Apparatus for detecting fluorescent light emitted from a sample, a biosensor system, and a detector for detecting supercritical angle fluorescent light

Abstract

An apparatus for detecting fluorescent light emitted from a sample comprises: a light source, which is configured to emit excitation light of an excitation wavelength towards a sample comprising fluorophores such that fluorescent light is induced; a photo-detector for detecting light incident on the photo-detector; and an interference filter arranged on the photo-detector, wherein the interference filter is configured to selectively collect and transmit light towards the photo-detector based on an angle of incidence of the light towards the interference filter, wherein the interference filter is configured to selectively transmit supercritical angle fluorescence from the sample towards the photo-detector and suppress undercritical angle fluorescence from the sample.

Claims

1. A biosensor system comprising: an apparatus for detecting fluorescent light emitted from a sample, said apparatus comprising: a light source, which is configured to emit excitation light of an excitation wavelength towards a sample comprising fluorophores such that fluorescent light is induced; a photo-detector for detecting light incident on the photo-detector; an interference filter arranged on the photo-detector, wherein the interference filter is configured to selectively collect and transmit light towards the photo-detector based on an angle of incidence of the light towards the interference filter, wherein the interference filter is configured to selectively transmit supercritical angle fluorescence from the sample towards the photo-detector and suppress under critical angle fluorescence from the sample; a sample holder for receiving and preprocessing a fluidic sample, the sample holder being configured to be arranged in the apparatus for allowing the apparatus to excite fluorescence in the fluidic sample and collect and detect supercritical angle fluorescence from the fluidic sample; and a receiver for receiving the sample holder, wherein the receiver is configured to define a relation between the fluidic sample in the sample holder and the interference filter, wherein the receiver is configured to arrange the sample holder in the apparatus such that supercritical angle fluorescence is only guided through layers with a refractive index that is equal to or larger than a refractive index of the sample holder.

2. The biosensor system according to claim 1, wherein the interference filter comprises a plurality of layers of at least two materials having different refractive indices for selectively transmitting light in dependence of wavelength of the light and angle of incidence of the light.

3. The biosensor system according to claim 1, wherein the interference filter is integrally arranged on a substrate comprising the photo-detector.

4. The biosensor system apparatus according to claim 1, further comprising a lens, which is arranged between the interference filter and the photo-detector, wherein the lens is configured to redirect light from the interference filter towards being incident parallel to a normal of a surface of the photo-detector.

5. The biosensor system according to claim 1, wherein the light source is configured to emit a collimated light beam for exciting fluorophores in the sample and wherein the apparatus is configured to receive the sample between the light source and the interference filter such that emitted light from the light source is transmitted through the sample before being collected by the interference filter.

6. The biosensor system according to claim 2, wherein the apparatus further comprises a mask arranged on the interference filter, wherein the mask is configured to block transmission of light of the excitation wavelength.

7. The biosensor system according to claim 1, wherein a capping layer is arranged on the interference filter, the capping layer forming a surface for receiving the sample and defining a desired critical angle for light from the sample being arranged on the capping layer.

8. The biosensor system according to claim 1, wherein the light source comprises a waveguide having a total internal reflection surface for transporting light by total internal reflection in the waveguide, wherein the light source is configured for exciting fluorophores in the sample by evanescent light escaping the total internal reflection surface.

9. The biosensor system according to claim 1, further comprising a lens for focusing excitation light towards a point in the sample.

10. The biosensor system according to claim 9, further comprises a scanning element for scanning the focused light over the sample for imaging of the sample.

11. The biosensor system apparatus according to claim 1, wherein the photo-detector comprises an array of photo-sensitive areas, wherein each photo-sensitive area separately detects an amount of light incident on the photo-sensitive area.

12. The biosensor system according to claim 1, further comprising a processing unit, which is configured to receive information of detected light from the photo-detector and to determine a biological measurement based on the received information.

13. An apparatus for detecting fluorescent light emitted from a sample, said apparatus comprising: a light source, which is configured to emit excitation light of an excitation wavelength towards a sample comprising fluorophores such that fluorescent light is induced; a photo-detector for detecting light incident on the photo-detector; and an interference filter arranged on the photo-detector, wherein the interference filter is configured to selectively collect and transmit light towards the photo-detector based on an angle of incidence of the light towards the interference filter, wherein the interference filter is configured to selectively transmit supercritical angle fluorescence from the sample towards the photo-detector and suppress undercritical angle fluorescence from the sample, and wherein a capping layer is arranged on the interference filter, the capping layer forming a surface for receiving the sample and defining a desired critical angle for light from the sample being arranged on the capping layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above, as well as additional objects, features and advantages of the present inventive concept, will be better understood through the following illustrative and non-limiting detailed description, with reference to the appended drawings. In the drawings like reference numerals will be used for like elements unless stated otherwise.

(2) FIG. 1 is a schematic view illustrating supercritical angle fluorescence.

(3) FIG. 2 is a chart illustrating intensity of supercritical angle fluorescence as a function of distance of a fluorophore from a surface.

(4) FIG. 3 is a schematic view of an apparatus according to a first embodiment.

(5) FIG. 4 is a chart illustrating transmission by an interference filter in dependence of an angle of incidence of light.

(6) FIG. 5 is a schematic view of an apparatus according to a second embodiment.

(7) FIG. 6 is a schematic view of a biosensor system.

DETAILED DESCRIPTION

(8) An apparatus for detecting fluorescent light emitted from a sample is provided. The apparatus is configured to detect supercritical angle fluorescent light. Before embodiments of the apparatus will be described in detail, supercritical angle fluorescence will be briefly described.

(9) Referring now to FIG. 1, fluorophores 100 can be described by dipoles that preferentially emit fluorescent light in certain axes depending on an orientation of the dipole. In addition to this propagating component, fluorophores 100 emit an evanescent non-propagating component. This non-propagating component becomes propagating when the fluorophore 100 is at or below a distance of one wavelength from an interface 102. This is called supercritical angle fluorescence (SAF) and is emitted above a critical angle (corresponding to total internal reflection) of the interface between two materials at different sides of an interface. For example, if the fluorophore 100 is present in a solution of a sample arranged in a sample holder, the critical angle is defined by a relation of refractive index between the solution 104 (forming a first material on one side of the interface 102) and a material of the sample holder 106 (forming a second material on an opposite side of the interface 102). The material of the sample holder may typically be glass or plastic and may be selected in order to define a refractive index so as to select a critical angle formed in the interface 102.

(10) The SAF may thus propagate into the material of the sample holder 106 and may be further transferred through a second interface 108 between the sample holder 106 and another medium 110, if the refractive index of the other medium 110 is the same or higher than a refractive index of the material of the sample holder 106, i.e. total internal reflection will be avoided in an interface of the sample holder 106 and the other medium 110. A surface of the sample holder 106 or a surface of a detector for collecting SAF may thus be treated with a refractive index matching medium 110 in order to ensure that the supercritical angle fluorescent light may be collected. For instance, the surface treatment may be provided by means of a viscous material, such as an oil, so that it may be ensured that there is no air gap between the sample holder 106 and the detector.

(11) The intensity of SAF can be as high as 50% of the total fluorescence emitted from a sample, if the fluorophores 100 are arranged close to a surface. As illustrated in FIG. 2, the intensity decreases with an almost exponential behavior with increasing distance (d) from the interface 102. Thus, if detection of light is selective to SAF and not disturbed by undercritical angle fluorescence, a very high sensitivity to fluorophores 100 arranged close to a surface is obtained.

(12) Referring now to FIG. 3, an apparatus 200 for detecting fluorescent light emitted from a sample will be described.

(13) The apparatus 200 comprises an interference filter 204. The interference filter 204 comprises a stack of layers for selectively transmitting light through the interference filter 204. The interference filter 204 may comprise a stack of alternating layers 204a, 204b of two different materials (having different refractive indices). In interfaces between layers in the interference filter 204, constructive interference may occur between light having traveled different path lengths through the interference filter 204 (different number of reflections in the interfaces between the layers). The refractive index and thickness of the layers 204a, 204b may thus control characteristics of light that will cause constructive interference. This implies that light that forms constructive interference in the interference filter 204 may be selectively transferred through the interference filter 204.

(14) The interference filter 204 may be designed such that light having a specific angle of incidence will be selectively transmitted through the interference filter 204. Hence, the interference filter 204 may selectively transmit SAF light, while suppressing undercritical angle fluorescence. Thus, the interference filter 204 may be configured to filter out SAF allowing the SAF to be detected with a high signal-to-noise ratio.

(15) As indicated in FIG. 4, the interference filter 204 may be designed such that a cut-off wavelength is defined, which is dependent on an angle of incidence of light on the interference filter 204. The chart in FIG. 4 illustrates the transmission by the interference filter 204 for different angles of incidence of light. Light incident at the critical angle θ.sub.c will thus be transmitted if the wavelength is equal to or larger than the fluorescence wavelength. Since the fluorescence wavelength is larger than the excitation wavelength, this also implies that excitation light will not be passed through the interference filter 204.

(16) However, as also indicated in FIG. 4, there is a difference in cut-off wavelength for s-polarized and p-polarized light. Thus, the interference filter 204 may be designed such that a cut-off wavelength for s-polarized light incident at the critical angle corresponds to the fluorescence wavelength.

(17) The apparatus 200 further comprises a photo-detector 206. The photo-detector 206 may be arranged to receive light having been transmitted by the interference filter 204.

(18) The photo-detector 206 may comprise at least one photo-sensitive area, which is configured to generate a response, such as an electric charge, in proportion to light incident on the photo-sensitive area. Thus, the photo-detector 206 may generate a measurement of intensity of light being transmitted through the interference filter 204.

(19) The photo-detector 206 may comprise a single or a few photo-sensitive areas, which may be configured to generally detect an intensity of SAF emitted by a sample. An output from the photo-detector 206 may then be used for quantitatively determining an amount of fluorophores 100 at a surface of the sample holder. However, the output may not necessarily determine an origin of the emitted light, such that the output may not be used for imaging of the sample. If a plurality of photo-sensitive areas is used, each may be configured to determine intensity of light emitted from a respective large region in the sample, such that the regions may be quantitatively compared.

(20) In an alternative embodiment, the photo-detector 206 may comprise an array of photo-sensitive areas. The array of photo-sensitive areas may be used for imaging of the sample, as each photo-sensitive area in the array may receive SAF originating from a specific part of the sample.

(21) The photo-detector 206 may for example be implemented as a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) image sensor. Analog output from the photo-sensitive areas may pass an analog-to-digital converter, such that the photo-detector 206 may output a digital representation of detected light. The digital representation is then suited to be transferred to other entities for processing the detected SAF.

(22) The interference filter 204 may be provided with a capping layer 208, which may protect the interference filter 204, e.g. against wear. The capping layer 208 may be formed on top of the interference filter 204. The apparatus 200 may be configured to receive a sample holder 106 such that the sample holder 106 is arranged on the capping layer 208. Alternatively, the sample holder 106 may be arranged directly on the interference filter 204.

(23) An upper surface of the capping layer 208 or (if no capping layer is provided) an upper surface of the top layer in the interference filter 204 may be treated such that a contact between the sample holder 106 and the interference filter 204 is formed through a refractive index matching medium, such as an oil. Thus, it may be ensured that SAF propagating through a wall of the sample holder 106 will be propagated further into the interference filter 204.

(24) The sample holder 106 may comprise walls, e.g. of glass or a plastic material, defining a cavity or space in which a fluidic sample may be provided. The sample holder 106 may thus function as a carrier for providing the sample in the apparatus 200.

(25) The sample holder 106 may comprise one or more pre-defined measurement sites 112. The sample holder 106 may thus comprise a first mask 114 arranged on an inner surface of a wall of the sample holder 106. The first mask 114 may have holes or apertures, allowing the sample to be in contact with a wall of the sample holder 106 such that SAF may be generated. The holes or apertures in the first mask 114 may thus form measurement sites 112. The first mask 114 may also be formed from a material blocking transmission of fluorescent light (and excitation light) in order to further prevent light not originating from measurement sites 112 to reach the photo-detector 206.

(26) The apparatus 200 may further comprise a light source 210, which is configured to emit excitation light towards a sample. As shown in FIG. 3, in a first embodiment, the light source 210 may be provided to emit light through the sample. Thus, the light source 210 and the interference filter 204 are provided on opposite sides of the sample.

(27) This arrangement of the light source 210 may be convenient, as placement of the light source 210 does not interfere with placement of the interference filter 204 and photo-detector 206. Thus, a very simple set-up may be used.

(28) Since light from the light source 210 is transmitted through the sample, fluorophores 100 in a bulk solution of the sample may be excited and emit fluorescent light. However, thanks to the interference filter 204 only selectively transmitting light having an angle of incidence equal with or larger than the critical angle, fluorescence from the bulk solution will not be transmitted through the interference filter 204 towards the photo-detector 206.

(29) The light source 210 may be a laser providing light of a specific wavelength, which constitutes an excitation wavelength for enabling fluorescent light to be transmitted by the fluorophores 100.

(30) The light source 210 may be associated with a lens or other optical component for focusing excitation light to a spot in the sample close to the inner surface of a wall of the sample holder 106. Thus, a confocal arrangement may be provided, wherein the fluorophores 100 close to the surface are selectively excited (and also SAF from these fluorophores is selectively detected over bulk fluorescence).

(31) The confocal arrangement may be used for scanning over a lateral surface at the inner wall of the sample holder 106, such that different regions of the sample may be sequentially excited.

(32) The capping layer 208 may comprise a second mask 212. Alternatively, a wall of the sample holder 106 may be provided with the second mask 212. The second mask 212 may selectively block transmission of the excitation wavelength, while transmitting the fluorescence wavelength. For instance, the second mask 212 may be configured to absorb or reflect light of the excitation wavelength. The second mask 212 may be configured to have an inverted pattern to the first mask 114. Alternatively, the second mask 212 may extend over an entire surface of the sample holder 206/interference filter 204.

(33) Since the excitation light is transmitted by the light source 210 directly towards the interference filter 204, the second mask 212 may be needed to avoid light from the light source 210 to leak through the interference filter 204. The blocking of the excitation light may be important as an intensity of the light transmitted by the light source 210 may be much stronger than an intensity of the SAF.

(34) The apparatus 200 may optionally further comprise a lens 214 between the interference filter 204 and the photo-detector 206. The lens 214 may direct light from the interference filter 204 towards being perpendicularly incident on the photo-sensitive area(s) of the photo-detector 206. Thus, the lens 214 may improve an intensity of light (number of photons) reaching the photo-detector 206 and also may improve the response to incident light (likelihood of an incident photon triggering a reaction in the photo-sensitive area).

(35) The photo-detector 206 and the interference filter 204 may be formed as an integrated detector package, optionally with the lens 214 arranged there between.

(36) The interference filter 204 may thus be formed directly on top a substrate comprising the photo-detector 206. This may be achieved by monolithically integrating the interference filter 204 on top of the photo-detector 206, such that the layers of the interference filter 204 are sequentially formed on the substrate comprising the photo-detector 206. Alternatively, the interference filter 204 may be separately manufactured before being integrated on top of the photo-detector 206.

(37) According to an alternative embodiment, the photo-detector 206, the lens 214 and the interference filter 204 may be manufactured and arranged in the apparatus 200 as separate components. However, in such case, a refractive index matching medium needs to be provided between each of the components in order to prevent total internal reflections in any of the surfaces (which would otherwise severely affect detection of SAF).

(38) Further, in an embodiment, the interference filter 204 may be configured to be exchangeable in the apparatus. The interference filter 204 may be adapted to selectively transmit SAF of a specific wavelength and incident on the interference filter 204 at a specific angle. Thus, by changing the interference filter 204, the apparatus 200 may be changed to work for a different wavelength of SAF and/or a different angle of incidence of the SAF on the interference filter 204.

(39) Thus, the apparatus 200 may be delivered with a set of interference filters 204 such that the apparatus 200 may be selectively configured to work for a set of different wavelengths of SAF and/or different angles of incidence of the SAF on the interference filter 204.

(40) As a different fluorescence wavelength often may require different excitation wavelengths, the apparatus 200 may further comprise a light source 210 that may be controlled such that an appropriate excitation wavelength is emitted towards the sample. Alternatively, the apparatus 200 may comprise a set of parallel light sources 210 configured to emit different wavelengths, such that the appropriate light source 210 may be chosen depending on the desired excitation wavelength to be used.

(41) The apparatus 200 may comprise a housing 216, in which all components of the apparatus 200 are mounted. The housing 216 may provide a well-defined relationship between the light source 210 and the interference filter 204 and the photo-detector 206 so that a robust set-up for performing analyses on samples is provided.

(42) The housing 216 may also enclose all the components, which may imply that laser light emitted by the light source 210 is confined to the housing 216. Thus, the apparatus 200 may be used safely and a user of the apparatus 200 need not be specially trained in use of laser equipment.

(43) The apparatus 200 may further comprise a receiver for receiving a sample holder 106. The receiver may be movable to between a receiving position, in which the receiver may partially extend from the housing allowing the sample holder to be placed in the receiver, and an analysis position, in which the receiver may be arranged in the housing in order to place the sample holder 106 in a desired relation to the light source 210, the interference filter 204 and the photo-detector 206.

(44) The receiver may alternatively provide guiding surface(s) for guiding insertion of the sample holder 106 (e.g. through a slot in the housing 216) so that a sample is properly presented in an analysis position in the housing 216.

(45) It should be realized that components of the apparatus 200 may alternatively be separately manufactured and delivered. The apparatus 200 may thus be assembled when it is to be used. Thus, the light source 210, the interference filter 204 and the photo-detector 206 may each be separately manufactured.

(46) As mentioned above, in a specific embodiment, the interference filter 204 and the photo-detector 206 may form an integrated detector, which may be manufactured and sold as a separate piece of equipment. For advanced users, the integrated detector may then be used as a part in a self-assembled set-up allowing the user to make choices of light source and optical components to be used in the set-up.

(47) Referring now to FIG. 5, a second embodiment of the apparatus 300 will be described. The discussion of features and components of the first embodiment apply to a large extent also to the second embodiment and the discussion will therefore not be repeated here for brevity. Rather, only the features that differ between the first and second embodiments will be discussed.

(48) The apparatus 300 of the second embodiment uses an evanescent field for providing excitation of the fluorophores 100. Thus, the apparatus 300 defines a light path associated with the light source 310. The light path may provide a surface in which total internal reflection of the excitation light may occur. The sample is arranged close to the total internal reflection surface and an evanescent light field in the vicinity of the total internal reflection surface may thus reach the sample and excite fluorophores 100.

(49) As shown in FIG. 5, the apparatus 300 may comprise a waveguide 318, such that light from a light source 310 may propagate through the waveguide 318 by means of total internal reflection. As shown in FIG. 4, the waveguide 318 may be arranged as a layer above the interference filter 304. Further, a sample holder 106 may be arranged above the waveguide 318 in order to allow the evanescent excitation light to affect the sample. Alternatively, the waveguide 318 may form part of sample holder 106, such that the waveguide 318 may present a surface on which the sample is directly provided.

(50) Using evanescent light for excitation of fluorophores 100 implies that only fluorophores 100 close to a surface are excited. Further, the interference filter 304 ensures that only SAF is collected. This implies that the set-up of the second embodiment may provide a very good signal-to-noise ratio of detected light, as background noise may be very limited.

(51) The apparatus 200, 300 discussed above may advantageously be used in a biosensor system 400, as shown in FIG. 6. The biosensor system 400 may comprise a sample holder 406 which may be specifically prepared for preprocessing a sample. The sample holder 406 may thus e.g. comprise a substance for reacting with a sample, such that the reaction may then be observed by the apparatus 200, 300. The sample holder 406 may also comprise fluorophores 100 which may selectively attach to targets in the sample for allowing the targets to be observed in the analysis performed by the apparatus.

(52) A pre-processing of the sample in the sample holder 406 may depend on the desired analysis to be made. For instance, the pre-processing of the sample may operate to expose molecules of interest from within a cell or to protect the molecules of interest from other molecules that seek to destroy the molecules of interest when the molecules of interest leave the protection of the cell. The pre-processing may involve mixing detector antibodies (tagged with fluorophores) with the molecules of interest before the detector antibodies are brought in contact with capture antibodies that are bound to the surface of the sample holder.

(53) It should be realized that many other types of pre-processing of the sample may be performed in the sample holder 406 and that the pre-processing of the sample may depend on the specific type of analysis to be made.

(54) The biosensor system 400 may for instance be used for detecting (bio)molecules so as to determine presence of molecules of interest in a sample. Similarly, the biosensor system 400 may in other embodiments be used for detecting viruses in a sample. The biosensor system 400 may in yet other embodiments be used for cell membrane imaging.

(55) The biosensor system 400 may comprise a processing unit 420, which may be arranged within the housing of the apparatus 200, 300. The processing unit 420 may thus be configured to receive information from the photo-detector 206, e.g. in the form of a digital representation of detected light, so as to allow making an analysis. The processing unit 420 may thus be configured to determine a biological measurement based on the received information.

(56) The processing unit 420 may alternatively be remote from the apparatus 200, 300. The apparatus 200, 300 may thus comprise a communication unit for wired or wireless communication with the processing unit 420. The communication unit may communicate the digital representation of detected light, or may communicate a pre-processed representation.

(57) The processing unit 420 may be any type of unit able to process information. The processing unit 420 may be a general-purpose processing unit, such as a central processing unit (CPU), which may be loaded with a computer program product in order to allow the processing unit 420 to perform the desired operations. The processing unit 420 may alternatively be a special-purpose circuitry for providing only specific logical operations. Thus, the processing unit 420 may be provided in the form of an application-specific integrated circuit (ASIC), an application-specific instruction-set processor (ASIP), a field-programmable gate array (FPGA), or a digital signal processor (DSP).

(58) In the above the inventive concept has mainly been described with reference to a limited number of examples. However, as is readily appreciated by a person skilled in the art, other examples than the ones disclosed above are equally possible within the scope of the inventive concept, as defined by the appended claims.

(59) For instance, a sample holder may be integrated with the interference filter and the photo-detector for providing a complete chip prepared for receiving a sample. The sample holder may be fused to the interference filter, such that refractive index matching medium is not needed between the interference filter and the sample holder. However, if the sample holder is only used once (to avoid need of rinsing the sample holder), it may be desired to have an inexpensive, disposable sample holder. For such applications, having a sample holder integrated with the interference filter and the photo-detector may be less advantageous.