AU2018101853A4 - Autosynchronous fluorescence microscopy system - Google Patents

Abstract

A time gated luminescence detection system comprising a housing enclosure comprising an optical exit aperture and an optical view aperture, said exit and view apertures being located on opposing sides of said enclosure and being optically aligned with an aperture axis; an optical excitation source mounted within said enclosure and configured to generate an optical excitation beam; a planar rotor; said rotor comprising: a plurality of rotor blades each said rotor blade comprising at least a partially reflective portion; a rotational activation motor mounted within said enclosure for providing rotational motion to said rotor in a rotor plane to sequentially place said system between an excitation phase and a detection phase; a reflector mounted within said enclosure, said reflector configured to receive an excitation beam generated by said optical source and further to direct said excitation beam to said at least partially reflective portion of a blade of said rotor when said system is in an excitation phase. a) U) S. 0ob

Description

AUTOSYNCHRONOUS FLUORESCENCE MICROSCOPY SYSTEM

Field ofthe Invention [0001] The present invention relates to time gated luminescence detection schemes and in particular to apparatus and methods for providing a time gated luminescence detection system.

[0002] The invention has been developed y for use as apparatus methods, and systems for provision of auto-synchronous time gated luminescence detection schemes and microscope systems and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

Background ofthe Invention [0003] Any discussion of the background art throughout the specification should in no way be considered as an admission that such background art is prior art, nor that such background art is widely known or forms part of the common general knowledge in the field in Australia or worldwide.

[0004] All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

[0005] Fluorescence is the name given to the process of light emission following a transition between energy levels that occur without a change in the electron spin state.

It is typically a short-lived phenomenon with excited-state lifetimes measured in nanoseconds for most common fluorophores. Phosphorescence on the other hand describes excited state transitions that involve a change in spin-state, and when these transitions are spin-forbidden, emission lifetimes can extend for several thousand times longer than fluorescence decay.

2018101853 29 Nov 2018 [0006] Fluorescence based techniques provide a powerful means for both the qualitative and quantitative detection of bio-molecules. Fluorescence methods afford a sensitive means for the detection of single molecules, however, fluorescent probes (also referred to as fluorescent markers), used to “label” a particular feature in a sample (for example a particular organism or bio-molecule such as Giardia in a water sample) to determine the presence and/or number of features in the sample, lose much of their discriminatory power in the presence of autofluorescence. Organic and inorganic autofluorophores are ubiquitous in nature and some materials fluoresce with great intensity, obscuring or diminishing the visibility of synthetic fluorescent probes. Spectral selection techniques (emission and excitation filters) can reduce the problem but are not always applicable due to the abundance and spectral range of autofluorescence.

[0007] Using techniques such as time-resolved fluorescence microscopy (TRFM), it is possible to spatially discriminate fluorescent regions that differ by less than a nanosecond in fluorescence lifetime. TRFM techniques operate in the frequency domain, usually employing a sinusoidal modulation of the excitation source to induce a phase (φ) delayed modulation in fluorescence intensity from which fluorescence lifetime can be determined using Δφ and modulation frequency parameters (see Figure 1A).

[0008] Time-gated luminescence (TGL) techniques operate within the time-domain and are directed towards detection of events that occur at much longer time-scales (phosphorescence).

[0009] For TGL operation as shown in Figure 1B, the detector is gated off whilst a brief pulse of light is used to excite emission from the sample. The detector is maintained in the off-state for a resolving period (gate-delay) whilst short-lived (<1 ps) fluorescence fades beyond detection. The detector is then enabled to capture luminescence in the absence of autofluorescence, greatly increasing the signal-to-noise ratio. As can be seen, pulse fluorometry or time-gated luminescence techniques operate on much longer timescales. Fluorescence lifetime constants can be determined by observing intensity in the fluorescence signal after different gate delays. A key advantage of TGL techniques is the suppression of short-lived fluorescence to increase the signal-to-noise ratio (SNR).

[0010] Whilst it is possible to discriminate probe fluorescence from autofluorescence using TRF techniques, a simpler and much less costly TGL instrument can be employed

2018101853 29 Nov 2018 if a suitable luminescent probe is available. For example, lanthanide (Eu³⁺ or Tb³⁺) chelate luminescent probes have exceptionally long phosphorescence lifetimes (τ) reaching milliseconds for some compounds. Other compounds that have found wide application forTGL include the platinum and palladium (copro)porphyrins with lifetimes ranging from 10 to 1000 ps depending on their environment. The very large difference in τ between typical autofluorophores and the luminophores used for TGL has helped ensure useful results were gained even with simple instruments relying upon chopper-wheels.

[0011] The substantial increase in SNR afforded by TGL techniques is a critical factor when searching for rare target organisms encountered in autofluorescent environments. For example, the detection of Cryptosporidium oocysts in drinking water requires the filtration of large volumes of water and results in a matrix of mineral particles, algae, desmids and plant matter that is strongly autofluorescent. TGL microscopy has been demonstrated to greatly suppress this background and simplify the detection of both Giardia and Cryptosporidium, two important waterborne pathogens. There are instances where the detection of rare-event signals using conventional fluorescence techniques is exceedingly difficult (or impossible) and consequently, where TGL microscopy has greatest utility.

[0012] Luminescent probes based on the lanthanides Eu³⁺ and Tb³⁺ were described in the 1960’s but effective immunofluorophores using these compounds were not reported until the early 1980’s. TGL microscopes were built to exploit these novel compounds, however various deficiencies in the instrumentation and luminescent probes resulted in relatively insensitive instruments. As technologies matured, improvements were made both in instrument design and probe quality.

[0013] With reference to Figure 1B, TGL techniques employ an optical (light) excitation pulse 1 at a suitable wavelength (e.g. in the ultraviolet range of the optical spectrum) to excite photon emission from the sample. The excitation pulse 1 ideally terminates abruptly (<1 ps) whilst the detector is maintained in the off-state for a predetermined time (the resolving period or gate delay 3), the duration of which is designed to permit short-lived autofluorescence 5 to decay. After the gate-delay 6 has elapsed, the detector is gated on to initiate the start of the acquisition period 7 to detect the

2018101853 29 Nov 2018 fluorescence signal from the luminescent label probe, free of background noise from autofluorophores in the sample.

[0014] In 1988, Soini et al. [Soini E J, Pelliniemi L J, Hemmila I A, Mukkala V M, Kankare J. J., Frojdman K., Lanthanide chelates as new fluorochrome labels for cytochemistry, Journal of Histochemistry and Cytochemistry 36(11) 1988 p. 1449-51] described a europium chelate that could be easily bound to bio-molecules to permit them to “re-test the old idea of time-resolved fluorescence microscopy in immunohistology and cytology'’. Using steady state excitation, it was shown that Eu-antibody labelled histology sections were visibly luminescent to the naked eye and would likely provide a means to improve signal to noise ratio under TGL conditions. The following year, Beverloo et al. [Beverloo, Η. B., Vanschadewijk, A., Vangelderenboele, S. and Tanke, H. J., Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy, Cytometry 11, p. 784-792, (1990).] described a Xenon flashlamp excited TGL microscope synchronized to a chopper wheel. Phosphorescence persists for orders of magnitude longer than prompt fluorescence and makes feasible the use of mechanical choppers for visualizing the phenomenon and the majority of early TGL instruments employed chopper wheels to isolate the excitation and detection states in a TGL cycle.

[0015] As described above, the gate-delay is the intervening period between termination of the excitation pulse and commencement of the acquisition phase (see Figure 1B). For most phosphorescent labels, decay follows single exponential kinetics (lo=lte^_t/T) and emission decays substantially as the gate-delay interval approaches the luminescence lifetime. Unfortunately, the gate-delays typical for chopper based switching mechanisms are quite long (about 20 to 500 ps) in comparison to the emission decay time of the fluorescent labels and therefore the fluorescence has typically decayed quite significantly before the detection state is able to commence. Also, conventional time-gated luminescence microscopes incur substantial losses both during the excitation and emission/detection states as light passages through the dichroic filter cube. The dichroic mirror in the filter cube also has significant limitations on the particular wavelengths that are allowed to be either reflected on to the sample or to pass through to be detected due to the limitations in coating designs. Thus, such systems have severe limitations on the number of allowed wavelengths and the reflection/transmission bandwidth of the design wavelengths that are able to be utilised

2018101853 29 Nov 2018 for the detection system. Such dichroic filter cubes are also expensive and complex to interchange causing significant barriers to the use of the TGL microscope system for different excitation sources or luminescent probes with different operating wavelengths.

[0016] Electronic shutters may be used to overcome the gate-delay limitation of chopper-wheel systems, for example ferro-electric liquid crystals (FELC) which rotate the plane of light polarization in response to an applied voltage and can serve as fast optical shutters. A TGL microscope was constructed by Verwoerd et al. in 1994 [Verwoerd N P, Hennink E J, Bonnet J, Van der Geest C R, Tanke H J, Use of ferro-electric liquid crystal shutters for time-resolved fluorescence microscopy, Cytometry 16(2) 1994 p. 113-7] in which the emission-plane chopper was replaced with two crossed LC shutters. For excitation, a Xenon-arc lamp was interrupted by a mechanical chopper to generate pulsed output; gating of the LC shutters was synchronized to the chopper wheel position. Whilst effective, the FELC shutters imposed a substantial insertion loss with transmission reduced to just 15% when fully open. A further limitation of chopper excitation schemes arises from the relatively slow rise and fall time of the pulse (for example, in the systems described by Verwoerd et al, the rise/fall time was 50 to 100 ps at a chopper rotation speed of 3,800 rpm). Excitation pulses with slow falling edges force extension of the gate-delay that leads ultimately, to a loss in SNR. The gain achieved by switching rapidly in the emission plane with the FELC shutter was offset by the slow falling edge of the excitation pulse.

[0017] Since 1994, the majority of the improvements in TGL microscope detection systems have been obtained by improvements in either the excitation source—flashlamp, visible and ultraviolet (UV) lasers, or light emitting diodes (LEDs)—and/or the detectors used to capture the fluorescent light with increasingly improved signal-to-noise ratio—image-intensified gated charge coupled device (CCD) and electron multiplying charge coupled device (EMCCD) detectors have been particularly successful as described in the inventor’s earlier patent application PCT/AU2005/001606.

[0018] The basic requirements for TGL microscopy are relatively straightforward, a pulsed excitation source and a gated detector. However, the common features of the prior art TGL microscope systems to provide these basic requirements has been the dual shutter system (either using chopper wheels, electronic shutters, or a combination of both) similar to that depicted in Figures 2A and 2B of the inventor’s earlier filed

2018101853 29 Nov 2018 patent, US 8,779,390 [Connally ‘390], incorporated herein by reference. As previously described, this type of system requires that the phase of each of the excitation- and emission-side shutters are precisely synchronised with both the excitation source and the detector acquisition state. To achieve this, complex electronic phase matching circuitry and control systems are required, which are both expensive to implement and maintain. Furthermore, the detector systems that must be used with these TGL microscope systems are prohibitively expensive (typical cost of a TGL EMCCD detector at the time of writing is in the range of about $20,000 to $45,000).

[0019] The prohibitive cost of such systems means that they are not available on a large scale since only the most well-funded research facilities can purchase and maintain such items.

[0020] With reference to the inventor’s earlier filed patent, US 8,779,390 [Connally ‘390], Figure 2A of the present application depicts an auto-synchronous fluorescence detection method and system described therein, the system comprising a device 5 including a rotating unit 10 (see Figures 3A to 3B of Connally ‘390) comprising a plurality of vanes 11, each comprising an arcuate reflective surface 13 for directing an excitation beam onto a sample during a portion of each revolution of the device 5 while time-gated fluorescence signals is permitted to pass in the space 15 between the vanes 11 to an observer whilst the sample is not illuminated by the excitation beam.

[0021] In the arcuate reflective surface embodiments of the Connally rotor device 10, the incident UV excitation beam is perpendicular to the rotation axis of the rotor. The beam strikes the broad reflective distal faces with a collimated beam of light about 5 mm in diameter and is reflected at 90°so that the exc itation beam exits the device parallel to the rotor’s rotation axis.

[0022] By reference to Figure 2B, the exit beam 21 is distorted by reflection from the arcuate rotor face 11 into an oval shape 25 compared with the circular cross-sectional profile of the original beam 23. The distance from the exit aperture of the prior art arcuate rotor 10 to the top surface of the objective is 50 mm, the objective aperture is 5 mm and light not contained within this narrow cone cannot enter the objective. As a consequence, more than 65% of the available excitation energy is lost, resulting in weaker luminescence emission from the sample. Loss is calculated on the basis of an oval spot profile 25 measuring 14.7 mm by 5 mm versus the energy contained in a 5

2018101853 29 Nov 2018 mm cone. A further effect of this design flaw is the uneven illumination across the slide, whereby target organisms labelled with luminescent probe appear much dimmer on the periphery of the field of vision compared with those located in the centre.

[0023] A further disadvantage of the rotor 10 of Connally ‘390 is vibration of the rotor 10 during operation. The mass of rotor 10 of prior art device depicted in Figure 2A, without the addition of drive control magnets, is about 1.2 g and about 3.5 g with magnets fitted. It is a difficult task to ensure the rotor 10 is perfectly balanced once fitted with magnets as the individual variation in magnet weight can be as much as 150 mg. Angular momentum is directly proportional to mass times velocity so that the periodic vibration increases in strength with angular velocity; an imbalance of 150 mg translates to a vibration force of 3.53 x 10^-2 kg.m².sec^-1 at a frequency of 250 Hz. When fitted to a fluorescence microscope system, prior art rotor 10 sits just above the objective so that any vibration is readily coupled directly into the microscope which is irreconcilable with an application where zero vibration can be tolerated at high magnifications.

[0024] Connally ‘390 details an opto-mechanical device comprising rotor 10 designed for insertion into a filter slot present on fluorescence microscopes such as the Olympus BX51 model. This filter slot present in the Olympus microscope is intended for use with the Differential Interference Contrast (DIC) filter and permits the insertion of an optical filter into the main optical axis of the microscope between the objective and the eyepiece. The width and height of the DIC filter slot constrains the dimensions of the device and also the manner in which desired functions may be implemented.

[0025] The design purpose of the device is to act as a temporal filter to suppress the problem of natural or intrinsic fluorescence that is commonly observed by microscopists utilizing conventional fluorescence microscopy. This intrinsic fluorescence (also known as auto-fluorescence) is observed in many natural products or biological fluids I substrates and is characterized by emission across a broad range of the visible spectrum which often prevents suppression through the use of monochromatic spectral filters.

[0026] A second characteristic feature of the emission is the very short fluorescence lifetime; the interval that a molecule spends in the excited state prior to returning to the ground state via the emission of a photon. This interval is typically measured, for

2018101853 29 Nov 2018 example, B-phycoerythrin (present in red algae and cryptophytes) has a fluorescence lifetime of 7.8 ns.

[0027] Insertion of the Connally ‘390 device including rotor 10 into the DIC filter slot provides a means to interpose a high-speed rotor gating system into the optical path. The first stage of temporal gating begins with transmission of a high intensity beam of ultra-violet light (sourced from the device) that exits the device to reach the slide containing the sample to be analysed. The UV light excites fluorescence from the sample that is blocked due to the position of the rotor 10 from returning through the microscope system to reach the observer’s eye. As the rotor continues forward on its trajectory it is no longer able to reflect the excitation beam onto the slide and simultaneous to that, the optical axis is opened to permit long-lived luminescence to reach the detection apparatus or eye.

[0028] In the instance that a microscopist wishes to analyse a sample for a specific organism or biomolecule, it is necessary to attach a luminescent molecule that has a lifetime of at least 50 ps or longer. Ideally a lanthanide chelate with a lifetime of several hundred microseconds is utilized with the prior art device comprising rotor 10 to ensure that signal brightness is sufficient for easy detection of the desired target. There are many lanthanide chelates commercially available that can satisfy this role as discussed above.

[0029] Connally ‘390 also describes an auto-synchronous fluorescence detection embodiment comprising a chopper wheel (see Figures 21A and 21B of Connally ‘390) having a plurality of openings, however in these embodiments, the excitation source beam is not aligned with the axis of the observable fluorescence signal such that this embodiment is not suitable for integration into existing microscope devices which only permit a single optical pathway for both excitation and emission detection of fluorescent media.

[0030] Connally ‘390 also describes an auto-synchronous fluorescence detection embodiment comprising a planar reflective surface (see Figures 18A and 19A of

Connally ‘390). In each of the Connally ‘390 embodiments, however, the movement of the vanes between the excitation and detection states of the system result in the excitation beam being scanned across the face of the sample rather than directed to a single spot of the sample under test. The disadvantages of this is that the scanned

2018101853 29 Nov 2018 excitation beam will excite more auto-flourophores in the sample causing increased signal noise; and decrease the residence time of the excitation beam on the observable portion of the sample resulting the observable sample receiving reduced radiation needed to activate the luminescent probe. Also, designs utilising a moving planar surface suffer the disadvantage that the reflective face is positioned at the correct angle for exciting the fluorophores in the sample only at the point when the centre line of the reflector is aligned with the excitation source. At any other time in the motion of the reflective surface, the angle of incidence is not precisely 90 degrees with respect to the reflective face and therefore the excitation beam will not be correctly reflected into the objective of the microscope for viewing the excited fluorescence.

[0031] Therefore, there is a need for improved autosynchronous TGL systems that are simple to both implement and use.

Definitions [0032] The following definitions are provided as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. For the purposes of the present invention, additional terms are defined below. Furthermore, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms unless there is doubt as to the meaning of a particular term, in which case the common dictionary definition and/or common usage of the term will prevail.

[0034] For the purposes of the present invention, the following terms are defined below.

2018101853 29 Nov 2018 [0035] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element.

[0036] The term “about” is used herein to refer to quantities that vary by as much as 30%, preferably by as much as 20%, and more preferably by as much as 10% to a reference quantity. The use of the word ‘about’ to qualify a number is merely an express indication that the number is not to be construed as a precise value.

[0037] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

[0038] Any one of the terms “including” or “which includes” or “that includes” as used herein is also an open term that also means “including at least” the elements/features that follow the term, but not excluding others. Thus, including is synonymous with and means comprising.

[0039] In the claims, as well as in the summary above and the description below, all transitional phrases such as “comprising”, “including”, “carrying”, “having”, “containing”, “involving”, “holding”, “composed of’, and the like are to be understood to be open-ended, i.e. to mean “including but not limited to”. Only the transitional phrases “consisting of” and “consisting essentially of” alone shall be closed or semi-closed transitional phrases, respectively.

[0040] The term “real-time”, for example “displaying real-time data”, refers to the display of the data without intentional delay, given the processing limitations of the system and the time required to accurately measure the data.

[0041] The term “near-real-time”, for example “obtaining real-time or near-real-time data” refers to the obtaining of data either without intentional delay (“real-time”) or as close to real-time as practically possible (i.e. with a small, but minimal, amount of delay whether intentional or not within the constraints and processing limitations of the of the system for obtaining and recording or transmitting the data.

2018101853 29 Nov 2018 [0042] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. It will be appreciated that the methods, apparatus and systems described herein may be implemented in a variety of ways and for a variety of purposes. The description here is by way of example only.

[0043] Also, various inventive concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0044] The phrase “and/or”, as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined,

i.e. elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e. “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0045] As used herein in the specification and in the claims, “of should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e. the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of”, or, when used in the claims, “consisting of” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either”, “one of’, “only one of’, or “exactly one of”. “Consisting

2018101853 29 Nov 2018 essentially of”, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0046] As used herein in the specification and in the claims, the phrase “at least one”, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B”, or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0047] For the purpose of this specification, where method steps are described in sequence, the sequence does not necessarily mean that the steps are to be carried out in chronological order in that sequence, unless there is no other logical manner of interpreting the sequence.

[0048] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Summary of the Invention [0049] It is an object of the present invention to overcome or ameliorate at least one or more of the disadvantages of the prior art, or to provide a useful alternative.

[0050] According to a first aspect of the present invention, there is provided a time gated luminescence detection system. The system may comprise a housing enclosure.

2018101853 29 Nov 2018

The enclosure may comprise an optical exit aperture and an optical view aperture. The exit and view apertures may be located on opposing sides of the housing. The exit and view apertures may optically aligned with an aperture axis.

[0051] According to a particular arrangement of the first aspect, there is provided a time gated luminescence detection system comprising a housing enclosure, the enclosure comprising: an optical exit aperture and an optical view aperture located on opposing sides of the enclosure; wherein the exit and view apertures optically aligned with an aperture axis.

[0052] According to a further arrangement of the first aspect, there is provided a time gated luminescence detection system comprising: a housing enclosure comprising an optical exit aperture and an optical view aperture, said exit and view apertures being located on opposing sides of said enclosure and being optically aligned with an aperture axis; an optical excitation source mounted within said enclosure and configured to generate an optical excitation beam; and a planar rotor; said rotor comprising: a plurality of rotor blades each said rotor blade comprising at least a partially reflective portion; a rotational activation motor mounted within said enclosure for providing rotational motion to said rotor in a rotor plane to sequentially place said system between an excitation phase and a detection phase; and a reflector mounted within said housing enclosure, said reflector configured to receive an excitation beam generated by said optical source and further to direct said excitation beam to said at least partially reflective portion of a blade of said rotor when said system is in an excitation phase; wherein when said system is in said excitation phase, at least one blade of said rotor is positioned intermediate said exit and view apertures and aligned with said aperture axis to receive said excitation beam from said reflector and direct said excitation beam through the exit aperture to a sample location; and wherein in said excitation phase, said reflective portion is adapted to direct said optical excitation beam through said exit aperture to said sample location wherein at least a portion of said directed excitation beam is substantially aligned with said aperture axis.

[0053] The system may further comprise an optical excitation source mounted within the housing and configured to generate an optical excitation beam.

[0054] The system may further comprise a planar rotor. The rotor may comprise a plurality of rotor blades each rotor blade comprising at least a partially reflective portion.

2018101853 29 Nov 2018 [0055] The system may further comprise a rotational activation motor mounted within the housing. The motor may be configured for providing rotational motion to the rotor in a rotor plane to sequentially place the system between an excitation phase and a detection phase. In use, the motor may be adapted to spin the rotor at a rate of between about 8,000 revolutions per minute (rpm) to about 28,000 rpm.

[0056] The system may further comprise a reflector mounted within the housing. The reflector may be configured to receive an excitation beam generated by the optical source. The reflector may be adapted to direct the excitation beam to the at least partially reflective portion of a blade of the rotor when the system is in an excitation phase.

[0057] The rotor may comprise, 1, 2, 3, 4, 5, 6, 7, 8 9 10 or more equi-circumferentially spaced blades.

[0058] The reflector and the at least partially reflective portion of the blades may be highly reflective for ultraviolet, visible or infrared optical wavelengths. The reflector and the at least partially reflective portion of the blades may be highly reflective for optical wavelengths in the range of 150 nm to 2000 nm. The reflector and the at least partially reflective portion of the blades may be highly reflective for optical wavelengths in the range of 150 nm to 400 nm. The reflector and the at least partially reflective portion of the blades may be highly reflective for optical wavelengths in the range of 300 nm to 1000 nm. The reflector and the at least partially reflective portion of the blades may be highly reflective for optical wavelengths in the range of 800 nm to 2000 nm. The reflector and the at least partially reflective portion of the blades may be highly reflective for optical wavelengths in the range of 150 nm to 800 nm. The reflector and the at least partially reflective portion ofthe blades may be simultaneously reflective for a plurality of wavelengths and may be simultaneously reflective for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wavelengths. The reflector and the at least partially reflective portion of the blades may be selectively reflective for a plurality of wavelengths. The selective reflector and the at least partially reflective portion of the blades may comprise an optical coating suitable for providing selective reflectivity at a plurality of desired wavelength. The optical coating may be a multi-layer coating. The coating may be an interference coating. The reflector and the at least partially reflective portion of the blades may be configured to reflect a narrow band of the optical spectrum centred about the wavelength(s) the reflector is configured to reflect. The reflective characteristics of the reflector may be

2018101853 29 Nov 2018 different from the reflective characteristics ofthe at least partially reflective portion ofthe blades. The reflective characteristics of the reflector may be substantially equal to the reflective characteristics of the at least partially reflective portion of the blades. The bandwidth of the spectrum about the wavelength(s) which the reflector The reflective characteristics of the reflector may be different from the reflective characteristics of the at least partially reflective portion of the blades are configured to reflect may be in the range of 0.01 to 50 nm, 0.01 to 40, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, or 0.01 to 1, 0.01, 0.5, 0.01 to 0.1, 0.01 to 0.05, 0.05 to 50, 0.05 to 40, 0.05 to 30, 0.05 to 25, 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.05, 0.05 to 0.1, 0.1 to 50, 0.1 to 40, 0.1 to 30, 0.1 to 25, 0.1 to 20, 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, or 0.1 to 1, 0.1, 0.05, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 25, 0.5 to 20, 0.5 to 15, 0.5 to 10, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, or 0.5 to 1 nm, and may be about 0.01, 0.02, 0.03, 0.04, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nm or more.

[0059] According to a further particular arrangement of the first aspect, there is provided a time gated luminescence detection system comprising: a housing comprising an optical exit aperture and an optical view aperture, the exit and view apertures being located on opposing sides of the housing and being optically aligned with an aperture axis; an optical excitation source mounted with the housing and configured to generate an optical excitation beam; a planar rotor; the rotor comprising: a plurality of rotor blades each the rotor blade comprising at least a partially reflective portion; a rotational activation motor mounted within the housing for providing rotational motion to the rotor in the rotor plane to sequentially place the system between an excitation phase and a detection phase; a reflector mounted within the housing, the reflector configured to receive an excitation beam generated by the optical source and direct the excitation beam to a reflecting portion of a blade of the rotor when the system is in an excitation phase.

[0060] When the system is in the excitation phase, at least one blade of the rotor may be positioned intermediate the exit and view apertures and may be aligned with the aperture axis to receive the excitation beam from the reflector and direct the excitation beam through the exit aperture to a sample location.

2018101853 29 Nov 2018 [0061] In the excitation phase, the reflective portion may be adapted to direct the optical excitation beam through the exit aperture to the sample location wherein at least a portion of the directed excitation beam may be substantially aligned with the aperture axis.

[0062] When the system is in the detection phase, none of the plurality of blades of the rotor may be positioned intermediate the exit and view apertures. Additionally, when the system is in the detection phase none of the plurality of blades of the rotor may be aligned with the aperture axis such that an optical signal originating from the sample location may be permitted to be viewed by an observer at an observer location through the exit and view apertures.

[0063] The system may further comprise a rotor sensor mounted within the enclosure and positioned so as to sense the presence or absence of each rotor blade as it passes adjacent the sensor. When the sensor detects the presence of a rotor blade, the sensor may generates a synchronisation signal. The system may further comprise a microcontroller adapted to receive the synchronisation signal from the sensor and to provide a switching signal to the excitation source. The switching signal may be adapted to turn the excitation source to an active state for generation of the excitation beam whenever a rotor blade is positioned intermediate the exit and view apertures and aligned with the aperture axis to receive the excitation beam and direct the excitation beam through the exit aperture to the sample location.

[0064] The microcontroller may be adapted to switch the excitation source to an active state for generation of the excitation beam such that a portion of the excitation beam is incident upon an blade edge of one or more of the plurality of rotor blades. The blade edge may comprise one or more of either a leading edge, or a trailing edge of one or more of the rotor blades.

[0065] When a portion of the excitation beam may be incident upon a blade edge of one or more of the plurality of rotor blades such that the excitation beam portion may also be incident upon the sample location, the autofluorophores present in a sample at the sample location may be excited to generate a fluorescence signal, wherein a portion of the fluorescence signal may be visible to an observer at the observer location during the excitation phase of the system.

2018101853 29 Nov 2018 [0066] The enclosure may be adapted to be coupled to a microscope device. The microscope device may comprise an optical microscope axis. When coupled to the microscope device, the aperture axis may be substantially aligned with the microscope axis. The microscope device may be a fluorescence microscope device.

Brief Description of the Figures [0067] Notwithstanding any other forms which may fall within the scope of the present invention, a preferred embodiment I preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1A shows a graph depicting a time-resolved fluorescence detection system;

Figure 1B shows a graph depicting a time-gated fluorescence detection system;

Figure 2A shows a rotor unit of a prior art autosynchronous fluorescence detection system comprising an arcuate reflector surface;

Figure 2B shows a schematic depiction of the excitation beam spot size obtained with the rotor unit of a prior art autosynchronous fluorescence detection system of Figure 2A.

Figure 3 shows a schematic representation of an improved autosynchronous TGL system according to an embodiment 100 of the invention as disclosed herein;

Figure 4A shows a schematic representation of an excitation phase of the autosynchronous TGL system of Figure 3;

Figure 4B shows a schematic representation of a detection phase of the autosynchronous TGL system of Figure 3;

Figure 5 shows an underside view of a rotor according to an embodiment of the invention as disclosed herein;

Figure 6 shows a representative graph of sensor synchronisation signal and power signal to the excitation source according to an embodiment of the invention as disclosed herein; and

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Figure 7 shows a rendered longitudinal cross-sectional view of the gated auto-synchronous luminescence detector system 100 disclosed herein.

Detailed Description [0068] It should be noted in the following description that like or the same reference numerals in different embodiments denote the same or similar features.

[0069] Referring to Figure 3, disclosed herein is a time gated luminescence detection system 100 comprising a housing enclosure 101 comprising an optical exit aperture 160 and an optical view aperture 165, the exit and view apertures (160 and 165) being located on opposing sides of enclosure 101 and being optically aligned with an aperture axis 181. System 100 further comprises an optical excitation source 110 mounted within enclosure 101 and configured to generate an optical excitation beam 111. System 100 further comprises a planar rotor 150 comprising a plurality of rotor blades 151 each rotor blade 151 comprising at least a partially reflective or highly reflective portion 155 (refer to Figure 5). System 100 further comprises a rotational activation motor 105 mounted within enclosure 101 for providing rotational motion to rotor 150 in a rotor plane to sequentially place system 100 between an excitation phase (Figure 4A) and a detection phase (Figure 4B). In particular arrangements, for example, as depicted in Figure 3, rotor plane is inclined within enclosure 101 at an angle of about 22.9° below the horizontal, however it will be appreciated by the skilled addressee that the specific inclination angle will be dependent upon the angle of inclination of other components with enclosure 101. For instance, in the particular arrangement depicted in Figure 3 a rotor plane inclination angle of about 22.9° corresponds with an inclination angle of about 19° below the horizontal of optical source 110 when coupled with an internal planar reflector 120 positioned on a reflector plane inclined at about 12.5° above the horizontal within enclosure 101. It will also be appreciated that the horizontal distance between the internal housing components will also dictate the inclination angle of the rotor plane and the other internal components in a conventional manner in order to ensure the excitation beam 111 is aligned with the optical aperture axis 181 when exiting housing 111 through exit aperture 160.

[0070] In use, the motor 105 of the embodiments disclosed herein is typically configured to spin the rotor at a rate of between about 8,000 revolutions per minute (rpm) to about 28,000 rpm. In the presently disclosed arrangement wherein the rotor

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150 of Figure 5 comprises two rotor blades 151, in use, the system e performs two excitation/detection cycles per each full rotation of rotor 150. For example, at a rotation speed of about 467 rotations per second (-28,000 rpm), the system 100 performs 933 excitation/detection cycles per second or 1.07 milliseconds per time gated luminescence detection operation.

[0071] System 100 further comprises a reflector 120 mounted within enclosure 101, reflector 120 configured to receive excitation beam 111 generated by optical source 110 and further to direct excitation beam 111 to at least partially reflective portion 155 of a blade 151 of rotor 150 when the system is in an excitation phase. Reflector 120 in the present arrangements is a planar reflector, however in further arrangements, reflector 120 may be provided with a prescribed curvature adapted, for example, to modify the beam spot profile of excitation beam 111 from source 110. For instance, source 110 may comprise an LED source which may generate an excitation beam 111 has an elliptical profile. Reflector 120 may be provided with a prescribed curvature in order to modify the beam profile to be more uniformly circular. As will be appreciated, a curved reflector may have either a spherical or aspherical curvature in accordance with requirements and may have different curvatures along perpendicular axes of the mirror plane (particularly when used to circularize an elliptical beam from LED source 110).

[0072] As will be described in further detail below, the system 100 shown in Figure 3 provides an improved autosynchronous TGL system 100 according to an embodiment of the invention as disclosed herein.

[0073] System 100 comprises a housing enclosure 101. Enclosure 101 is adapted to be compatible for insertion into a filter slot present on existing fluorescence microscopes such as, for example, the BX51 model of microscope provided by Olympus. Of course, it will be appreciated that variations in the system 100 and particularly enclosure 101 may be provided to ensure compatibility with alternative microscope options from other manufacturers.

[0074] Mounted within enclosure 101 of system 100, a motor 105 is configured such that a rotor 150 mounted to motor 105 is rotated about rotation axis 107 when in operation.

2018101853 29 Nov 2018 [0075] As can be seen in Figure 5 showing an underside view of rotor 150, rotor 150 is a planar disk comprising a plurality of blades 151. Mounted upon each blade 151 of rotor 150 there is provided a planar reflective surface 155. Rotor 150 is configured to be centrally mounted to motor 105 and thus to rotate about central rotor rotation axis 107. Referring back to Figure 3, an excitation beam 111 from an optical excitation source 110 mounted in enclosure 101 - when reflected off planar reflective surface 155 of rotor 150 onto a sample 121 for analysis located at sample location 120 such as a microscope sample slide - does not experience the spatial dispersion (of the excitation beam 111 at sample location 120) seen in the prior art with arcuate reflecting surfaces (e.g. surfaces 13 of Connally prior art rotor device 10 seen in Figure 2A). Also mounted within enclosure 151 of system 100 is a lens 130 configured to collimate excitation beam 111 from excitation source 110, and direct it to sample location 120 via intermediate reflections from first mirror 140 and reflective surfaces 155 of rotor 150 and to exit enclosure 101 through exit aperture 160, and onto sample location 120. Similarly with respect to reflector 120, lens 130 may be either a spherical or aspherical lens, and may have different optical focussing power along perpendicular axes of the lens surface for use, for example, to assist with circularizing an elliptical beam 111 from a LED source 110.

[0076] As can be seen in Figure 5, rotor 150 also optionally comprises guard bands 157. Guard bands 157 may be absorbing, non-reflective or both absorbing and non-reflective with respect to excitation beam 111 from optical source 110. Use of guard bands 157 enables the use of a continuous wave optical excitation source 110 in particular arrangements of system 100 in accordance with requirements. Effectively, the use of the guard bands 157 autonomously turns a continuous wave excitation source 110 into a quasi-continuous-wave source which is automatically synchronised with the excitation state(s) of the rotor 150.

[0077] By reflecting the excitation beam from planar reflective surfaces 155 of rotor 150 the problem of dispersion related to reflection from an arcuate surface (such as curved reflector 13 of prior art rotor device 10) is overcome in the arrangements of the autosynchronous fluorescence detection systems disclosed herein.

[0078] System 100 further comprises a rotor sensor 170 for synchronising the rotor rotation with the excitation beam 111 generated by excitation source 110. Excitation

2018101853 29 Nov 2018 source 110 is preferably an ultraviolet (UV) LED source particularly adapted for emitting excitation beam 111 having an optical wavelength particularly tuned for absorption by the fluorescent probe marker molecules used to label particular constituents in a sample placed at sample location 120. However, the excitation source may be adapted to output an emission beam having any suitable wavelength for efficient activation of probe molecules for a particular application in detecting a desired organism or impurity in a sample 121.

[0079] In use, excitation source 110 is configured to emit an excitation beam 111 to excite the fluorescent probes a sample as sample location 120. In particular arrangements, rotor sensor 170 is configured to provide a synchronisation signal between rotor 150 and excitation source 110. For example, sensor 170 in particular embodiment is configured to provide an “ON” signal (for example, a sensor “low” voltage, 0) to source 110 whenever one of the planar reflective surface 155 associated with each of the plurality of blades 151 or rotor 150 is in optical alignment with the excitation communication path 115 between source 110 and sample location 120 (i.e. as depicted in Figure 4A). Further, sensor 170 is configured to provide an “OFF” signal (for example, a sensor “high” voltage, O) to source 110 whenever the planar reflective surfaces 155 associated with each of the plurality of blades 151 or rotor 150 is not in optical alignment with the excitation communication path 115 between source 110 and sample location 120 (i.e. as depicted in Figure 4B).

[0080] It is essential to have very accurate (microsecond) control of the UV LED excitation source 110 with relation to the position of rotor blades 151 to enable the system 100 to operate in time-gated luminescence mode. It is also possible for the instrument to very quickly switch into conventional fluorescence mode by changing the switching timing of excitation source 110 so that some of the excitation beam 111 is incident upon an edge (either leading or trailing edges, or both) to be reflected off the partially closed or open rotor blades. This effect is very useful for switching out of time-gated mode for a brief interval (say 100 ms) to see if a cell nucleus is present as a small portion of the excitation beam 111 reflected from a blade edge would be incident upon the sample 121 to excite any autofluorophores or florescent probe molecules present in sample 121 which would thus be visible to an observer at observer location 200. In a typical arrangement, cells of sample 121 under test would be stained with a fluorescent marker such as a dye (for example, DAPI) that makes DNA glow blue under

2018101853 29 Nov 2018

UV excitation such that the nucleus of a cell in sample 121 can be identified. Due to its short fluorescence lifetime, this blue glow would only be visible in non-TGL mode.

Therefore, the ability to rapidly switch out of TGL-mode into conventional fluorescence mode to check that the sample 121 under examination has a nucleus present therein is a particularly useful feature of the presently disclosed system 100.

[0081] Secondly, in typical arrangements, the excitation source is chosen to be a high-power UV LED device capable of delivering up to about 3W of UV output for an electrical input to excitation source 110 input of about 4A at about 3.6V. This corresponds to a power input of about 14 Watts in a very small source device 110. Since, in normal operation according to the embodiments and arrangements disclosed here, the UV LED source 110 is pulsed for about 1/3 of each rotation cycle of rotor 150, the amount of heat required to be dissipated by the enclosure 101 is only of the order of about 4.2 W which can safely be dissipated in the enclosure 101 of system 100 when the enclosure is formed from a suitably thermally conducting material such as aluminium or the like.

[0082] A representation of the sensor synchronisation signal 501 from sensor 170 and power signal 503 to the excitation source 110 in a particular arrangement is shown in Figure 6.

[0083] Excitation beam is collimated by lens 130. In alternate arrangements, lens 130 may be particularly adapted to focussing of excitation beam 111 from source 110 onto the sample location 120 such that a sample 121 receives an increased optical power density (i.e. optical power per unit area, W/cm²). Collimated (or focussed) beam 111 is initially directed onto first internal mirror 140 to be reflected upwards towards the underside of rotor 150 where reflective surface 155 are mounted on each blade 151 or rotor 150. The beam is reflected from mirror 140 at an appropriate angle to ensure that it strikes reflective surface 155 are mounted on each blade 151 or rotor 150 for reflection through exit aperture 160 and to sample 121 located on e.g. a microscope slide positioned at sample location 120.

[0084] Figure 3 includes optimised measurements for the angles of first mirror 140 and rotor 150 (spinning about motor axis 107 in operation) for a particular embodiment of the invention disclosed herein to ensure that excitation beam 111 from source 110 is configured to illuminate sample 121 whenever each of the plurality of reflective surface

2018101853 29 Nov 2018

155 are aligned with the optical communication path 115. The particular arrangement shown is configured to ensure that the final portion of the path 115 of excitation beam 111 is substantially perpendicular to sample location 120 and such that it is aligned with the aperture axis 181 of system 100 and the optical axis 181 of a microscope located at observer location 200. This is to ensure that the system is compatible with the typical fluorescence microscopes available which generally utilise the same optical pathway for both excitation of fluorophores in the sample as well as detection of the resulting fluorescence signal. In such microscope devices, the objective (e.g. objective 190 as seen in Figures 4A and 4B) serves to both focus the excitation beam into the tiny region of a sample under observation and also to focus and/or magnify the resulting fluorescence to make it visible to the observer. The microscope objective lens 190 (as seen in Figures 4A and 4B) of the microscope used for observing the fluorescence signals from the sample 121 defines the optical axis 181 of the microscope and is adapted to receive optical fluorescence signals emitted by sample 121 in response to the stimuli from absorption of excitation beam 111 by auto-fluorescing organisms and fluorescent probe molecules in sample 121.

[0085] The optical fluorescence signals emitted by sample 121 in response to the stimuli from absorption of excitation beam 111 includes short-lived auto-fluorescence 123 arising from autofluorophores in sample 221. Optical fluorescence signals emitted by sample 121 also included the.

[0086] In use, motor 105 rotates rotor 150 such that system 100 alternates between an excitation phase as seen in Figure 4A and a detection phase as seen in Figure 4B.

[0087] During the excitation phase of system 100, shown in Figure 4A, the blade 151 of rotor 150 comprising the particular planar reflective surface 155 involved in directing excitation beam 111 to the sample 121 is aligned with the optical path 115 intermediate the excitation source 110 and sample location 120. In this position blade 151 of rotor 150 also occludes view aperture 165 such that no optical signals originating from sample 121 at sample location 120 are able to enter microscope objective 190 such that it is visible at observer location 200.

[0088] During the detection phase of system 100, shown in Figure 4B, the blade 151 of rotor 150 has moved out of position occluding view aperture 165 such that reflective surface 155 has moved out of position so that no excitation light 111 from source 110 is

2018101853 29 Nov 2018 directed onto sample 121. System 100 may optionally include a beam dump 109 to trap any excess excitation light 111 from source 110 while system 100 is in a detection phase.

[0089] Also in the detection phase, optical signals originating from sample 121 at sample location 120 are now able to enter microscope objective 190 such that they are visible to an observer at observer location 200.

[0090] Due to the rapid decay of the auto-fluorescence signal 123, when system 100 is in an excitation phase, substantially all to 100% of the auto-fluorescence signal 123 is blocked from reaching microscope 190 and observer location 200 by the blade 151 of rotor 150 comprising the particular planar reflective surface 155 involved in directing excitation beam 111 to the sample 121. Then, as the rotor rotates out of position blocking the observer location 200 the system 100 moves into a detection phase whereby optical signals from sample 121 are visible by an observer at location 200 through microscope 190, the auto-fluorescence signal 123 has decayed to be significantly less than any long-lived fluorescence/luminescence 125 of probe molecules present in sample 121. In this manner, the only optical signals from sample 121 that are permitted to reach microscope 190 and observer 200 are the long-lived fluorescence/luminescence 125 of probe molecules present in sample 121 thus enabling detection of the desirable tagged molecules of interest present in sample 121.

[0091] In a particular construction arrangement of system 100, rotor 150 can be made to be about 30 mm in diameter but only about 0.2 mm thick, thus effectively reducing the mass of rotor 150 to about 480 mg. Note that a rotor 150 with this mass compares with a rotor only about 13% of the weight of prior art rotor device 10 of about 3.5 g. The modified design of rotor 150 enable use of commercial DC motor 105 to drive rotor 150 rather than the more complicated system of the prior art comprising use of coils acting on magnets embedded into the prior art rotor 10. This results in a significantly more compact, vibration free assembly of system 100 also possessing the further significant advantage that the excitation beam experiences no adverse spatial dispersion when incident upon the sample of interest.

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Example Arrangement of Improved Gated Auto-Synchronous Luminescence Detector [0092] As discussed above, Figure 3 shows a schematic depiction of the improved Gated Auto-Synchronous Luminescence Detector (GALD) system 100. System 100 in a present exemplary arrangement is also shown in rendered longitudinal cross-sectional view in Figure 7. As noted above, the enclosure 101 of system 100 is configured to be compatible with the differential interference contrast (DIC) filter slot present on existing fluorescence microscopes such as the Olympus BX51 model microscope devices. Accordingly, due to the very limited space available within the enclosure 101, it is necessary to mount the excitation source 110, for example a UV LED device, at an angle within enclosure 101 and partially intrude into the top plate of enclosure 101 to achieve the necessary orientation. If the angle of the UV LED source 110 is smaller than about 19°below horizontal, the excitation beam 111 will not be in alignment with the optical axis 181 of the microscope objective lens 190 located at observer location 200. If the excitation beam 111 incident on the sample and the detection of the resulting fluorescence signals from sample 121 are not aligned with the optical axis 181 of the microscope objective lens 190, then the system would not be compatible with the typical microscope devices used for fluorescence detection. If the excitation and detection pathways were separated, then two discrete optical systems would be required for the two optical pathways, each of which would require independent alignment. As would be appreciated, this would result in significantly increased cost and operating complexity to any fluorescence detection system. Any deviation from the mounting angles for each of the excitation source 110, first mirror 120 and inclination of rotor 150 tends to force the position of first mirror 120 further forwards to the point where it begins to occlude the exit aperture 160 which is clearly an undesirable configuration.

[0093] Motor 105 of the current embodiment is preferentially chosen to be a very low-profile motor device, for example, a motor manufactured by Maxon Motors (Switzerland), motor model: EC10; part number: 302000. Motor 105 is supported by a machined mounting block 108 which is mounted within enclosure 101 such that the inclination angle of rotor 150 is configured to reflect excitation beam 111 along microscope optical axis 181 to sample location 120 when blades 151 are in the excitation phase position. The motor support in the present arrangement is also configured to mount synchronisation sensor 170 which may be, for example, an

2018101853 29 Nov 2018 infra-red retro-reflective sensor. In the present arrangement, in use, the sensor 170 projects light up to the surface of the blades 151 of rotor 150. In the present arrangement rotor blade 150 is formed from stainless steel. Although not polished, the blades 151 of rotor 150 are sufficiently reflective to trigger the sensor 170 to generate a synchronisation signal as the periphery of the rotor blades 151 rotates into position near sensor 170. On detection of a return signal, sensor 170 generate a synchronisation signal such as, for example, a logic LOW signal, whenever a rotor blade 151 segment is situated near the sensor and this allows a microcontroller (not shown) to deduce the position of the rotor blades 151 and thereby synchronize the UV LED excitation source 110 to switch on at the correct time.

[0094] Notwithstanding the above, it is still possible to use a constant source of illumination (continuous wave excitation source 110) and rely upon non-reflective guard bands 157 (as shown in Figure 5) at the leading and trailing edges of the rotor blades 151 to automatically synchronize the excitation phase of system 100 in operation with the detection phase and a gated phase where the prompt autofluorescence 123 excited in sample 121 is blocked from reaching view aperture 165 and observer 200 by guard bands 157. Alternate methods of embodying guard bands 157 to that shown in Figure 5 are also possible to generate ‘virtual’ guard bands, for example, the black non-reflective regions e on the edges of the rotor blades 151 of the presently disclosed embodiments of rotor 150, could be replaced with bevelled regions 157 configured so as to reflect the excitation beam 111 away from the principal optical aperture axis 181. Alternatively, virtual guard bands may be implemented in the drive software for the excitation source 110, which may, for example be implemented by accurately controlling the LED on and off switching timing.

[0095] A useful feature of the embodiment described in Figure 7 whereby the position of rotor 150 is known with high accuracy and the UV LED source 110 is activated by a microcontroller (not shown) arises from the ability to switch the UV LED source 110 on prior to the rotor blade 151 fully occluding the aperture 160. This can be done as the rotor blade 151 advances across the aperture 160 (closing of the aperture) and similarly as the rotor blade 151 trailing edge exits (opening of the aperture 160). The resultant effect is to scatter light in excitation beam 111 from the trailing or receding edges of the rotor blades 151 whilst the aperture 160 is partly open and the sample 121 under analysis is visible to the observer 200. This scattered light permits the observer 200 to

2018101853 29 Nov 2018 briefly see a small amount of short-lived fluorescence 123 as well as the long-lived phosphorescence 125. The microcontroller permits fine adjustment of the switching timing of excitation source 110 relative to the position of the blades 151 of rotor 150 so the effect (visible short-lived fluorescence 123) can be as large or small as required.

[0096] It is the nature of time-gated luminescence imaging that in the absence of a target (labelled organism or biomarker) being present in a sample 121 under test, the observer 200 will not be able to see anything through the microscope since the field of view is uniformly devoid of light. This effect can be particularly disconcerting to a microscopist manually examining a sample 121 for the presence of a labelled pathogen or biomarker since it is unclear if the microscope is still in focus and working properly since nothing is visible, or if indeed there really is nothing in the sample to see. The ability to permit a low level of light leakage of the excitation beam 111 to render a small amount of visible short-lived fluorescence 123 by the aforementioned method is a valuable means of providing visual feedback to the microscopist that everything is working correctly and the microscope is correctly focussed on the sample location 120.

[0097] The fine control of the timing of excitation source 110 also provides another valuable feature to the microscopist. Photobleaching is a well-known phenomenon whereby fluorescent agents (both natural and synthetic) lose their activity (brightness) due to photolytic damage. In those instances when a target cell has relatively few luminescent molecules attached to it, fading can occur within seconds using conventional fluorescence microscopy. This effect is greatly ameliorated in the system 100 disclosed herein by switching the UV LED source 110 for only a small duration of time, such that the ON time is a small fraction of the total time between switching events. The normal relationship between the IR sensor signal 501 and the UV LED source switching current 503 is shown in Figure 6, wherein the LOW state of signal 501 for the IR sensor 170 indicates detection of the presence of a blade 151 of rotor 150 in position for an excitation phased of system 100 as discussed above; and the LOW state for the UV switching waveform 503 indicates the UV LED source 110 is ON. In this waveform capture it can be seen that under normal conditions the UV LED has a duty cycle of about 33%. The duty cycle can be reduced to about 10% with only a relatively small drop in observed brightness, but with a substantially reduced risk of photobleaching the target.

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Interpretation

In Accordance With [0098] As described herein, ‘in accordance with’ may also mean ‘as a function of and is not necessarily limited to the integers specified in relation thereto.

Embodiments [0099] Reference throughout this specification to “one embodiment”, “an embodiment”, “one arrangement” or “an arrangement” means that a particular feature, structure or characteristic described in connection with the embodiment/arrangement is included in at least one embodiment/arrangement of the present invention. Thus, appearances of the phrases “in one embodiment/arrangement” or “in an embodiment/arrangement” in various places throughout this specification are not necessarily all referring to the same embodiment/arrangement, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments/arrangements.

[0100] Similarly, it should be appreciated that in the above description of example embodiments/arrangements of the invention, various features of the invention are sometimes grouped together in a single embodiment/arrangement, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment/arrangement. Thus, the claims following the Detailed Description are hereby expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment/arrangement of this invention.

[0101] Furthermore, while some embodiments/arrangements described herein include some but not other features included in other embodiments/arrangements, combinations of features of different embodiments/arrangements are meant to be within the scope of the invention, and form different embodiments/arrangements, as would be

2018101853 29 Nov 2018 understood by those in the art. For example, in the following claims, any of the claimed embodiments/arrangements can be used in any combination.

Specific Details [0102] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

Terminology [0103] In describing the preferred embodiment of the invention illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar technical purpose. Terms such as “forward”, “rearward”, “radially”, “peripherally”, “upwardly”, “downwardly”, and the like are used as words of convenience to provide reference points and are not to be construed as limiting terms.

Different Instances of Objects [0104] As used herein, unless otherwise specified the use of the ordinal adjectives “first”, “second”, “third”, etc., to describe a common object, merely indicate that different instances of like objects are being referred to, and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

Scope of Invention [0105] Thus, while there has been described what are believed to be the preferred arrangements of the invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the scope of the invention. Functionality may be added or deleted from the block

2018101853 29 Nov 2018 diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.

[0106] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Industrial Applicability [0107] It is apparent from the above, that the arrangements described are applicable to the mobile device industries, specifically for methods and systems for distributing digital media via mobile devices.

[0108] It will be appreciated that the methods and systems described/illustrated above at least substantially provide a time gated luminescence detection system.

[0109] The time gated luminescence detection system described herein, and/or shown in the drawings, are presented by way of example only and are not limiting as to the scope of the invention. Unless otherwise specifically stated, individual aspects and components of the a time gated luminescence detection system may be modified, or may have been substituted therefore known equivalents, or as yet unknown substitutes such as may be developed in the future or such as may be found to be acceptable substitutes in the future. The time gated luminescence detection system may also be modified for a variety of applications while remaining within the scope and spirit of the claimed invention, since the range of potential applications is great, and since it is intended that the present time gated luminescence detection system be adaptable to many such variations.

Claims (5)

1. A time gated luminescence detection system comprising:

a housing enclosure comprising an optical exit aperture and an optical view aperture, said exit and view apertures being located on opposing sides of said enclosure and being optically aligned with an aperture axis;

an optical excitation source mounted within said enclosure and configured to generate an optical excitation beam; and a planar rotor; said rotor comprising:

a plurality of rotor blades each said rotor blade comprising at least a partially reflective portion;

a rotational activation motor mounted within said enclosure for providing rotational motion to said rotor in a rotor plane to sequentially place said system between an excitation phase and a detection phase; and a reflector mounted within said housing enclosure, said reflector configured to receive an excitation beam generated by said optical source and further to direct said excitation beam to said at least partially reflective portion of a blade of said rotor when said system is in an excitation phase;

wherein when said system is in said excitation phase, at least one blade of said rotor is positioned intermediate said exit and view apertures and aligned with said aperture axis to receive said excitation beam from said reflector and direct said excitation beam through the exit aperture to a sample location; and wherein in said excitation phase, said reflective portion is adapted to direct said optical excitation beam through said exit aperture to said sample location wherein at least a portion of said directed excitation beam is substantially aligned with said aperture axis.

2. A system as claimed in Claim 1, wherein, when said system is in said detection phase, none of said plurality of blades of said rotor are positioned intermediate said exit and view apertures, and none of said plurality of blades of said rotor are aligned with said aperture axis such that an optical signal originating from said sample location is permitted to be viewed by an observer at an observer location through said exit and view apertures.

3. A system as claimed in either Claim 1 or Claim 2, further comprising a rotor sensor mounted within said enclosure and positioned so as to sense the presence or

2018101853 29 Nov 2018 absence of each said rotor blade as it passes adjacent said sensor, such that, when the sensor detects the presence of a rotor blade, the sensor generates a synchronisation signal.

4. A system as claimed in Claim 3, further comprising:

a microcontroller adapted to receive said synchronisation signal from said sensor and to provide a switching signal to said excitation source;

wherein said switching signal is adapted to turn said excitation source to an active state for generation of said excitation beam whenever a rotor blade is positioned intermediate said exit and view apertures and aligned with said aperture axis to receive said excitation beam and direct said excitation beam through said exit aperture to said sample location.

5. A system as claimed in Claim 4, wherein said microcontroller is adapted to switch said excitation source to an active state for generation of said excitation beam such that a portion of said excitation beam is incident upon an blade edge of one or more of said plurality of rotor blades; wherein said blade edge comprises one or more of either a leading edge, or a trailing edge of one or more of said rotor blades.