Imaging device

Abstract

A fluorescence lifetime imaging device for imaging the fluorescent output of a target fluorophore at an imaged subject spaced from the imaging device by a spacing. The imaging device comprises a light source configured to emit excitation light for fluorescent excitation of the target fluorophore. A first optical sensor is configured to detect returned excitation light scattered from the imaged subject, and a second optical sensor is configured to detect fluorescent light emitted by the fluorophore in response to excitation thereof by the excitation light, and to output a corresponding fluorescence intensity detection output. An image data generating unit is configured to generate data describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of said detection of returned excitation light corresponding to a time-of-flight of excitation light across said spacing between the light source and the imaged subject.

Description

[0001] IMAGING DEVICE

[0002] Field of the Invention

[0003] The present invention relates to imaging devices, and particularly, although not exclusively, to imaging device for fluorescence lifetime imaging, such as on distant objects and / or not-flat objects.

[0004] Background

[0005] Fluorescence lifetime imaging exploits the knowledge that a fluorescence lifetime of a fluorescent molecule is a measure of how long the molecule (known as a fluorophore) remains in its excited state before returning to the ground state by emitting a fluorescence photon. The emission of a fluorescence photon is a quantum mechanical process that is inherently random and occurs at a time within a probability distribution of times that can be described by an exponentially decaying mathematical function. The characteristic time constant of this decay is known as the fluorescence lifetime. Such lifetimes are in the range of a few picoseconds (1-10 ps) to several tens of nanoseconds (10-100 ns). Fluorescence lifetime imaging combines many fluorescence lifetime measurements made across a spatially distributed subject, into a single image of lifetimes whereby each image pixel may be encoded (e.g., by pixel colour and / or intensity) to produce a spatially distributed (i.e., two-dimensional) image showing a map of the spatial distribution of a fluorescent molecule.

[0006] Methods for imaging fluorescence lifetimes employ, for example, time-of-flight (ToF) imaging via a camera. ToF cameras typically drive LEDs with a sinusoidal light intensity modulation to illuminate an imaged subject. A phase shift between the intensity modulation of the illumination light and returned light from the illuminated sample is used for distance measurement. An example is disclosed by Ayush Bhandari et al. in: Optica Vol.2, No.11 , November 2015, p965: “Blind and reference-free fluorescence lifetime estimation via consumer time-of-flight sensors". This describes a method of using a ToF camera for imaging fluorescence lifetimes in which two categories of device are used for fluorescence imaging: one working in the time domain, and one working in the frequency domain. Patent application document US2015 / 0173621 A1 describes the use of a CMOS fluorescence imaging chip within the frequencydomain method, to detect skin cancer.

[0007] Homulle H. A. R. et al. in: Biomed Opt. Express, 11 April 2016, pp1179-814: “Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements." describe a prototype device for fluorescence-guided surgical oncology using low resolution photon synchronous detection imaging. Here, excitation light is collimated with fluorescence light and separated by a dichroic mirror.

[0008] In an article by David S. Kittle et al. in Nature Scientific Reports; 8 December 2016, “Realtime optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation" there is a discussion of the importance of the time-resolved fluorescence spectroscopy for differentiation of tumours and normal (non-tumour) tissue during surgical procedures. This article discusses the importance of time resolution needed to resolve mixed organic fluorescence dyes. Lorenzo Palombi, et al. describe a LIDAR sensor in OPTICS EXPRESS, 17 June 2013, Vol. 21 , No. 12, p14736: “A fluorescence LIDAR sensor for hyper-spectral time-resolved remote sensing and mapping” . This LIDAR sensor is described as being used as an optical spectrometer.

[0009] These disclosures indicate to the inventors the need to improve sensitivity and robustness of devices for fluorescence imaging. The present invention has been devised in light of the above considerations.

[0010] Summary of the Invention

[0011] In a first aspect, the invention may provide a fluorescence lifetime imaging device for imaging the fluorescent output of a target fluorophore at an imaged subject spaced from the imaging device by a spacing, wherein the imaging device comprises: a light source configured to emit excitation light for fluorescent excitation of the target fluorophore; a first optical sensor configured to detect returned excitation light scattered from the imaged subject; a second optical sensor configured to detect fluorescent light emitted by the fluorophore in response to excitation thereof by the excitation light, and to output a corresponding fluorescence intensity detection output; an image data generating unit configured to generate data describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of said detection of returned excitation light corresponding to a time-of-flight of excitation light across (e.g., back and forth) said spacing between the light source and the imaged subject. For example, the time-of-flight may be a time-of-flight from the light source to the second optical sensor via the imaged subject (i.e., a round trip time) or may be a time-of-flight from the light source to the imaged subject (i.e., outward trip time).

[0012] In this way excitation light may illuminate, bathe or irradiate on area of a distant subject containing a target fluorophore(s), and fluorescence intensity detection outputs may be appropriately timed or time- adjusted according to a time of occurrence of detection of returned excitation light so that account can be taken of a time interval between emission of excitation light towards the target fluorophore and subsequent fluorescent light emission in response to absorption of the excitation light. Consequently, the time of initial absorption of the excitation light by the target fluorophore(s) can be determined whatever the size of the spacing between the imaging device and the target fluorophore(s). Fluorescence characteristics of the target fluorophore(s), such as a fluorescence lifetime or a decay constant / rate, may be determined accurately even when the size of the spacing between the imaging device and the target fluorophore(s) changes over time.

[0013] The device may be implemented as a portable device or wearable device operable to observe an environment surrounding a user / wearer. The device may be applied to fluorescence imaging, fluorescence lifetime imaging, fluorescence lifetime imaging microscopy, time-of-flight imaging, or timegated imaging etc.

[0014] The first optical sensor may comprise a first optical filter arranged such that returned excitation light scattered (e.g., by diffuse and / or specular reflection) by the subject may be detected after first passing through the first optical filter, which may be configured with an optical pass characteristic (e.g., pass band, low-pass or high-pass) encompassing wavelengths of light corresponding to a fluorescence excitation wavelength of the target fluorophore(s), but excluding wavelengths of light corresponding to a fluorescence emission wavelength of the target fluorophore(s). This provides that the first optical sensor may be responsive to returned excitation light scattered by the subject and insensitive to light generated by fluorescence emission from the target fluorophore(s).

[0015] The second optical sensor may comprise a second optical filter arranged such that returned fluorescence light emitted (e.g., by a fluorescence process of the fluorophore(s)) by the subject may be detected after first passing through the second optical filter, which may be configured with an optical pass characteristic (e.g., pass band, low-pass or high-pass) encompassing wavelengths of light corresponding to a fluorescence emission wavelength of the target fluorophore(s), but excluding wavelengths of light corresponding to a fluorescence excitation wavelength of the target fluorophore(s). This provides that the second optical sensor may be responsive to fluorescence light generated by fluorescence emission from the target fluorophore(s) and insensitive to returned excitation light scattered by the subject.

[0016] The device could be used to target any fluorophore. The construction may require only to change a filter and / or the light source. Examples of possible applications include, but are not limited to:

[0017] - Medical applications, tumour / cancer screening, minimization of cutting area during surgery, bacteria or fungi disease detection.

[0018] - Environmental applications, pollution visualisation, bacteria and fungi visualisation. Forensic applications, drug visualisation or other chemical markers visualisation, low content blood or semen visualisation.

[0019] - Tumour / cancer tissue imaging for fluorescence-guided surgical oncology and for cancer screening application. This includes use in hospitals and medical clinics.

[0020] - Crop disease monitoring, pollution monitoring, crime scene investigation are examples of other potential use.

[0021] The device may be small and / or portable suitable for mass production. It has advantage over mass spectrometry cancer detection by providing live visualisation of the affected area. The invention may be more favourable for surgeons because they see tumour / cancer area giving them possibility for planning and minimizing cutting area and time necessary for the operation. The device may be a head- mounted / mountable device so as, e.g., no to occupy a user’s (surgeon) hands and providing undisturbed vision. Use of a fluorescent dye may be obviated, with consequential patient health benefits.

[0022] The second optical sensor may be configured to be activated to detect fluorescent light emitted by the fluorophore, and the image data generating unit comprises a trigger unit configured to delay said activation of the second optical sensor until said occurrence of detection by the first optical sensor.

[0023] Excitation light may be provided in pulsed or intensity-modulated form. A pulsed or modulated laser light source may be used. The light source may be configured to scan excitation light over a selected target containing fluorophores. An illuminated point on a target may scatter excitation light and emit fluorescent light. Scattered light may be first detected by a first detector responsive to excitation light (e.g., and unresponsive to fluorescence light) with the result triggering a second detector responsive to fluorescence light (e.g., and unresponsive to excitation light) to begin detecting / responding to fluorescence light. The second detector responsive to fluorescence light may thereby start generating fluorescence intensity data after a suitable time. By triggering the operation / activation of the fluorescence detector conditional upon detection of scattered excitation light, a precise timing of when to start to record fluorescence intensity data at each object point may be achieved for distant not-flat objects that normally would be affected by time delays (timing errors) due to the travel time of light at the speed of light. The trigger may be selectively further delayed by user-selectable additional time delay amounts that allow users to record fluorescence intensity data for different time intervals from within a fluorescence intensity lifetime spectrum of a fluorophore.

[0024] Such data acquisitions in different time intervals may be differentiated to distinguish between fluorophores emitting light with similar wavelength but different lifetimes. Processed data may be assembled into images showing fluorescence or differential fluorescence at given time intervals.

[0025] The image data generating unit may comprise a time adjustment calculating unit configured to: determine a time interval between said emission of excitation light and said detection of returned excitation light; determine a time-of-flight value of the excitation light across said spacing between the light source and the imaged subject, based on the time interval; and, adjust a time associated with a detection of said fluorescent light by the second optical sensor according to said time-of-flight value.

[0026] Accordingly, the device may calculate the time interval (delay) and simply correct the times associated with the recorded fluorescence detection data later in ‘post-processing’ rather than delaying operation of the fluorescence detector according to a ‘trigger’.

[0027] Pixels from the second optical sensor (e.g., camera) may be corrected according to a delay (resulting from light speed) measured or determined using the first camera optical sensor.

[0028] By correcting the times associated with detected fluorescence intensity values from pixels from the second optical sensor (e.g., individual pixels of a second camera photosensor chip) with use of distance information from the first optical sensor (e.g., corresponding individual pixels of a first camera photosensor chip registering scattered light, a fluorescence time may be corrected for delays caused by the separation distance between the light source and the imaged subject, and the finite value of the speed of light. Fluorescence images in different time intervals may be differentiated in order to distinguish between fluorophores emitting light with similar wavelength but different lifetimes.

[0029] The data describing fluorescence lifetime images may describe providing fluorescence lifetime images or differential fluorescence lifetime images for distant, not-flat objects. The light source may be configured to generate an intensity-modulated excitation light output and the image data generating unit is configured to determine a phase shift of a modulation of an intensity of said detected fluorescent light relative to a phase of said modulation of intensity of the excitation light.

[0030] The light source may be configured to generate a pulsed excitation light output and the image data generating unit configured to determine a fluorescence intensity decay rate of an intensity in said detected fluorescent light.

[0031] The light source may comprise an LED light source The first optical sensor may comprise an excitation light filter configured to transmit excitation light of the target fluorophore and to not transmit fluorescence light. The second optical sensor may comprise a fluorescence light filter configured to transmit fluorescence light of the target fluorophore and to not transmit excitation light.

[0032] The light source may comprises a laser configured to output said excitation light as a laser beam, and further comprising a scanning unit configured to move the direction of the laser beam in a scanning movement for illuminating a plurality of parts of the imaged subject in succession according to the scanning movement.

[0033] In a second aspect, the invention may provide a camera comprising a fluorescence lifetime imaging device as described above and further comprising an image data recording medium for recording said generated data describing fluorescence lifetime images. The camera may comprise a display device comprising a processor and a display screen in which the processor is configured to receive the data describing fluorescence lifetime images and to drive the display screen to display the images according to the received data. The display device may be a head-mountable device, or may a computer display (PC) screen, a television (TV) screen or similar configuration instead of head-mounted displaying device.

[0034] Fluorescence lifetimes of molecules are typically in the range of between several pico seconds (e.g., 1 ps to 10ps) to up to several tens of nanoseconds (e.g., 10ns to 100ns). The first optical sensor and / or the second optical sensor, in any aspect, preferably are configured to record intensity data with pico-second (ps) time resolution. For example, an ability to record an individual measurement spanning a duration of time (e.g., integration time) not exceeding about 100ps or preferably not exceeding 10ps. This enables accurate fluorescence lifetime measurements to be made with sufficient accuracy and can be achieved, for example, with the use of commercially available time of flight cameras or other commercially available fast optical sensors / detectors, as would be readily apparent to the person of ordinary skill in the art.

[0035] In a third aspect, the invention may provide a head-mounted imaging device comprising a fluorescence lifetime imaging device as described above.

[0036] In a fourth aspect, the invention may provide a method for imaging the fluorescent output of a target fluorophore at an imaged subject spaced from the imaging device by a distance or spacing, wherein the method comprises: by a light source, illuminating the subject with excitation light for fluorescent excitation of the target fluorophore; by a first optical sensor, detecting returned excitation light scattered from the imaged subject; by a second optical sensor, detecting fluorescent light emitted by the fluorophore in response to excitation thereof by the excitation light, and outputting a corresponding fluorescence intensity detection output; generating data describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of said detection of returned excitation light corresponding to a time-of-flight of excitation light across said distance or spacing between the light source and the imaged subject.

[0037] In the method, the second optical sensor may be configured to be activated to detect fluorescent light emitted by the fluorophore, and the method may comprise delaying said activation of the second optical sensor until said occurrence of detection by the first optical sensor.

[0038] The method may comprise: determining a time interval between said emission of excitation light and said detection of returned excitation light; determining a time-of-flight value of the excitation light across said spacing between the light source and the imaged subject, based on the time interval; and, adjusting a time associated with a detection of said fluorescent light by the second optical sensor according to said time-of-flight value.

[0039] The method may comprise, by the light source, generating an intensity-modulated excitation light output, and determining a phase shift in a modulation of an intensity in said detected fluorescent light relative to a corresponding phase of the modulated excitation light.

[0040] The method may comprise, by the light source, generating a pulsed excitation light output, and determining a fluorescence intensity decay rate of an intensity in said detected fluorescent light.

[0041] The method may comprise, by the light source, outputting said excitation light as a laser beam, and moving the direction of the laser beam in a scanning movement for illuminating a plurality of parts of the imaged subject in succession according to the scanning movement.

[0042] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0043] Summary of the Figures

[0044] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figures 1A, 1B and 1C shows schematically the stages of optical excitation of, and subsequent fluorescence emission from, a target fluorophore in a subject imaged by a fluorescence lifetime imaging device.

[0045] Figure 2 shows schematically a fluorescence lifetime imaging device.

[0046] Figure 3 shows images of a subject imaged by a fluorescence lifetime imaging device.

[0047] Figure 4 shows schematically a fluorescence lifetime imaging device.

[0048] Figure 5 shows schematically a head-mountable fluorescence lifetime imaging device.

[0049] Figure 6 shows schematically a head-mountable fluorescence lifetime imaging device in use.

[0050] Figure 7 shows schematically a head-mountable fluorescence lifetime imaging device in an exploded view.

[0051] Figure 8 shows schematically detected signals corresponding to excitation light and fluorescence output, both modulated according to a modulation of intensity.

[0052] Detailed Description of the Invention

[0053] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0054] Figures 1A to 1C show schematically the stages of optical excitation of, and subsequent fluorescence emission from, a target fluorophore in a subject imaged by a fluorescence lifetime imaging device in terms of a measurement from a single point 4 on the subject 24.

[0055] A fluorescence lifetime imaging device 1 is configured for imaging the fluorescent output 20 of a target fluorophore 26 at an imaged subject spaced 24 from the imaging device by a spacing, D. The imaging device comprises an illuminator unit 3 comprising a light source 2 configured to emit excitation light 14 for fluorescent excitation of the target fluorophore 26. A first optical sensor 8 of the illumination unit is configured to detect returned excitation light (16, 18) scattered from the imaged subject 24. A second optical sensor 6 of the illumination unit is configured to detect fluorescent light (20, 22: see Fig.1 B) emitted by the fluorophore 26 in response to excitation thereof by the excitation light 14, and to output a corresponding fluorescence intensity detection output signal for input to an image data generating unit 5. The image data generating unit 5 is configured to generate data (38, 40) describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of the detection of returned excitation light 18 corresponding to a time-of-flight of excitation light across (e.g., back and forth) the spacing, D, between the light source 12 and second optical sensor 8 via the imaged subject 24.

[0056] At a start time 28 recorded by the image data generating unit 5 (see Fig.lC), a fast pulse of excitation light 14 is sent from the source 2 towards distant subject 24. An area 26 of the subject containing fluorophores is activated at a given point / position 4 thereon where absorption of the excitation light occurs. From the activation point 4 simultaneously a portion (16, 18) of the excitation light 14 is scattered whereas the of the excitation light impinging at that point is absorbed and re-emitted as fluorescence light (20, 22: see Fig.l B).

[0057] The first sensor 8, which is configured to generate a detection signal 30 in response to a detection of returned excitation light 18, and the time 30 of such detection is recorded by the image data generating unit 5 (see Fig.lC).

[0058] The image data generating unit 5 is configured to perform a time-of-flight measurement 32 to determine distance, D, to measured point 4 from the light source 2. This is done by driving the light source 2 to emit a pulse of excitation light 14 towards the subject 24, and recording the time 28 of pulse emission. A part of reflected excitation light 18 will return to be detected by the first sensor (excitation (E) light detector) 8 which responds to the returned light by generating a detection signal to which the image data generating unit 5 is responsive by recording the time 30 of signal detection. The interval of time, At, between the recorded time of pulse emission and the recorded time of returned excitation light detection is then used by the image data generating unit 5 to perform a time-of-flight measurement.

[0059] By knowing how much time has elapsed, At, for completion of an initial round-trip travel of excitation light from the light source 2 of the imaging device 1 to its first sensor (excitation (E) light sensor) 8, a point in time for the start of generation of fluorescence light at / by the fluorescing point 4 of the fluorophore 26 (in response to the first arrival there of photons of the excitation pulse 14) is determined relative to the recorded time of pulse emission. This allows the image data generating unit 5 to take into account the delay of the fluorescence light arrival to the second detector 6.

[0060] The elapsed time interval, At, is then allocated 34 to be a subsequent time adjustment setting to be applied 7 by the image data generating unit 5 to the recorded times of all subsequent fluorescence light detection signals generated by the second sensor (fluorescence (F) light sensor) 6 that follow the recorded time 30 of initial fluorescence signal detection. It is to be noted that the recorded times of all subsequent fluorescence light detection signals are times measured relative to the recorded start time 28 (time t = tO) at which a fast pulse of excitation light 14 was sent from the source 2 towards distant subject

[0061] 24.

[0062] In other words, a fluorescence detection signal generated at relative detection time: t3 = t2 - to, (i.e., if t2 and tO are some local clock time) is adjusted by application 7 of the time adjustment setting, At, such that t3 - (t3)* = t2 - tO - At. Here At = t1 - tO, is the time interval between the recorded time, tO (times 28, 36), of pulse emission and the recorded time, t1 (times 30, 38), of returned excitation light detection.

[0063] Thus, by knowing fluorescence light delay, At, due to the separation distance, D, the image data generating unit 5 is configured to estimate the time 38 from which fluorescence light intensity emissions begin to occur relative to the recorded start time 28, 36 (time t = tO) at which a fast pulse of excitation light 14 was sent from the source 2 towards distant subject 24. Without this information, an accurate determination of fluorescence lifetimes of fluorophores, or other characteristics of a fluorophore determined from the temporal variation of its fluorescent emissions, is not possible across an imaged subject for which the separation distance, D, changes from point to point across its imaged surface (e.g., non-flat). Consequently, an accurate image of fluorescence lifetimes of the fluorophore across the nonfloat imaged surface of the subject cannot be obtained. The invention therefore addresses this problem as described above.

[0064] In addition, by suitably adjusting the time adjustment setting, any time segment or part (38, 40) of the lifetime of the fluorescence signal that it is desired to measure may be selected for measurement by the image data generating unit 5, since an accurate estimate of the start time 38 of the fluorescence signal is known. Adjustment of the time adjustment setting may be implemented by controlling the second detector 6 (fluorescence (F) detector) to be initiated after a correspondingly adjusted time lapse (At* > At; e.g., such that: t3 —> (t3)** = t2 - tO - At*) following the initial detection of returned excitation light 18 by the first sensor 8 (excitation (E) sensor). Furthermore, if there are two or more different fluorophores 26 present in the sample 24 that emit fluorescent light of same wavelength, but have different respective lifetimes, 42, 44, then the time adjustment setting may also be adjusted in this way to register a time / moment 40 during which a luminescence signal 42 from a chosen one of the fluorophores is detected to be stronger than a luminescence signal 42 from another one of the fluorophores. Comparison of fluorescence intensities for one fluorophore (i.e., one detection time interval 40) against the other fluorophore (i.e., the other detection time interval 38) for analytical purposes. Alternatively, or in addition, fluorescence intensity detection time interval 40 may be sequentially moved along fluorescence time scale by sequentially increasing the size of the time adjustment setting, At, to perform fluorescence lifetime scanning in order to obtain a greater portion of (e.g., the whole of) the fluorescence intensity-vs-time spectrum.

[0065] The first sensor 8 comprises an optical filter 10 configured with a transmission spectrum arranged to pass optical wavelengths of excitation light 18, and to prevent passage of optical wavelengths of fluorescent light 22. The second sensor 6, which is configured to generate a detection signal in response to a detection of fluorescence light 20, comprises an optical filter 12 configured with a transmission spectrum arranged to pass optical wavelengths of fluorescence light 18, and to prevent passage of optical wavelengths of excitation light 16. Typically, the photosensitive element of each of the first sensor and the second sensor has a broad light spectrum sensitivity, allowing cheaper elements / components to be used, and the use of filters is preferable in these circumstances at least. Either filter may be, for example, a bandpass dichroic filter. In this way excitation light sensor 8 can detect only excitation light 18 and fluorescence light sensor 6 can detect only fluorescence light 20.

[0066] Figure 2 schematically illustrates another example of an illumination unit 100 of a fluorescence lifetime imaging device, according to an embodiment of the invention. The illumination unit 100 is configured for imaging the fluorescent output 20 of a target fluorophore 26 at a plurality of location 4 on an imaged subject 24 spaced from the imaging device by a spacing, D. The device comprises a light source 102 in the form of a laser (e.g., an LED laser) configured to output the excitation light as a laser beam 14 for fluorescent excitation of the target fluorophore 26. The device 100 further comprises a scanning mirror unit 104 comprising a moveable mirror configured to receive the light 14 from the laser at a reflective surface of the mirror and to reflect the received light in a direction towards the imaged subject 24. The scanning mirror unit is arranged to controllably rotate and tilt (i.e., in both azimuth and altitude) the mirror in such a way to move the direction in which the reflected laser beam 14 is directed towards the subject 24. The device 100 may, for example, be configured to control the scanning mirror unit to scan the direction of the laser beam in a scanning movement 105 (e.g., a raster scanning movement) for illuminating a plurality of parts of the imaged subject 24 in succession according to the scanning movement.

[0067] The mirror within the scanning mirror unit 104 may be a dichroic mirror, designed to filter the laser light 14 received from it such that the reflected laser light is substantially free of unwanted spectral tails (or at least they are sufficiently reduced in intensity). The dichroic properties of some dichroic mirrors may change with the angle of incidence / reflection of incoming and reflected light. A dichroic mirror may comprise a plurality of separated reflective layers. Under large angles of light incidence, this separation appears larger as viewed along the line of incidence. That may change the properties of some dichroic mirrors. For example, when looking at a dichroic mirror on the one hand at a viewing angle perpendicular to the plane of the mirror, and on the other hand at a non-perpendicular viewing angle, significant differences in colour may be seen with the naked eye. In preferred examples, to reduce these effects, a narrow angle of scanning may be used, that does not reduce the cleaning effect upon laser light by a dichroic mirror. Alternatively, the dichroic filter may be located at the output of the laser in a position maintaining a perpendicular angle of incidence of the laser light, and to a non-dichroic scanning 104 mirror may then also be used. This dichroic property of the mirror may be advantageous if the laser light produced by the laser 102 is not clean enough (i.e., sufficiently narrow band, or having the appropriate output spectrum) to perform fluorescence measurement due to spectrum tails overlapping with fluorescence light that is significantly less intense than excitation light. This dichroic property of the mirror may address, or reduce, this problem. The scanning mirror unit 104, by its controlled movement causes the light bam 14 to move from a given measurement point 4 on the imaged subject, to the next desired location at an area on the sample 26 wanted by the user to be swept / scanned by the scanning action of the laser beam 14. A first optical sensor (E) 120 is configured to detect returned excitation light 18 scattered from the imaged subject 24. A second optical sensor (F) 112 of the illumination unit is configured to detect fluorescent light 20 emitted by the fluorophore 26 in response to excitation by the excitation light 14. The second optical sensor 112 outputs a corresponding fluorescence intensity detection output signal for input to an image data generating unit (e.g. item 5, Fig.lC) to generate data (38, 40; 5, Fig.lC) describing fluorescence lifetime images based on the fluorescence intensity detection output. The data is according to a time of occurrence of the detection of returned excitation light 18 corresponding to a time-of-flight of excitation light across (e.g., back and forth) the spacing, D, between the light source 102 and the second optical sensor 112 via the imaged subject 24.

[0068] From each measurement point 4 upon the imaged subject scattered excitation light will be generated and some of that scattered light 18 will be returned to reach a first optical sensor 112 (excitation light detector, E). At a certain moment in time, following emission of the excitation light 14, fluorescence light 20 will be emitted by the excited fluorophore and part of that fluorescent light will reach the second optical sensor 112 (fluorescence light detector, F). Both the first and the second light sensors comprise a dichroic filter at their optical input, via which input light is received by the sensor, having a transmission spectrum (e.g., bandpass) for either excluding fluorescence light 20 (filter 10, in the case of the first optical sensor 120) or for excluding excitation light 18 (filter 12, in the case of the second optical sensor 112). In addition, optical elements (e.g. collimating lens or lenses) 106 are provided for receiving the filtered light transmitted by the respective dichroic filter (12, 10), followed by a respective image intensifier unit 110, for example comprising microchannel plates (MCP) and a respective signal readout unit (‘MCP Readout’) for receiving an output signal from a respective MCP and generating an output signal (e.g., a fluorescence intensity detection output, e.g., a timing signal as discussed below). The generated output signal are for use by the image data generating unit to generate data describing fluorescence lifetime images based on the fluorescence intensity detection output according to a time of occurrence of detection by the first light sensor 120 of returned excitation light corresponding to a time-of-flight of excitation light across said spacing between the light source and the imaged subject.

[0069] The illumination unit 100 further includes a delay unit 116 configured to account for a time-of-flight of excitation light. The delay unit is configured to receive light detection signals output by the first optical sensor 120, and to control the operation of the second optical sensor accordingly. In particular an output signal 118 output from the first optical sensor in response to detection of returned excitation light from the imaged subject 24, is output as a timing signal 118 for use in controlli ng / timing the activation (i.e., switching ‘on’ to a detecting mode) of the second optical sensor 112.

[0070] When using the time-domain method, discussed below, a pulse of excitation light from the light source generates scattered light and fluorescence light which reach the two respective optical sensors. The first optical sensor generates a timing signal 118 that goes to the delay unit 116. The delay unit comprises a thresholder configured to compare the measured excitation light intensity value (derived from, or conveyed by, the received signal 118) to a pre-set intensity threshold value. Only if the threshold is exceeded then the timing / trigger signal is initiated, as discussed herein, to initiate recording by the second optical sensor at an appropriate time point and for a variable time interval.

[0071] Following a first time, or moment, of emission of excitation light 14 from the light source 102, the delay unit 116 configured to delay activation of the second optical sensor until a subsequent first time, or moment, of subsequent receipt of a portion 18 of that excitation light that is returned to the illumination unit 100 by scattering from the imaged subject 24. This delay of time corresponds to a time-of-flight delay, At, of excitation light across distance D separating the illumination unit 100 from the images subject 24. A trigger signal 114 is then output by the delay unit for input to the second optical sensor 112 to activate that sensor (i.e., switch it ‘on’) such that fluorescence intensity signals may be output by it with which to record fluorescence light intensity. This commences the recording of fluorescence data (i.e., intensity and corresponding measurement time values) at the appropriate time position (38, Fig .1 C). The trigger signal 114 may, for example, be configured to open a photocathode 108 of the second optical sensor 112, so as to apply a technique known in the art as “time gating”. The delay unit may also be configured to apply a further / additional user-input time delay value, or succession of values, corresponding to selected positions 40 along the time axis to permit fluorescence intensity measurements to be obtained at desired times following onset of fluorescent emissions by the fluorophore 26.

[0072] Figure 3 and Figure 4 schematically illustrate an implementation of the invention to a camera comprising optical sensors in the form of two-dimensional (2D) photosensitive arrays, such as CCD photosensor arrays 312, 314 for example. Consider a tetrahedral object 202 containing fluorescent material, consider illuminating the object with fluorescence excitation light 204, and consider any two points upon that object, spaced apart 208 at different distances, D, from the illumination unit (1 , 100). Photons from the source (2, 102) to the photosensor would take path 208 twice the length of the path taken by fluorescence light from the same points 208. In other words, excitation light 204 must travel out to, and back from, the illuminated points 208 in order to be detected, whereas fluorescence light 206 would have to be transverse only once.

[0073] The two-dimensional (2D) photosensitive arrays of the time-of-flight cameras 312, 314 may then generate data 312, 318 representing 3-dimensions where two dimensions X and Y are spatial and third dimension contains time-of-flight data (for excitation light: At = 2D / c; for fluorescence light: At = D / c, where c is the speed of light) that is may be translated to a distance dimension (for excitation light: D = cAt / 2; for fluorescence light: D = cAt), between the camera and any given imaged point of the imaged subject corresponding. Comparing time-of-flight data 212 (At) generated according to returned excitation light and time-of-flight data 218 generated according to fluorescent light, a two-times (2X) scaling along time-of- flight axis can be expected. Figure 3 shows an example of this in terms of time-of-flight data and 2D spatial data associated with imaging a tetrahedral object 202 and a hemispherical object. The time-of- flight values for the tetrahedral object 214 and the hemispherical object 216 derived from excitation light 212, is scaled by a factor of two (2) relative to the time-of-flight values for the tetrahedral object 220 and the hemispherical object 222 derived from fluorescence light 218. The image data generating unit may use the time-of-flight values, At, derived from excitation light 212 for generating data describing a 2D image 224 of fluorescence lifetimes for the two objects. In other words, the times of detection of fluorescence data are corrected to account for the time-of-flight delays since the moment of excitation light emission to provide the corrected timings, for example 38 or 40 of Fig. 1 C, from which to calculate the data representing florescence lifetime images 224, and thereby fluorescence from 3D objects can be projected onto two dimensions (2D) as 2D objects 226, 228.

[0074] Figure 4 shows schematic of implementation of a lifetime fluorescence imaging device. The camera comprises a pulsed excitation light source 310 comprising one or more light-emitting diodes (LED) mounted upon cooling fins 308, and configured to generate pulses of excitation light. Focusing optics 324, such as one or more lenses are disposed over the LEDs to receive excitation light emitted by the LEDs and to collimate, focus or otherwise shape the output excitation light as desired for efficient illumination of the imaged subject 24, or for higher illuminating efficiency of regions of interest upon it. Excitation light may be subject to filtering via an optical filter 326 disposed upon the one or more lenses 324. The optical filter 326 may be a bandpass dichroic filter designed to pass only excitation light if the LED optical output light spectrum is too broad for fluorescence imaging use. The spectral tails of the LEDs excitation light spectrum might overlap with fluorescence spectral bands and are also preferably removed by the filter 326.

[0075] The camera also comprises two imaging array optical sensor units. An excitation light imaging array optical sensor unit (320, 316, 312) comprises a first photosensitive imaging pixel array 312, a lens 316 and an excitation bandpass filter 320, and is configured to generate from each imaging pixel of its imaging pixel array a respective detection signal in response to detection of returned excitation light that has scattered from the imaged subject. 24. A fluorescence light imaging array optical sensor unit (314, 318, 328) comprises a second photosensitive imaging pixel array 314, a lens 318 and an excitation bandpass filter 328, and is configured to generate from each imaging pixel of its imaging pixel array a respective detection signal in response to detection of fluorescence light that has been emitted by a fluorophore 26 at the imaged subject.

[0076] The bandpass filter 320 of the excitation light imaging array optical sensor unit is in the form of a bandpass dichroic filter configured to transmit photons of excitation light and to not transmit photons of fluorescence light. The bandpass filter 328 of the fluorescence light imaging array optical sensor unit in the form of a bandpass dichroic filter configured to transmit photons of fluorescence light and to not transmit photons of excitation light.

[0077] To avoid photons of different wavelengths than those desired from leaking onto one of the two imaging array optical sensor units, a black coated protection barrier 322 may be disposed around the excitation light source 310 and / or lenses 324 thereof so as to reside between the light source / lenses and the excitation light imaging array optical sensor unit and / or the fluorescence light imaging array optical sensor unit. The dark material barrier may also be configured for protecting light leakage between the dichroic filters of the respective two imaging array optical sensor units. Black coated protection may be disposed around camera lenses 316, 318. Data from the two imaging array optical sensor units is then processed in with a processing unit 306 to obtain data describing a 2D image 224 as described above with reference to figure 3. A 2D image display unit 304 comprises an OLED, LCD or micro-LED array, etc. and is configured to receive image data from the processing unit 306 and to generate a 2D image described by that received data. If the device is implemented as a head-mounted device, corrective optics unit 302 is provided and positioned within close proximity to the display unit 304, to provide the necessary corrective optics to allow close-proximity viewing of the 2D image generated by the display when head-mounted.

[0078] Figures 5, 6 and 7 show views of a head-mounted device in schematic form. Referring to Figure 5, the display part of the device intended for close proximity to one (not both) user eye is depicted as 402. Two imaging array optical sensor units, a fast light source and additional standard vision camera are depicted collectively as residing in one unit 404. A data processing unit 406 is mounted on adjustable band 408 that can fix the device on a user’s head. A power source unit 410 unit is separately attached and can be detached (while still providing power) to reduce device weight for long time operation, for example medical screening test. Figure 6 is shows the lifetime fluorescence camera device mounted on user’s head 412. The display 402 stays in close proximity to one eye leaving the other eye undisturbed.

[0079] Figure 7 shows an exploded view revealing the internal construction of lifetime fluorescence camera device. The power source 410 is, in this illustration, alternatively provided as a plurality of standard lithium batteries, that can be charged. The charging could be performed during camera operation, e.g., during long period of use, for example medical screening tests. Three cameras are presented, 512, 510 and 514, comprising the two imaging array optical sensor units described above, and the additional standard vision camera 514 provided for generating live-view imaging data for display on the display unit 402 as white-light natural / normal human vision images. Views from this camera could be enhanced by adding lifetime fluorescence images processed by unit 306. That would allow to see exactly where fluorescence region is with respect to standard human vision view. The data processing unit 406 may be configured to combine image data 224 describing the lifetime fluorescence with data describing images of the live display from the additional standard vision camera 514 thereby providing a natural view of the measured fluorophore distribution superimposed on the live-view natural images, for viewing by the user.

[0080] Dichroic filters for the excitation light imaging array optical sensor unit 320, and for the fluorescence light imaging array optical sensor unit 328 are combined with excitation light source 326 and protection for standard human vision camera protective glass 516. Corners of each filter and glass should be blackened to avoid unwanted light leakage.

[0081] A fast data processing board 306 from two imaging array optical sensor units comprises an FPGA or dedicated chip. Its main function is as an image data generating unit configured to generate data describing fluorescence lifetime images based on fluorescence intensity detection output according to a time of occurrence of the detection of returned excitation light corresponding to a time-of-flight of excitation light across the spacing between the light source and the imaged subject. Bandpass dichroic filter 320 is designed to pass only excitation light. Bandpass dichroic filter 328 is designed to pass only fluorescence light. Close distance eye displaying device 402 comprises items 302 and 304 described above. The adjustable head mount device 408 is provided with the lithium battery pack 502 with charging unit such that the device can also work while charging batteries.

[0082] A casing 504 holds the two imaging array optical sensor units and the standard natural-view camera 514 - a standard camera to provide live vision for the scene, and filters, fast LEDs and head radiator therefor.

[0083] Optical lenses 506 for the LEDs, black material 508 in the form of an insert (to prevent light leakage noted above) are shown. An imaging array optical sensor unit 510 with lens and light shielding to be placed on the front of the bandpass excitation filter (Cf. 312, 316 and 320) together with an imaging array optical sensor unit 512 with lens and light shielding to be placed on front of bandpass fluorescence filter (Cf. 314, 318 and 328). A transparent glass shield 516 is provided for standard camera 514, which could comprise an antireflective coating.

[0084] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0085] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0086] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0087] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0088] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0089] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%. Examples

[0090] EXAMPLE 1

[0091] Frequency Domain

[0092] Figure 8 shows a schematic representation of a simple process of frequency-domain analysis of modulated excitation light and fluorescent light signals generated by the first and second optical sensors, respectively, in response to the illumination of a subject 24 with excitation light 14.

[0093] The frequency-domain method is based on a phase measurement of a relative phase difference, (p, between e.g., a periodic signal 602 corresponding to detected returned excitation light that has scattered from the imaged subject 24, and a correspondingly periodic signal 604 corresponding to detected fluorescence light.

[0094] The light source 2, 102, 310, is modulated, for example, with a sinusoidal modulation as:

[0095] E(t) = Eo{1+Asin(wt)}

[0096] The returned part of the scattered excitation signal 602, corrected fortime-of-flight delays, reflects this modulated intensity form as: l(t) = lo{1+Asin(ojt)}

[0097] Here, the quantity I is the optical intensity (or a measurement signal representing it) , the quantity A is modulation factor, and co is modulation frequency. The fluorescence signal 604, corrected fortime-of- flight delays, also reflects this modulated intensity form as:

[0098] F(t) = Fo{1+Asin(cot - a)}

[0099] Which also exhibits a relative phase-shift a.

[0100] Here, F is the optical intensity, (or a measurement signal representing it). Importantly, it can be shown that the phase shift, a, has a value that is dependent upon the fluorescence lifetime, T, of the fluorophore that produced the fluorescence, as follows: a = Arctan(cor).

[0101] This means that the fluorescence lifetime of the fluorophore 26 may be determined to be:

[0102] T = tan(a) / oj.

[0103] The value of the phase shirt, a, may be estimated by determining the neighbouring time points at which the sinusoidal modulation term within each of l(t) and then F(t) each becomes zero such that: l(t) = Io

[0104] F(t + a) = Fo

[0105] This is represented by notional reference line 605 in Figure 8. Time Domain

[0106] Alternatively, in the time-domain method, the light source is configured to emit a short pulse of excitation light:

[0107] E(t) = Eo6(t)

[0108] Here, 6(t) is the Dirac Delta function which notionally represents the short pulse duration. In response to the excitation pulse, the fluorophore is excited to fluoresce and substantially (or effectively / practically) immediately after excitation, the intensity of fluorescent emission by the fluorophore begins to decay exponentially with a decay rate determined by the fluorescence lifetime, T, of the fluorophore that produced the fluorescence, as follows:

[0109] F(t) = Fo.exp(-t / r)

[0110] By fitting an analytical function of this form to the detected fluorescence signals, one may determine a value for the fluorescence lifetime, T, of the fluorophore. If two different fluorophores are present, then the fitting formula becomes:

[0111] F(t) = Fo.exp(-t / r1) + Go.exp(-t / r2)

[0112] Here Fo and Go are constants and T1 and T2 are the respective fluorescence lifetimes of the two different fluorophores. In the time-domain, the methodology may comprise making repeated measurements of the time between the initial excitation of the fluorophore and fluorescence light intensity at each pixel of a detector. Data describing a histogram of light intensity (or photon count) versus detection time after the initial excitation may be generated. The fitting of the exponential decay curve, noted above, to the histogram associated with each image pixel may then be performed to obtain a value of the fluorescence lifetime, T, of the fluorophore at a spatial location imaged by an individual pixel. Lifetime images may be displayed using an arbitrary colour encoding each lifetime value.

[0113] In a first method, the time-domain process is employed by the device. The light source (2, 102, 310) is driven to emit short pulses of excitation light 14 with which to excite fluorescence in the target fluorophore 26. The light pulses are separated in time by a pre-set time interval during which the light source does not emit excitation light. During this time interval, fluorescence intensity measurements are made with which to measure the time-decay of the fluorescence light output intensity of the excited fluorophore as it decays from its excited state to its unexcited state.

[0114] In a case of low fluorescence efficiency, the fluorescence data collection can be extended to multiple cycles of excitation light pulse emission and subsequent fluorescent intensity measurement, which is known in the art as Time-Correlated Single Photon Counting (TCSPC) and would be readily understood ad available to the person of ordinary skill in the art..

[0115] In a second method, the frequency domain process is employed. This process, the light source (2, 102, 310) is driven to emit a continuous light output comprising an intensity modulated to vary periodically with a pre-set modulation frequency. This periodic excitation light 14 excites fluorescence in the target fluorophore 26 which responds to be similarly periodically varying in time. The phase of the modulating intensity of detected fluorescence at a given moment in time, is then compared to the phase of the modulating intensity of the excitation light signal at that same moment in time, and a phase shift in fluorescent light signal is determined relative to the phase of the concurrent excitation light signal.

[0116] Modulated excitation light (e.g., laser light) may comprise an intensity modulation in the form of a sinusoidal waveform. In this implementation both the first and the second optical sensors would work continuously recording scattered and fluorescent light concurrently over an extended period of time. A relative phase shift, as discussed above, may then be determined by a process shown in the simple example above, or by a process using a more sophisticated method such as disclosed in reference [1], below: Ayush Bhandari et al. : Optica Vol.2, No.11 , November 2015, p965: “Blind and reference-free fluorescence lifetime estimation via consumer time-of-flight sensors" . From this phase shift, a value of the fluorescence lifetime, T, of the fluorophore may be estimated. Because signal of scattered light and fluorescence light originates from the same point, the estimate of the fluorescence lifetime, T, for the fluorophore at the associated measured point 4 on the subject 24, it is not affected by light speed delays.

[0117] In both methods, two separate optical sensors are used, one for recording fluorescence and the other to allow distance related information to be recorded. The use of two optical sensor systems is able to account for large differences in intensity between returned excitation light and emitted fluorescence light. As such, each of the two optical sensors may be separately optimize for recording only photons from the dedicated relevant optical bandwidth. Different signal amplification methods can be used for each of them. This allows more versatility and better results since it avoids compromises and limitations inherent in using one optical sensor to do both jobs. Use of scattered light as a trigger is to compensate for fluorescence excitation / detection delays caused by the finite value of the speed of light during measurement of distant not flat objects.

[0118] The first optical sensor is dedicated to record scattered light from the target. It uses a dichroic band pass filter that passes scattered light but not fluorescent light. The second optical sensor uses a dichroic band pass filter that passes fluorescence light but not scattered light. The photon detector(s) in each optical sensor may comprise a semiconductor diode, a photomultiplier tube PMT or a microchannel plate MCP. The first optical sensor may comprise a photosensor diode due to higher luminosity expected for scattered return light, with second optical sensor (fluorescence detector) comprising more sensitive elements based on PMT or MCP. Optical lenses can be used to increase sensitivity (light-gathering power) to detect distant objects via the optical sensor.

[0119] References

[0120] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0121] [1] Ayush Bhandari et al. : Optica Vol.2, No.11 , November 2015, p965: “Blind and reference-free fluorescence lifetime estimation via consumer time-of-flight sensors" [2] Homulle H. A. R. et al. : Biomed Opt. Express, 11 April 2016, pp1 179-814: “Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements."

[0122] [3] David S. Kittle et al. : Nature Scientific Reports; 8 December 2016, “Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation"

[0123] [4] Lorenzo Palombi, et al. : OPTICS EXPRESS, 17 June 2013, Vol. 21 , No. 12, p14736: “A fluorescence LIDAR sensor for hyper-spectral time-resolved remote sensing and mapping”.

[0124] [5] US2015 / 0173621 A1

Claims

Claims:1 . A fluorescence lifetime imaging device for imaging the fluorescent output of a target fluorophore at an imaged subject spaced from the imaging device by a spacing, wherein the imaging device comprises: a light source configured to emit excitation light for fluorescent excitation of the target fluorophore; a first optical sensor configured to detect returned excitation light scattered from the imaged subject; a second optical sensor configured to detect fluorescent light emitted by the fluorophore in response to excitation thereof by the excitation light, and to output a corresponding fluorescence intensity detection output; an image data generating unit configured to generate data describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of said detection of returned excitation light corresponding to a time-of-flight of excitation light across said spacing between the light source and the imaged subject.

2. A fluorescence lifetime imaging device according to any preceding claim wherein the second optical sensor is configured to be activated to detect fluorescent light emitted by the fluorophore, and the image data generating unit comprises a trigger unit configured to delay said activation of the second optical sensor until said occurrence of detection by the first optical sensor.

3. A fluorescence lifetime imaging device according to any preceding claim wherein the image data generating unit comprises a time adjustment calculating unit configured to: determine a time interval between said emission of excitation light and said detection of returned excitation light; determine a time-of-flight value of the excitation light across said spacing between the light source and the imaged subject, based on the time interval; and, adjust a time associated with a detection of said fluorescent light by the second optical sensor according to said time-of-flight value.

4. A fluorescence lifetime imaging device according to any preceding claim wherein the light source is configured to generate an intensity-modulated excitation light output and the image data generating unit is configured to determine a phase shift of a modulation of an intensity of said detected fluorescent light relative to a phase of said modulation of intensity of the excitation light.

5. A fluorescence lifetime imaging device according to any preceding claim wherein the light source is configured to generate a pulsed excitation light output and the image data generating unit configured to determine a fluorescence intensity decay rate of an intensity in said detected fluorescent light.

6. A fluorescence lifetime imaging device according to any preceding claim wherein said light source comprises a laser configures to output said excitation light as a laser beam, and further comprising a scanning unit configured to move the direction of the laser beam in a scanning movement for illuminating a plurality of parts of the imaged subject in succession according to the scanning movement.

7. A camera comprising a fluorescence lifetime imaging device according to any preceding claim and further comprising an image data recording medium for recording said generated data describing fluorescence lifetime images.

8. Head-mounted imaging device comprising a fluorescence lifetime imaging device according to any preceding claim.

9. A method for imaging the fluorescent output of a target fluorophore at an imaged subject spaced from the imaging device by a spacing, wherein the method comprises: by a light source, illuminating the subject with excitation light for fluorescent excitation of the target fluorophore; by a first optical sensor, detecting returned excitation light scattered from the imaged subject; by a second optical sensor, detecting fluorescent light emitted by the fluorophore in response to excitation thereof by the excitation light, and outputting a corresponding fluorescence intensity detection output; generating data describing fluorescence lifetime images based on said fluorescence intensity detection output according to a time of occurrence of said detection of returned excitation light corresponding to a time-of-flight of excitation light across said spacing between the light source and the imaged subject.

10. A method according to claim 9 wherein the second optical sensor is configured to be activated to detect fluorescent light emitted by the fluorophore, and the method comprises delaying said activation of the second optical sensor until said occurrence of detection by the first optical sensor.11 . A method according to any of claims 9 to 10 comprising: determining a time interval between said emission of excitation light and said detection of returned excitation light; determining a time-of-flight value of the excitation light across said spacing between the light source and the imaged subject, based on the time interval; and, adjusting a time associated with a detection of said fluorescent light by the second optical sensor according to said time-of-flight value.

12. A method according to any of claims 9 to 11 comprising, by the light source, generating an intensity- modulated excitation light output, and determining a phase shift in a modulation of an intensity in said detected fluorescent light relative to a corresponding phase of the modulated excitation light.

13. A method according to any of claims 9 to 12 comprising, by the light source, generating a pulsed excitation light output, and determining a fluorescence intensity decay rate of an intensity in said detected fluorescent light.

14. A method according to any of claims 9 to 13 comprising, by the light source, outputting said excitation light as a laser beam, and moving the direction of the laser beam in a scanning movement for illuminating a plurality of parts of the imaged subject in succession according to the scanning movement.

Images