WO2009052836A1

WO2009052836A1 - A method and apparatus for illuminating a sample

Info

Publication number: WO2009052836A1
Application number: PCT/DK2008/050263
Authority: WO
Inventors: Frans Ravn
Original assignee: Chemometec A/S
Priority date: 2007-10-26
Filing date: 2008-10-27
Publication date: 2009-04-30

Abstract

The present invention relates to an apparatus to illuminate a sample. The apparatus comprises of a sample plane having an illumination region onto which the sample is arranged; an excitation unit having a light source to generate an excitation light; and a lens unit, having a micro lens array where the micro lens array comprises a plurality of lenses arranged in a two dimension arrangement, to receive the excitation light and generate an illumination light that is directed towards the illumination region. The lens unit produces a homogeneous illumination light that is projected on the illumination region of the sample plane with high illumination efficiency.

Description

A METHOD AND APPARATUS FOR ILLUMINATING A SAMPLE

Field of the invention

The present invention relates to illuminating a sample. More particularly, the present invention relates to a method and apparatus for homogenously illuminating a sample with high illumination efficiency.

Description of the related art

Photoluminescent analysis of a sample, such as biological material, is based on illuminating a sample with light (excitation) of a given wavelength and collecting light emitted (emission) from the sample or from parts or components of the sample, at another substantially different wavelength. The difference in wavelength between the excitation light and emission light (generally called Stoke's shift) is a property of the sample being analysed, more generally a property of the photoluminescent molecules present in the sample. If the Stoke's shift is great enough to allow substantial optical separation of the excitation and emission light, then it is feasible to use the method of photoluminescence for the analysis of the sample.

The intensity of photoluminescent emission (e.g. phosphoresce or fluorescence) is typically lower than that of the excitation light, usually by an order of several magnitudes. The fact that the emission is detected against "darkness" makes the method well suited, since many of the commercially available detectors show low response to "darkness" but respond considerably well to light, e.g. photons, striking the detector. Nevertheless, improved sensitivity, e.g. expressed as increased emission, is typically a favourable property and therefore, there exist currently numerous methods for improving sensitivity.

Increase in the intensity of the excitation light generally increases the amount of emitted light because the probability of generating the emission light is proportional to the number of photons interacting with photoluminescent molecules of the sample. One of the often used methods for increasing intensity of the excitation light is using lasers for excitation. The lasers are usually available in different configurations and produce emission of high intensity, e.g. expressed as energy flux. Furthermore, lasers enable focussing the excitation light easily onto a small area of the sample, thus generating a high light density, e.g. expressed as emitted energy per area. Another method for illuminating a photolumincscent sample is to use a dispersive light source, such as a lamp or a light emitting diode, as an excitation light source. The dispersive light soorce emits light in virtually all directions. In order to use such a light source in an application, such as fluorescence microscope, it is necessary to focus the excitation light onto the sample. Such focusing produces an image of the light source, which may be of dimensions comparable to the field of view of the microscope. The advantage of such light sources is that they emit a dispersed light, thus illuminating a considerably larger area of the sample. Although, such light sources have high degree of efficiency, defined by the fraction of light striking the sample material, but these light sources suffer from producing illumination of relatively poor degree of homogeneity over the surface of the sample.

Figure 1 illustrates a typical prior art apparatus that is used to illuminate a sample and the result of such illumination.

Figure 1A illustrates an excitation unit having an imaginary light source to generate an excitation light. The light source shows a substantial structure in its light emitting elements (101), comprising 4 square regions emitting light evenly. The light source is separated and surrounded by passive elements (102), which emit no detectable light In present scenario, the light source measures 1 x 1 mm, and the angular emission is from 440 to -40 deg. Figure 1B shows a prior art apparatus, simulating the use of the light source

(103, identical to item 101) for illuminating the sample material. The prior art apparatus consists of a collimating unit (104). One such unit may be a biconvex aspheric lens with a focal length f = 8.5 mm and a diameter D ≈ 12 mm (e.g. Melles Griot LAG000). Further, the prior art apparatus consists of a wavelength separation unit (105) typically used in fluorescence analysis for the elimination of light at wavelengths where emission is expected. The apparatus also consists of a focussing unit (106). One such unit may be plano-convex lens with a focal length f - 12 mm and a diameter D - 12 mm (e.g. Edmund Optics part no 45084) and finally a sample plane (107), which is to be illuminated. The apparatus shows a number of randomly selected rays and illustrates the path of these rays through the prior art apparatus.

Figure 1C shows a simulated image of the light source, as it would appear on the sample plane. Figure 1C shows a strong similarity with the light source illustrated in Figure IA. Emittance structure of the light source is easily identified in the image. It is typical to use a difraser element, such as difruser, to suppress or eliminate the identifiable emittance structure in the image. However, this affects the size of the image and causes blurring, resulting in broadening of the illumination and thus loss of effective light and/or decreased homogeneity of the illumination light.

Summary of the invention

One embodiment of the invention relates to an apparatus to illuminate a sample. The apparatus comprises of a sample plane having an illumination region onto which the sample is arranged; an excitation unit having a light source to generate an excitation light; and a lens unit, having a micro lens array where the micro lens array comprises a plurality of lenses arranged in a two dimension arrangement, to receive the excitation light artd generate an illumination light that is directed towards the illumination region. The lens anil produces a homogeneous illumination light that is projected on the illumination region of the sample plane with high illumination efficiency. Another embodiment of the invention relates to a method for illuminating a sample. The method includes arranging a sample on a sample plane having an illumination region; generating an excitation light using an excitation unit having a light source; and generating an illumination light, directed towards the illumination region, using a lens unit that comprises of a micro lens array having a plurality of lenses arranged in a two dimension arrangement The ϊens unit produces a homogeneous illumination light that is projected on the illumination region of the sample plane with high illumination efficiency.

Brief description of the drawings The embodiments of the invention, together with its advantages, may be best understood from the following detailed description taken in conjunction with the accompanying figures in which:

Figure 1 illustrates a typical prior art apparatus that is used to illuminate a sample and result of such illumination- Figure 2 illustrates an apparatus that is used to illuminate a sample using a lens unit according to one of the embodiments of the invention.

Figure. 3 illustrates the comparison of results of the prior art apparatus and the apparatus of present invention for the illumination of sample material Figure, 4 illustrates an implementation according to one embodiment of the invention.

Figure. 5 illustrates size of illumination region according to one embodiment of the invention. Figure. 6 illustrates a micro lens array element according to one embodiment of the invention.

Figure 7 illustrates in A) an arrangement of the apparatus where the units are arranged on, or substantially on the optical axis of a photoluminescent imaging system, and in B) an arrangement where the units are arranged substantially off the optical axis of a photohiminescent imaging system according to embodiments of the present invention.

Detailed description of the Invention

The object and advantages of the present invention will become apparent to those skilled in the art upon reference to the detailed description taken in conjunction with ihe provided figures.

Figure 2 illustrates an embodiment of an apparatus to illuminate a sample. The apparatus comprises of a sample plane (205) having an illumination region onto which the sample is arranged; as excitation unit (201) having a light source to generate an excitation light; and a tens unit (206). The lens unit has a micro lens array, which comprises of a plurality of lenses arranged in a two dimension arrangement The leas unit receives the excitation light and generates an illumination light that is directed towards the illumination region. The lens unit produces a homogeneous illumination light that is projected on the illumination region of the sample plane with high illumination efficiency.

In one embodiment of the invention, fee illumination region, i.e, area of the sample material that is illuminated is at least 0,5mm², such as 1.0mm² or greater, preferably 2.0mm² or greater, more preferably 4.0mm² or greater, more preferably 8.0mm² or greater, more preferably 16.0mm² or greater. In another embodiment of the invention, the size of the illuminatioii region is at least 2 times larger than the size of emittance area of the light source, preferably 4 times larger, more preferably 10 times larger, more preferably 20 times larger.

One often preferred aspect of embodiments of the present invention is that regions of the sample material, not under investigation, are substantially not exposed to the illumination light. In other words, only the illumination region is exposed to the illumination light. This is often a desirable property, preferably where the illumination results in alteration of chemical and/or physical property of the sample material, leading to fading of the fluorescent signal. In one embodiment of the invention, the sample being analyzed contains a particle. The particle or material contained on or within the particle is photoluminescent active, generating fluorescence when illuminated with the illumination light.

In another embodiment of the invention, the particle is labelled with a photoluminescent active material, preferably with a fluorescent material, to produce a photoluminescence when illuminated with the illumination light.

The sample for illumination is selected from both cultured cell and primary cell, such as a mammalian cell, yeast cell, fungus and bacterium.

In order to obtain an illumination with high efficiency and low variation for the illumination of photoluminescence sample material, the structure of light emitting part (emittance element) of the light source is of essential importance. The light source is usually selected from a dispersive light source, an emitting diode, a laser diode and a laser.

In several highly preferred embodiments of the present invention, power light emitting diodes (LED) light source with homogeneous emittance element, as can be found in several commercially available semiconductor light sources (e.g. LZ1-

00G105 from LedEgin, XLamp 7090 LEDs from Cree, Luxeon K2 from Lumiled,

NS6B083T from Nichia or the Golden Dragon (LD W51M) from Osram) is used.

Typically, the emittance element of the light source is of substantial physical size, such as for instance greater than 0.5mm², or even greater than 1.0mm².

When light sources having light emittance elements of distinct structures are used, such as lamp or a light emitting diode (LED), or in general light sources that form an image or structure when focused, then it is possible to illuminate considerably large sample material, while maintaining both highly homogeneous illumination, e.g. defined by variation in illumination energy across the sample under investigation, and good efficiency of the emitted light, e.g. defined by the ratio of the illumination light to emitted light The present invention offers substantial improvement of several applications of photoluminecence, such as fluorescent microscopy. By generating a high homogeneous illumination of the sample, it is possible to perform high quality photolumineccnce analysis in a faster and simple manner, thus allowing the use of more simple and robust instrumental design.

In several preferred embodiments, the image of the light source produced by a lens substantially resembles the region of the sample material which is under investigation, preferably by closely resembling the height/width ratio of the detected photolυminescent region or image.

Several preferred embodiments of the invention use a single light source, e.g. emitting diode, laser diode or laser as a light source, while other equally preferred embodiments use a plurality of light sources, preferably is a single assembly. In many of these embodiments, it is preferred that the two or more light sources arc identical in optical properties. However, in other equally preferred embodiments, at least two light sources are different in their optical properties. Generally, two or more identical light sources are able to generate a greater flux of light, while two or more different light sources add flexibility with respect to properties such as wavelength of the emitted light.

In an embodiment of the invention, the light source is arranged in a light source parallel to the illumination region with an accuracy variation in position with respect to the light source by lmm or lower.

In other preferred embodiments, the excitation unit comprises of a plurality of light sources, preferably in a single assembly.

In preferred embodiments of the invention, the light source(s) is arranged relative to at least one other unit and/ or the sample plane. The arrangement comprises of relative movement of the light source to at least one other unit and/ or the sample plane by lmm or less. Such arrangement affects homogeneity of the illumination light over the illumination region, preferably where an ideal arrangement results in the minimum illumination variation.

In an embodiment of the invention, the lens unit focuses the excitation light from the light source, such as a dispersive light source, onto the sample plane. The lens unit comprises of a plurality of lenses. It is often preferred that such lenses are a number of substantial identical lenses, preferably lenses arranged in a lens array, such as micro lens arrays.

Several preferred embodiments of the present invention use two substantially identical micro lens arrays, arranged in a pair with opposite orientation. However, other equally preferred embodiments use a single element, comprising micro lens arrays on two opposite surfaces. The single element of the micro lens arrays, including an upper array and a lower array, is preferably produced as one piece by moulding together both upper array and lower array using a moulding method, i.e. by moulding a substantially optically transparent material, such as glass or polymer. Example 5 later in this section explains the single element one piece lens unit comprising both upper array and lower array in detail.

A preferred property of light transmitted through the micro lens array, is high degree of parallel excitation light, and therefore in several preferred embodiments of the present invention, the micro lens array includes one or more lens, which increases the degree of parallelism of the light leaving the light source and entering the micro lens array.

In one embodiment of the invention, the excitation light focused onto the micro lens array has a divergence angle of 0.1mrad or less, preferably 0.05mrad or less, more preferably 0.02mrad or less. In other embodiment of the invention, each of the lenses in the plurality of lenses of the micro lens array produces an image similar in dimensions to the region of the sample being analysed, preferably where the image produced is substantially rectangular. The plurality of lenses comprises of at least 4 lenses, preferably more than 4 lenses, more preferably more than 50 lenses elements, and even more preferably more than 100 lenses, such as 150 lenses or 200 lenses. hi other embodiment of the invention, the lenses are semi-spherical lenses arranged in an array, preferably where two said arrays are arranged in a series, thus forming an array of lenses. Each array of the two said arrays includes the plurality of lenses. In particular, each array includes at least 4 lenses, preferably more than 4 lenses, more preferably more than 50 lenses elements, and even more preferably more than 100 lenses, such as 150 lenses or 200 lenses, as described above.

The dimension of the lens is in the range from 0.5 to 3mm, preferably in the range from 1 to 2mm and the size of the micro lens array is less than 20mm, the size being the diameter of the lens array in the direction perpendicular to an excitation- sample axis, where excitation-sample axis is defined by optical path of the excitation light, when the excitation light is directed directly from the light source to the sample plane. In other embodiment of the invention, the micro lens array is housed in a casing comprising means for arranging and fixing the micro lens array. The casing may be made of cast polymer,

In other embodiment of the invention, the pattern of the lenses is preferably similar to the shape of beam of the excitation light

In several preferred embodiments of the present invention, using micro leas array elements, the micro lens arrays on both sides is substantially identical in form, and preferably also in position, while in other equally preferred embodiments the form and/or position of the elements of the micro lens arrays are substantially different, preferable where such difference enhances illumination efficiency and/or reduces illumination variation and/or integrates an optical feature of a separate optical dement, such as a lens, into the optical effect of the micro lens array element.

When issing a number of identical lenses it is often preferred that such lenses are arranged in a single element, e.g. a micro lens array.

In addition to using a micro lens array several preferred embodiments of the present invention further includes a collimating unit having one or more lenses (an array of lenses) to receive the excitation light and generate a collimated excitation light, preferably where the purpose of such one or more lens is to coUimate light from the light source and/or to increase the spatial angle through which excitation light front the excitation unit is collected.

One often preferred property of using a number of identical lenses or lens elements, such as micro lens array, is that it is possible to eliminate the imaging of any structure of the light source, such as a filament of a lamp. If such structure is imaged on the sample, the intensity of illuminated light varies in accordance with the imaged structure of the light source. Such structure can not be eliminated by conventional imaging optics and typically would require methods such as defocusing or diffusing of light, where the result of such methods would generally result in reduction of illumination efficiency. Several preferred embodiments of the present invention include micro lens arrays which are comprised in a single element, preferably an element produced by casting optically transparent material, preferably a polymer material in a mould.

When producing a micro lens array dement by casting polymer materia!, it is often preferred to use considerably small thickness. Such small thickness is typically obtained by using small lenses, with lens of 3mm or less in diameter, such as 2mm or less in diameter, even lmm or less, such as 0.5mm or less. Typically, such micro tens array element are less than 10mm in thickness, such as 8mm or less, such as 5mm or less, even 3mm or less. Depending on the method used to produce the mould used lor casting, it is often of interest to use lenses of certain diameter, such as lenses of 0.5mm diameter or more, such as lmm or more, such as 2mm or more, such as 3mm or more. Thus, it is often preferred that the diameter of the lenses is in the range from 0.5 to 3mm, preferably in the range 1 to 2mm.

In several preferred embodiments of the present invention, the size of the micro lens array is less than 20mm, such as 15mm or less, such as 10mm, in certain embodiments even smaller, such as 8mm or less, such as 5mm or less, the size being the diameter of the lens array in the direction perpendicular to the major axis of illumination.

In other embodiment of the invention, the lens unit comprises of a first micro lens array in opposite orientation with a second micro lens array. The selection of the first micro lens array and the second micro lens array is based on enhancing illumination efficiency and/or reducing illumination variation and/or integrating an optical feature of a separate optical element into the optical effect of the lens array.

Several preferred embodiments of the present invention include methods for production, which may place the light source relative to at least one of the optical component, in such a manner that satisfactory/ predetermined illumination efficiency and/or variation in illumination intensity is obtained.

This is preferably obtained by including means which allow the light source to be placed in a plane parallel to the illumination region, with accuracy in position of better than 1 mm, such as better than 0.5mm, or even better man 0.2mm, such as with accuracy of 0.1mm or better. Also several embodiments include methods for production, which can place the light source and at least one optical element (unit and/ or sample plane) in position relative to each other, in a direction parallel to the main direction of illuminated light. This is preferably obtained by including means that allow the light source and/or at least one optical element to be placed relative to each other, with accuracy in position of better than 0.1 mm, such as better than 0.5mm, or even better than 0.2mm, such as with accuracy of 0.1mm or better. The arrangement affects homogeneity of the illumination light over the illumination region, preferably where an ideal arrangement results in the minimum illumination variation.

One preferred embodiment of the present invention allows the light source to be placed close to the sample material being analysed. This allows the construction of compact optical system, since the illumination means, including light source and optical components can be shorter than 100mm in length along the optical axis of the system, preferably shorter than 60mm, more preferably shorter than 40mm, preferably even shorter than 20mm, such as shorter than 15mm.

In an embodiment of the invention, a wavelength separation unit is provided to receive the illumination light and to define a wavelength band and polarity of the illumination light. The wavelength separation unit is a spectral filter means selected from an interference filter, absorbing filter, and excitation filter.

In yet another embodiment of the invention, a focussing unit to focus the illumination light onto the illumination region of the sample plane is provided. The focussing unit is selected from a lens and array of lenses.

In another embodiment of the invention, a detector to detect emission light signals emitted from the sample to obtain data is provided. Further a processor is provided to process the obtained data, thereby analysing the sample.

Several preferred embodiments of the present invention have the units are arranged on, or substantially on the optical axis of a photohmύnescent imaging system, preferably in arrangement where the flux of the illumination light has a general direction diτect!y towards the detector unit of an imaging system. Other equally preferred embodiments of the present invention have the illumination means arranged substantially off the optical axis of an imaging system, preferably in an arrangement that includes a dichroic mirror reflecting the includes a dichroic mirror reflecting emission light from the sample onto an imaging system. The units refer to the excitation unit, collimating unit, lens unit, wavelength separation unit, and focussing unit or a selection of such units, such as the excitation unit, and lens unit including the sample plane. Typically, in several preferred embodiments of the invention, the size of the apparatus is shorter than 100mm in length along the excitation-sample axis, preferably shorter than 60mm, more preferably shorter than 40mm, preferably even shorter than 20mm, such as shorter than 15mm. According to several preferred embodiments of the invention, the illumination efficiency is higher than 75%, preferably higher than 80%, more preferably higher than 85%, more preferably higher than 90%, and even more preferably higher than 95%. According to several preferred embodiments of the invention, the homogeneity of the illumination, defined by illumination variation in the imaged region of the sample materia! is less than 0.50, preferably less than 0.40, more preferably less than 0.30, more preferably less than 0.20, more preferably less than 0.15, more preferably less than 0.10, more preferably less than 0.05. The illumination efficiency and illumination variation is obtained without use of a difruser element and also without substantial defbcusing of the units of the illumination means.

In other embodiment of the invention, a method for illuminating a sample is provided. The method includes arranging a sample on a sample plane having an illumination region; generating an excitation light using an excitation unit having a light source; and generating an illumination light, directed towards the illumination region, using a lens unit that comprises of a micro lens array having a plurality of lenses arranged in a two dimension arrangement. The lens unit produces a homogeneous illumination light to be projected on the illumination region of the sample plane with high illumination efficiency.

In one embodiment of the invention, the particle in the sample is labelled with a photolumincscent active material, preferably fluorescent material. The illumination region having the sample is exposed to the illumination light to produce a fluorescence signal from the sample, which is arranged on the sample plane. In one embodiment of the invention, the light source is placed parallel to the illumination region with an accuracy variation in position with respect to the light source of lmm or lower.

In other embodiment of the invention, the light source is arranged relative to at least one other optical element (unit and/ or the sample plane) of the apparatus. This includes moving the light source relative to at least one other optical element (unit and/ or the sample plane) by lmm or lesser for the arrangement. Such arrangement affects homogeneity of the illumination light over the illumination region, preferably where an ideal arrangement results in the minimum illumination variation. In other embodiment of the invention, collimated excitation light is generated from the excitation light using a coUiraating unit, directed towards the lens unit, from the excitation light using a collimating unit. The collimating unit includes an array of lenses. In other embodiment of the invention, spatial angle through which the excitation light from the excitation unit is collected is increased.

In other embodiment to the invention, arranging the lenses of the micro lens array are arranged in an array, preferably where two said arrays are arranged in a series, thus forming an array of lenses. Each array of the two said arrays includes the plurality of lenses. In particular, each array includes at least 4 lenses, preferably more than 4 lenses, more preferably more than 50 lenses elements, and even more preferably more than 100 lenses, such as 150 lenses or 200 lenses, as described above.

In other embodiment of the invention, the size of the micro lens array is less than 20mm, me size being the diameter of the lens array in the direction perpendicular to an excitation-sample axis, where excitation-sample axis is defined by optical path of the excitation light, when the excitation light is directed directly from the light source to the sample plane.

In other embodiment of the invention, the lens unit comprises of a first micro lens array in opposite orientation with a second micro lens array. A selection of the first micro lens array and the second micro lens array is made based on enhancing illumination efficiency and/or reducing illumination variation and/or integrating an optical feature of a separate optical element into the optical effect of the lens array.

In other embodiment of the invention, the micro lens array is housed in a casing comprising means for arranging and fixing the micro lens array.

In yet another embodiment of the invention, a wavelength band and polarity of the illumination light is defined using a wavelength separation unit. The wavelength separation unit is a spectral filter means selected from an interference filter, absorbing filter, and excitation filter. In another embodiment of the invention, focussing of the illumination light onto the illumination region of the sample plane is performed using a focussing unit. The focussing unit is selected from a lens and array of lenses.

In another embodiment of the invention, degree of parallelism of the illumination light entering the focussing unit is increased using the micro lens array. In another embodiment of the invention, emission light signals emitted from the sample are detected to obtain data. Further, the obtained data is processed to analyse the sample.

In one embodiment of the invention, the units are arranged on or substantially on the optical axis of a photolumincsccnt imaging system, preferably in an arrangement where the illumination light is directed directly towards a detector unit of an imaging system. However, in other embodiment of the invention, the units are arranged at substantially off the optical axis of a photoluminescent imaging system, preferably in an arrangement that includes a dichroic mirror reflecting emission light from the sample onto an imaging system.

The illumination efficiency, according to mis method, is higher than 75%, preferably higher than 80%, more preferably higher than 85%, more preferably higher than 90%, and even more preferably higher than 95%. The homogeneity of the illumination, defined by illumination variation in the imaged region of the sample material, according to this method is less than 0.50, preferably less than 0.40, more preferably less than 0.30, more preferably less than 0.20, more preferably less than 0.15, more preferably less man 0.10, more preferably less than 0.05.

The apparatus and method of the present invention is exemplified through several examples as presented below for better understanding of the present invention:

Example 1

Focusing of illumination Light by Micro Lens Array (Refer Figure 2) Figure 2A shows an arrangement according to an embodiment of the present invention, simulating illumination of a sample, similar to the system in Example 1, with the addition of a lens unit (206) having a micro lens array. The system contains an excitation unit (291) having a light source identical to the one in Figure IA, a coiliroating unit (202), such as a biconvex aspheric lens, a wavelength separation unit (203), a focussing unit (204), such as a plano-convex lens, and finally the sample to be illuminated placed on the sample plane (205). In this example, the micro lens array (206) is 12 mm in diameter, and the circumscribed square (12 by 12mm) is divided into 60 x 44 rectangular adjacent symmetrical biconvex lens elements with a thickness of 1.191 mm and lens radii of 0.4mm and -0.4mm respectively, each individual lens element is generating a small image of the light source and when all individual images are superimposed on the sample a rectangular shaped with bomogeny illumination is present

The system was subjected to simulation identical to the simulation of prior art apparatus and the result Is illustrated in Figure 2B, which shows the illumination of the sample. Figure 2B shows that the structure of the light source is substantially eliminated, substantially without loss of illumination efficiency due to broadening of the illumination. These properties are highly preferred when performing photølumήiecent analysis, such as fluorescence analysis.

Example 2 Comparison of Illumination (Refer Figure 3}

Figure 3 shows a graph, illustrating fee difference of simple illumination and illumination according to the present invention, when using a light source having substantial illumination structure. The comparison is based on the results of the prior art apparatus and the apparatus of the present invention as described above, The graph in Figure 3 shows intensity of the illumination as found in the prior art apparatus and the apparatus of the present invention and indicated in Figure 1C and 2B, along a horizontal line indicated by the arrows in the figures.

The illumination as found in the prior art apparatus is represented by the line 301, while the illumination as found in the apparatus of the present invention is represented by the line 302, The lines 303 represent the boundaries of the illumination as defined by magnification of the optical system.

The graph illustrates that line 302 has less spread outside the boundaries 303 than does line 301, which will give greater illumination efficiency. Further the variation in line 302 within the boundaries 303 is considerably less than the variation in line 301, both with respect to the structure in the emittance of the light source, present as a significant drop in illuminatiori in the centre and as significant drop-off in intensity towards the edges of the boundaries.

Example 3

Implementation. According to the Present Invention (Refer Figure 4) Figure 4 shows an implementation according to an embodiment of the present invention. It illustrates the apparatus, comprising an excitation unit (401) having a light source, for instance ML101J17-01 (Mitsubishi, Japan), LZ1-00xx05 or LZ1-

00xx03 (LedEngin, USA), specifically the selection of light source depends on the desired wavelength region and intensity. Further, it comprises a collimating unit (402), e.g. LAG000 (Melles Griot, USA), an array of micro lenses (403), e.g. double sided lens array element, a wavelength separation unit (404), typically interference and/or absorbing filters, in fluorescence analysis the filter is termed excitation filter, and finally a focusing component (405), e.g. a PCX og DCX lens with effective focal length of between 2 and 20 mm, where the effect of changing the focal length affects the size of the region illuminated by the light source. Finally the illustration shows the sample plane (406), onto which it is desired to project a relatively homogeneous light from the light source, e.g. illumination region, preferably with relatively high illumination efficiency.

Figure 4B shows the implementation according to the present invention, where simulated rays of light are drawn, demonstrating the optical operation of the system.

Example 4 Adaptation of illumination Region (Refer Figure 5) Figure 5 shows the illumination of the sample plane according to an embodiment of the present invention. The effect of changing focal length of the focusing unit (405 in Figure 4) afreets the effective size of the illumination region.

Example 5 Element of Micro Lenses (Refer Figure 6) Figure 6 shows a micro lens array element according to an embodiment of the present invention. The element shown in Figure 6A is a thermal plastic item, comprising means for arranging and fixing the element (601), here on the form of a rim, and two arrays of micro lenses, an upper array (602) and an array on the bottom (lower array, which is not shown). The single element of the micro lens arrays, including the upper array (shown, 602) and the lower array, is preferably produced as one piece by moulding together both upper array and lower array using a moulding method, i.e. by moulding a substantially optically transparent material, such as glass or polymer. The terminologies - the upper array and lower array - arc according to this example of the embodiment of the invention. Any person skilled in the art would appreciate that during one of the implementation embodiment as shown in Example 3, the said two arrays may act like front array (excitation light facing array) and rear array (array from where the excitation light leaves the lens unit). In other equally preferred embodiments, the two arrays may act like upper array and bottom array as well.

Each of the micro lens arrays consists of a number of lenses, such as 1.2 x 0.9 mm lenses of 1.7 mm radius, separated by a predetermined thickness, for instance about 5mm, which is the thickness of the micro lens element. As an example, the pattern of the micro lenses, shown in Figure 6B, is a 13 by 9 grid, which has been roughly shaped in a circular fashion, preferably similar to the shape of the beam of light, to reduce physical size, by removing 5 lenses from each corner.

Example 6 Arrangement of optical module (Refer Figure 7)

A typical embodiment according to present invention includes two or more optical units and Figure 7 shows the arrangement of these units. The units are an exciation unit (701), sample plane (702), detection unit (703) and an optical mirror (704). Figure 7A shows a preferred embodiment comprising an excitation unit, sample plane and detection unit, where all units are arranged on a parallel axes relative to one other. The excitation unit, typically comprises of a light source, focusing unit and wavelength separation unit (these items not shown in the figure) illuminating light onto the sample plane, where the sample is typically arranged in a perpendicular orientation relative to the main axes of light emitted from the other units. Typically signals from the sample compartment are emitted in all directions from but a fraction of these signals are collected by the detection unit, which in this arrangement is on, or substantially on, the same axes as the excitation unit and the sample plane. Figure 7B shows another equally preferred embodiment of an embodiment of the present invention, where signals from the sample plane are directed towards the detection unit by an optical mirror. This embodiment allows the detection module to be situation outside the axes formed between the excitation unit and sample plane, such as illustrated in Figure 7B, where the detector unit is in a perpendicular arrangement.

As Figure 7 only shows the general orientation of the different units, and not the position of different elements within the units, it would be appreciated by a person skilled in the art, that when including an optical mirror to direct the light off the optical axis, it can often be advantageous to include an optical component of tbc detection unit, such as one or more lens(es) between the sample unit and the optical unit Typically, the effect of such arrangement would be to assure a more parallel nature of the light emitted from the sample plane, detected in the detection unit, which may render more homogeneous relation of the excitation tight to a wavelength dependent property, such as filtration, and/or to increase the effective aperture of the detection module, by placing a collecting optic close to the image.

Embodiments of the present invention include two or more detection unit, for instance by combining the arrangement shown in Figure 7 A and B. This is typically done by including an optical mirror with wavelength dependent reflectance properties, such that reflectance/transmittance properties are substantially different for different wavelengths. Such embodiments allow simultaneous detection of two or more optical properties of the sample in the sample plane.

It should be appreciated that reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, it is emphasized and should be appreciated mat two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various portions of this specification are not necessarily all referring to the same embodiment Furthermore, the particular features, structures or characteristics may be combined as suitable in one or more embodiments of the invention.

Throughout the foregoing description, for the purposes of explanation, numerous specific details were set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without some of these specific details..

Accordingly, the scope and spirit of the invention should be judged in terms of the claims which follow.

Claims

We claim:

1. An apparatus to illuminate a sample, said apparatus comprising: a sample plane having an illumination region onto which the sample is arranged; an excitation unit having a light source to generate art excitation light; and a lens unit, having a micro lens array where the micro lens array comprises a plurality of lenses arranged in a two dimension arrangement, to receive the excitation light and generate an illumination light that is directed towards the illumination region; wherein, the lens unit produces a homogeneous illumination light to be projected on the illumination, region of the sample plane with a high illumination efficiency,

2. The apparatus according to claim 1, wherein the illumination region is at least 0.5mm². 3. The apparatus according to claim 1, wherein the illumination region is at least 2 times larger than the size of emittance area of the light source.

4. The apparatus according to claim 1 , wherein the sample contains a particle.

5. The apparatus according to claim 4, wherein the particle or material contained on or within the particle is photoluminescent active, generating fluorescence when illuminated with the illumination light.

6. The apparatus according to claim 4, wherein the particle is labelled with a photoluminesoent active material, preferably fluorescent material.

7. The apparatus according to claim 4. wherein the particle is selected from a mammalian cell, yeast cell, fungus and bacterium. 8. The apparatus according to claim 1, wherein the only illumination region is exposed to the illumination light, thereby not exposing the sample not under investigation to illumination, preferably where the illumination results in alteration in chemical and/or physical property of the sample, such as fading of fluorescent signal. 9, The apparatus according to claim 1, wherem the light source is selected from a dispersive light source, an emitting diode, a laser diode and a laser. 10. The apparatus according to claim 1, wherein the light source includes an emittasce element having an emittance area of greater than 0.5 mm², preferably greater than 1.0 mm².

11. The apparatus according to claim 1, wherein the light source is arranged in a light source parallel to the illumination region with an accuracy variation in position with respect to the light source by lmm or lower.

12. The apparatus according to claim 1, wherein the excitation unit comprises of a plurality of light sources, preferably in a single assembly.

13. The apparatus according to claim 1, wherein the light source is arranged relative to at least one other unit and/ or the sample plane.

14. The apparatus according to claim 13, wherein the arrangement comprises of relative movement of the light source to at least one other vnύt and/ or the sample plane by 1 mm or less,

15. The apparatus according to claim 14, wherein the arrangement affects homogeneity of the illumination light over the illumination region, preferably where an ideal arrangement resets in the minimum illumination variation.

16. The apparatus according to claim 1, further comprising a coilimating unit to receive the excitation light and generate a collimated excitation light

17. The apparatus according to claim 16, wherein the collimating unit includes an array of lenses.

18. The apparatus according to claim 16, wherein the eolhmating unit increases spatial angle through which the excitation light from the excitation unit is collected.

19. The apparatus according to claim 1, wherein the micro lens array increases degree of parallelism of the illumination light.

20. The apparatus according to claim 1, wherein the excitation light focused onto the micro lens array has a divergence angle of 0.1mrad or less, preferably 0.05mrad or less, more preferably 0.02mrad or less.

21. The apparatus according to claim 1, wherein each of fee lenses in the plurality of lenses of the micro lens array produces an image similar in dimensions to the region of the sample being analysed, preferably where the image produced is substantially rectangular. 22. The apparatus according to claim 1 , wherein the plurality of lenses comprises of at least 4 lenses, preferably more than 4 lenses, more preferably more than 50 lenses elements, and even more preferably more than 100 lenses, such as 150 lenses or 200 lenses.

23. The apparatus according to claim 1, wherein the lenses are semi-spherical lenses arranged in an array, preferably where two said arrays, each array having the plurality of lenses, are arranged in a series, thus forming an array of lenses.

24. The apparatus according to claim 1, wherein dimension of the lens is in the range from 3mm or lesser, preferably in the range from 1 to 2mm.

25. The apparatus according to claim 1, wherein size of the micro lens array is less than 20mm, the size being the diameter of the lens array in the direction perpendicular to an excitation-sample axis.

26. The apparatus according to claim 1, wherein the lens unit comprises of a first micro lens array in opposite orientation with a second micro lens array.

27. The apparatus according to claim 25, wherein selection of the first micro lens array and the second micro lens array is based on enhancing illumination efficiency and/or reducing illumination variation and/or integrating an optical feature of a separate optical element into the optical effect of the lens array. 28. The apparatus according to claim 1, wherein the micro lens array is housed in a casing comprising means for arranging and fixing the micro lens array.

29. The apparatus according to claim 28, wherein the casing is of cast polymer.

30. The apparatus according to claim I, wherein pattern of the lenses is preferably similar to the shape of beam of the excitation tight 31. The apparatus according to claim 1, further comprising a wavelength separation unit to receive the illumination light and to define a wavelength band and polarity of the illumination light.

32. The apparatus according to claim 31, wherein the wavelength separation unit is a spectral filter means selected from an interference filter, absorbing filter, and excitation filter.

33. The apparatus according to claim 1, further comprising a focussing unit to focus the illumination light onto the illumination region of the sample plane.

34. The apparatus according to claim 33, wherein the focussing unit is selected from a lens and array of lenses. 35. The apparatus according to claim 1, wherein size of the apparatus is shorter than 100mm in length along the excitation-sample axis, preferably shorter than 60mm, more preferably shorter than 40mm, preferably even shorter than 20mm, such as shorter than 15mm.

36. The apparatus according to claim 1, wherein the units are arranged on, or substantially on the optical axis of a photoluminescent imaging system, preferably in an arrangement where the illumination light is directed directly towards a detector unit of an imaging system. 37. The apparatus according to claim 1 , wherein the units are arranged substantially off the optical axis of a photoluminescent imaging system, preferably in an arrangement that includes a dichroic mirror reflecting emission tight from the sample onto an imaging system.

38. The apparatus according to claim 1 , wherein the illumination efficiency is higher than 75%, preferably higher than 80%, more preferably higher than 85%, more preferably higher than 90%, and even more preferably higher than 95%.

39. The apparatus according to claim 1 , wherein the homogeneity of the illumination, defined by illumination variation in the imaged region of the sample material is less than 0.50, preferably less than 0.40, more preferably less than 0.30, more preferably less than 0.20, more preferably less than 0.15, more preferably less than

0.10, more preferably less than 0.05. 40. The apparatus according claim 1, wherein the illumination efficiency and illumination variation is obtained without use of a diffuser element. 41. The apparatus according claim 1, wherein the illumination efficiency and illumination variation is obtained without substantial defbcusing of the units of the illumination means. 42. A method for illuminating a sample, said method comprising: arranging a sample on a sample plane having an illumination region; generating an excitation light using an excitation unit having a tight source; and generating an illumination light, directed towards the illumination region, using a lens unit that comprises of a micro lens array having a plurality of lenses arranged in a two dimension arrangement; wherein, the lens unit produces a homogeneous illumination light to be projected on the illumination region of the sample plane with a high illumination efficiency. 43. The method according to claim 42, wherein the illumination region is at least 0.5mm². 44. The method according to claim 42, wherein the illumination region is at least 2 times larger than the size of eminence area of the light source.

45. The method according to claim 42, wherein the sample contains a particle.

46. The method according to claim 45, wherein the particle or material contained on or within the particle is photoluminescent active, generating fluorescence when illuminated with the illumination light 47. The method according to claim 45, further comprising labelling the particle with a photoluminescent active material, preferably fluorescent material.

48. The method according to claim 45, wherein the particle is selected from a mammalian cell, yeast cell, fungus and bacterium.

49. The method according to claim 42, further comprising exposing only the illumination region to the illumination light, thereby not exposing the sample not under investigation to illumination, preferably where the illumination results in alteration in chemical and/or physical property of the sample, such as fading of fluorescent signal.

50. The method according to claim 42, wherein the light source is selected from a dispersive light source, an emitting diode, a laser diode and a laser.

51. The method according to claim 42, wherein the light source includes an eminence element having an emittance area of greater than 0.5 mm², preferably greater man 1.0 mm².

52. The method according to claim 42, further comprising arranging the light source in a light source parallel to the illumination region with an accuracy variation in position with respect to the light source of 1mm or lower.

53. The method according to claim 42, wherein the excitation unit comprises of a plurality of light sources, preferably in a single assembly. 4. The method according to claim 42, further comprising arranging the light source relative to at least one other unit and/ or the sample plane.

55. The method according to claim 42, further comprising moving the light source relative to at least one other unit and/ or the sample plane by lmm or lesser for the arrangement.

56. The method according to claim 55, wherein the arrangement affects homogeneity of the illumination light over the illumination region, preferably where an ideal arrangement results in the minimum illumination variation. 57. The method according to claim 42, further comprising generating a collimated excitation light, directed towards the lens unit, from the excitation light using a collimating unit

58. The method according to claim 57, wherein the coUimating unit includes an array of lenses.

59. The method according to claim 42, further comprising increasing spatial angle through which the excitation light from the excitation unit is collected. 60. The method according to claim 57, wherein the eollimated excitation light focused onto the micro lens array has a divergence angle of 0.1mrad or less, preferably 0.05mrad or less, more preferably 0.02mrad or less.

61. The method according to claim 42, wherein each of the lenses in the plurality of lenses of the micro lens array produces an image similar in dimensions to the region of the sample being analysed, preferably where the image produced is substantially rectangular.

62. The method according to claim 42, wherein the plurality of lenses comprises of at least 4 lenses, preferably more than 4 lenses, more preferably more than 50 lenses elements, and even more preferably more than 100 lenses, such as 150 lenses or 200 lenses.

63. The method according to claim 42, wherein the lenses are semi-spherical.

64. The method according to claim 42, further comprising arranging the lenses in an array, preferably where two said arrays, each array having the plurality of lenses, are arranged in a series, thus forming an array of lenses. 65. The method according to claim 42, wherein dimension of the lens is in the range from 3mm or lesser, preferably in the range from 1 to 2mm. 66. The method according to claim 42, wherein size of the micro lens array is less than 20mm, the size being the diameter of the lens array in the direction perpendicular to an excitation-sample axis. 67. The method according to claim 42, wherein the lens unit comprises of a first micro lens array in opposite orientation with a second micro lens array.

68. The method according to claim 67, further comprising selecting the first micro lens array and the second micro lens array based on enhancing illumination efficiency and/or reducing illumination variation and/or integrating an optical feature of a separate optical element into the optical effect of the lens array.

69. The method according to claim 42, further comprising housing the micro lens array in a casing comprising means for arranging and fixing the micro lens array.

70. The method according to claim 69, wherein the casing is of cast polymer.

71. The method according to claim 42, whereiti pattern of the lenses is preferably similar to the shape of bsam of the excitation, light.

72. The method according to claim 42, further comprising defining a wavelength band and polarity of the illumination light using a wavelength separation unit 73. The method according to claim 72, wherein the wavelength separation unit is a spectral filter means selected from an interference filter, absorbing filter, and excitation filter. 74. The method according to claim 42, further comprising focussing the illumination light onto the illumination region of the sample plane using a focussing unit 75. The method according to claim 74 wherein the focussing unit is selected from a lens and array of lenses.

76. The method according to claim 74, further comprising increasing degree of parallelism of the illumination light entering the focussing unit using the micro lens array. 77. The method according to claim 42, further comprising arranging the units on or substantially on the optical axis of a photoluminescent imaging system, preferably in an arrangement where me illumination light is directed directly towards a detector unit of an imaging system.

78. The method according to claim 42, further comprising arranging the units at substantially off the optical axis of a photαlumincseent imaging system, preferably in an arrangement that includes a dichroic mirror reflecting emission light from the sample onto an imaging system.

79. The method according to claim 42, wherein the ilhiininatiαn efficiency is higher than 75%, preferably higher than 80%, more preferably higher than 85%, more preferably higher than 90%, and even more preferably higher than 95%,

80. The method according to claim 42, wherein the homogeneity of the illumination, defined by illumination variation in the imaged region of the sample material is less than 0,50, preferably less than 0.40, more preferably less than 0.30, more preferably less than 0.20, more preferably less than 0.15, more preferably less than 0.10, more preferably less than 0.05.

81. The method according claim 42, wherein the illumination efficiency and illumination variation is obtained without use of a diffuses: element.

82. The method according claim 42, wherein the illumination efficiency and illumination variation is obtained without substantial defbcusing of the units.