with the help of an upright microscope.

33.- Use as claimed in claim 32, wherein the corpuscles are cells.

34.- Use as claimed in claim 32 or 33, wherein the corpuscles have a fluorescent pigment which absorbs at a first wavelength and emits at a second wavelength longer than the first wavelength.