Systems and methods for measuring chlorophyll fluorescence

Abstract

A system for measuring chlorophyll fluorescence is provided that includes at least one light source configured to emit substantially collimated or focused light to illuminate an area, an actuator configured to controllably direct light from the light source to enable the light source to illuminate a plurality of different areas, and an optical sensor configured to detect chlorophyll fluorescence from each illuminated area.

Description

[Technical field]

[0001] The present invention relates to a system and method for measuring chlorophyll fluorescence, in particular to an installation suitable for use in commercial greenhouses, vertical farms or field farms to measure chlorophyll fluorescence of plants located across a target area using optical analysis. [Background technology]

[0002] In plant growth, chlorophyll fluorescence is the phenomenon in which absorbed light is re-emitted by chlorophyll molecules in plants during de-excitation. Light absorbed by plants can generally be contributed to electron transfer, heat or fluorescence, the ratio of which can vary based on environmental factors and factors related to plant physiology. Measurement of chlorophyll fluorescence can be used as an indicator of several aspects of plant growth, including the rate of CO2 uptake by plants via photosynthesis (electron transfer through the photochemical system), the effects of biotic or abiotic stress and environmental changes (e.g., temperature, humidity, irrigation, carbon dioxide concentration) on plant growth efficiency. Thus, chlorophyll fluorescence is a useful technique for assessing crop yield, nutrition and quality.

[0003] Chlorophyll fluorescence is typically assessed by first exposing dark-adapted plants to a short flash of high-intensity light to saturate the photochemical processes in the plant that allow the fluorescence level to reach a maximum. Fluorescence then returns to a minimum level in the dark-adapted state. Subsequently, actinic light is applied and the saturating pulse is again applied. Fluorescence rises in response to the pulse to a new light-adapted maximum and then falls back to a light-adapted minimum. The ratio of these fluorescence levels provides various indicators of the plant's condition, including efficiency, i.e., the fraction of the incident light that contributes to useful electron transport.

[0004] An explanation of the theory behind chlorophyll fluorescence analysis can be found in E. Murchie and T. Lawson, "Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications", Journal of experimental botany, 64 13(2013):3983-98, DOI:10.1093 / jxb / ert208. Summary of the Invention [Problem to be solved by the invention]

[0005] In commercial plant growth, it is known to bathe seedbeds in light and optically monitor the plants for fluorescence. Proximity measurement techniques are also known, in which a pulse of light and subsequent fluorescence is performed through an optical fiber gripped or held close to the area of ​​interest. Both of these techniques have problems that it would be desirable to overcome. In particular, the former technique is very energy demanding due to the need for periodic bright light pulses, and is generally relatively insensitive to fluorescence. On the other hand, the proximity techniques typically require manual actions and may damage the plants being measured. The present invention aims to provide a system for performing chlorophyll fluorescence analysis in a commercial setting, which does not have the above-mentioned drawbacks associated with known chlorophyll measurement systems. [Means for solving the problem]

[0006] According to a first aspect of the present invention, there is provided a system for measuring chlorophyll fluorescence comprising at least one light source configured to emit substantially collimated or focused light to illuminate an area, an actuator configured to controllably direct light from the light source to enable the light source to illuminate a plurality of different areas, and an optical sensor configured to detect chlorophyll fluorescence from each illuminated area.

[0007] The system uses an operating light source that emits substantially collimated or focused light to illuminate a small area of ​​the crop canopy. The system is typically mounted above the crop and illuminates the canopy from above in a small area corresponding to the measurement target location. By illuminating a small area of ​​the canopy at a time, the light intensity threshold of saturation and measurement pulses can be reached at a much lower overall energy cost than systems that bathe the canopy in light pulses. By operating the directional light source, regions of interest can be measured over a larger area of ​​the crop canopy, ensuring that a large-scale image of crop growth can still be generated. Furthermore, operating the light source to illuminate a target area off the vertical axis, i.e. with some horizontal component, also allows for equally accurate measurements of targets below the canopy. This targeted measurement system can be performed without contact with the plant and without the need for a manual operator, thus offering a significant advantage over known systems that measure plant fluorescence.

[0008] It is understood that the present system does not require precise collimation of the light source, and in fact commercially available lasers have some degree of beam divergence. The term "substantially collimated" will be understood to encompass light sources having a beam divergence of 10° or less. Of course, more tightly collimated light is preferred, in fact beam divergence of 5° or less, more preferably 2° or less, even more preferably 1° or less, and most preferably 0.1° or less. Commercially available lasers typically have beam divergence much less than 0.1°, but as explained further below, the present system can be implemented with light sources having much looser collimated light.

[0009] As mentioned above, the system includes an actuator configured to controllably direct light from a light source to enable the light source to illuminate a plurality of different areas. This may typically involve an actuator configured to move the light source to receive the light from the light source. That is, by moving the entire light source, it is possible to change the area illuminated by the light from that light source. Alternatively or in addition, the actuator may be configured to move one or more optical elements, such as a mirror, relative to the light source to direct the light from the light source. For example, the use of a scanning mirror or a light steering microelectromechanical system may allow the light from the light source to be directed between different areas without moving the light source.

[0010] It is particularly preferred that the system includes an actuator configured to move the optical sensor and / or move one or more optical elements, such as a mirror, relative to the optical sensor to enable the optical sensor to detect chlorophyll fluorescence from each illuminated area. This is preferred as it allows the optical sensor to be aimed directly at the measurement point, as will be described in more detail below, and allows the use of sensors with smaller fields of view. Although this is preferred, a fixed optical sensor could instead be used, e.g. with a field of view covering the entire space accessible to the movable light source.

[0011] Typically, the actuator configured to move the optical sensor and / or move one or more optical elements relative to the optical sensor is the same actuator configured to direct the light from the light source, i.e. one actuator directs both the light source and the optical sensor. For example, the light source and the optical sensor may both be mounted on the same body or frame, and the body or frame is moved by the actuator to simultaneously direct both the light source and the optical sensor to different targets. Alternatively, the actuator may control a set of optical elements used to direct the light from the light source and to direct the light to the optical sensor. Although this is preferred, different actuators can also be used for the light source and the optical sensor. Note that this does not exclude using additional actuators to move the light source and the optical sensor relative to each other, for example to accommodate variable measurement distances, but typically it is simpler and therefore preferred that at least one light source and the optical sensor are fixed relative to each other.

[0012] As mentioned above, having a movable optical sensor allows the use of a sensor with a smaller field of view. In particular, the optical sensor may receive light from a field of view that is smaller than the total space accessible for illumination by at least one light source, preferably less than 10%, more preferably less than 5%, even more preferably less than 1%, and most preferably less than 0.5% of the total space accessible for illumination by the light source. The total space accessible by the light source defines the operating area and refers to a path along which the actuator can direct the light from the light source to illuminate different areas of the crop canopy, which may be a solid angle and within which the light can be directed to perform measurements. In absolute terms, the optical sensor may receive light from a field of view of 400 square degrees or less, preferably 200 square degrees or less, more preferably 50 square degrees or less, and most preferably 25 square degrees or less. A typical camera that may be used as an optical sensor may have a field of view of 18° x 12° or less, or more preferably a field of view of 6° x 4°. The use of optical sensors with a relatively small field of view is particularly preferred for several reasons. Significantly, this reduces the signal-to-noise ratio of the chlorophyll fluorescence measurements since the illuminated area covers a larger percentage of the sensor's field of view. Furthermore, a smaller field of view means less data to transfer and process when analyzing chlorophyll fluorescence measurements, speeding up the operation of the system and limiting power usage.

[0013] In many embodiments, the optical sensor preferably includes an imaging sensor, such as a camera. This is preferred for several reasons. First, it allows the location of the illuminated area within the field of view of the sensor to be determined, which in turn allows the distance to the measurement target to be determined, as described below. In addition, it also allows the system to build up an image of the canopy, which may be useful for assessing crop condition and identifying points of interest. Although an imaging sensor is preferred, the system may also use a non-imaging sensor and still obtain acceptable chlorophyll fluorescence measurements. Indeed, as described below, a non-imaging sensor may be preferred in some circumstances.

[0014] In some cases, it may be preferable to provide an optical sensor capable of a high measurement frequency, which allows a more precise measurement of the response curve of the target to the illuminating light. In this regard, a high measurement frequency may mean a frequency of at least 1 kHz, preferably at least 10 kHz, more preferably at least 100 kHz, and most preferably at least 1 MHz, which corresponds to a measurement time interval of at most 1 ms, preferably at most 100 μs, more preferably at most 10 μs, and most preferably at most 1 μs. Generally, a higher measurement frequency is preferred, since it provides a better resolution to the fluorescence response curve. Cameras are less preferred in this regard, since they typically provide a lower number of frames per second. Alternatively, optical sensors that are non-imaging and / or output analog signals may be used, since they typically allow a higher measurement frequency to be achieved. Suitable optical sensors may include silicon photodetectors and / or photodiodes.

[0015] One problem faced by chlorophyll fluorescence measurement systems is that they are often installed in settings that use pulse-width modulated glow lights. Such glow lights generally contain the same far-red wavelengths of light associated with chlorophyll fluorescence, and the pulse-width modulation of this light source causes flicker in any captured image of a plant. This background flicker makes it difficult to determine the percentage of light in any one image that is from chlorophyll fluorescence and the percentage that is simply reflected from the glow light. Traditionally, this has been addressed by modifying the glow light to eliminate the use of pulse-width modulation so that any background is constant in all images. However, systems using an imaging sensor may further include a controller configured to distinguish between a target area, including an area illuminated with light from at least one light source, and at least one background area in the measurement image, and the controller is further configured to adjust the detected intensity in the target area based on the detected intensity in the background area. The target area is typically a bright spot in the image that can be identified based on its brightness as well as its location and shape, i.e., typically has a location that is limited to a specific area depending on the distance to the target. Computer vision techniques can be used to more accurately delineate the target area, which may include image subtraction, contouring, averaging, or noise removal. Any points in the image outside this area may be expected to contain primarily reflected light from the glow light, and therefore may be used to establish a background intensity level in the target area. Note that the advantage of this technique is made possible by the way in which the system only illuminates a small area of ​​the target; known techniques that bathe the target in light do not provide an instantaneous comparison with the unilluminated background area.

[0016] In these embodiments, the background area may be substantially the entirety of the measurement image outside the target area. This may simplify the calculation of background compensation. Alternatively, the controller may be configured to select a portion of the measurement image outside the target area as the background area, preferably based on detected intensity across the measurement image. For example, this may include computer vision techniques that focus on identifying areas of the same plants or the same leaves as in the target area, may be based on intensity and proximity in the image to the target area, or may target specific vertical or horizontal portions of the image to avoid background differences caused by, for example, a rolling shutter.

[0017] Adjusting the detected intensity in the target area may typically include the controller calculating an average background intensity over the background area and subtracting the average background intensity from the detected intensity in the target area. For example, the average background intensity may be calculated by summing the intensity of each pixel in the background area and dividing by the number of pixels. Alternatively, the 50th percentile may be taken as the median background pixel intensity. The background value may then be subtracted from each pixel in the target area to compensate for artifactual background illumination.

[0018] Another advantage of using an imaging sensor, especially for systems where the light source includes a laser, is that it can improve the quality of the chlorophyll fluorescence measurement by compensating for the laser illumination profile. Preferably, the system further includes a controller configured to identify a target area in the measurement image of the imaging sensor, the target area being a portion of the area illuminated by the one or more light sources, preferably the laser is configured to illuminate a spot-shaped area, and the target area is a central portion of the illuminated spot-shaped area. Since lasers typically do not have a flat-top illumination profile, some portions of the area illuminated by the laser light may not reach the saturation threshold. Thus, the imaging sensor can enable the controller to isolate portions of the illuminated area where this threshold is met, and chlorophyll fluorescence measurements can be detected from this area. The target area can be pre-determined based on the characteristics of the laser, or can be determined directly from the measurement image based on detection of reflected laser light or on the detected chlorophyll fluorescence profile.

[0019] As mentioned above, there are various advantages to using both imaging and non-imaging sensors. Therefore, in some embodiments, it may be preferable to use both imaging and non-imaging sensors. Preferably, the optical sensor, i.e. the optical sensor that detects chlorophyll fluorescence, preferably actuated by an actuator, is a non-imaging sensor such as a photodiode, and the system further comprises a non-imaging sensor such as a camera. The non-imaging sensor preferably outputs an analog signal and / or has a constant frequency of at least 1 kHz, preferably at least 10 kHz, more preferably at least 100 kHz, most preferably at least 1 MHz. In this embodiment, the non-imaging sensor preferably has a small field of view, as defined above. With respect to a non-imaging sensor, the field of view refers to the area from which light impinges on the sensor, which can be controlled by one or more optical elements, such as a lens, associated with the non-imaging sensor. Preferably, the non-imaging sensor or one or more optical elements, such as a mirror, associated with the non-imaging sensor are movable by an actuator (preferably the same actuator that directs the light from the light source) to enable the optical sensor to detect chlorophyll fluorescence from each illuminated area. The imaging sensor preferably has a wider field of view than the non-imaging sensor and can be used to find illuminated areas, e.g., crop canopies, in a space accessible for illumination by a light source and perform visual assessment of the crop. For example, the non-imaging optical sensor can have a field of view of 400 square degrees or less, preferably 200 square degrees or less, more preferably 50 square degrees or less, and most preferably 25 square degrees or less, while the imaging optical sensor can have a field of view of at least 1000 square degrees, preferably at least 2000 square degrees, more preferably at least 5000 square degrees, and most preferably at least 10000 square degrees. The imaging sensor can be substantially fixed in the system, e.g., to provide a fixed image of the crop canopy. However, alternatively, both the imaging sensor and the non-imaging sensor or the optical elements associated therewith can be actuated by the same or different actuators to direct light to each illuminated area.The imaging sensor may be used to determine the distance to a measurement target based on the location of the illuminated area within the field of view, as described below.

[0020] Preferably, the system further comprises an optical filter arranged to block reflected light from the at least one light source and to allow the optical sensor to detect light resulting from chlorophyll fluorescence. This may be done by placing the filter in front of the optical sensor (i.e. between the optical sensor and the area illuminated by the light source) or otherwise in the light path between the target area and the optical sensor. Typically, the light source emits light having a different wavelength or range of wavelengths than that re-emitted as chlorophyll fluorescence. For example, the light source may be 450 nm and the fluorescence may be in the range of 650 nm to 750 nm. Providing an optical filter to filter the wavelength or range of wavelengths emitted by the light source allows for a more accurate measurement of chlorophyll fluorescence. The wavelengths re-emitted in the fluorescence are longer than those emitted by the light source, and therefore preferably the filter may be a long pass filter, preferably configured to block light having a wavelength of about 650 nm or less, or preferably configured to block light outside the range of 650 nm to 750 nm, preferably outside the range of 650 nm to 700 nm or 700 nm to 750 nm. The use of separate bandpass filters, 650nm-700nm and 700nm-750nm, independently movable in the optical path of the sensor may allow separate PSI and PSII fluorescence measurements. A particular preferred type of filter is an interference filter. Interference filters are inexpensive, but the wavelengths that are filtered out depend on the angle of incidence. Because only a small area is illuminated with light, light from within this area will be incident on the filter at substantially the same angle of incidence, meaning that the interference filter may be partially effective. This is particularly true when the optical sensor is movable, since pointing the sensor at the illuminated area would mean that light will always be incident on the sensor from substantially the same direction, regardless of where the measurement is being made in the community.

[0021] In some cases, it may be preferable to be able to selectively use filters. For example, an optical filter may be movable relative to the optical path of the light impinging on the optical sensor to selectively block reflected light from at least one light source, preferably the optical filter may be located on a filter wheel. This allows the system to take additional measurements to assess the condition of the crop. For example, it may be desirable to detect the amount of incident light reflected from a target location or build an accurate color picture of the canopy to infer more about the condition of the crop. Alternatively, it may be preferable to switch between different filters, such as 650nm-700nm and 700nm-750nm bandpass filters, as described above.

[0022] As alluded to above, another advantage of the system is that it may build up an image of the crop canopy height. This may be achieved by providing a controller configured to identify the target distance based on the position, size and / or shape of the illuminated area in the field of view of the optical sensor. For example, if the light source is a laser with a 3 mm spot size, this may be used to infer the distance to the laser spot based on the size of the spot in the field of view of the optical sensor. Alternatively or additionally, the position of the spot in the field of view of the optical sensor may vary depending on the distance to the target based on that as a result of parallax and may also be used to identify the distance to the target. For example, if activation of the light source changes the relative direction in which the light source and the optical sensor are pointing, the position of the actuator may also need to be identified to identify the distance to the measurement target. Finally, the shape of the spot may be used to identify distance variations over the target area, such as the angle of the target relative to the beam direction. For example, a circular spot may suggest a target perpendicular to the beam, while an elliptical spot may suggest a tilt with one side of the target further from the light source than the other side, and the magnitude of this tilt may be identified by the elongation of the spot. This distance and tilt can be factored into the chlorophyll fluorescence measurements to compensate for the drop in detected intensity with increasing distance or slope, and as another measure of plant growth or to build a 3D model of the canopy. Additionally, the light source output is preferably adjusted based on the position, size and / or shape of the illuminated area within the optical sensor's field of view. This may enable the system to compensate for the drop in light energy density with increasing distance or steeper slope.

[0023] Preferably, the actuator has two degrees of freedom to allow at least one light source (and optical sensor, if also directed by the actuator) to illuminate different areas across two dimensions. The system preferably operates by making chlorophyll fluorescence measurements across a two-dimensional array of measurement locations. Other embodiments may have only one direction of movement, by measuring areas along only one direction or using a line-focused laser sweep across the canopy.

[0024] The system can operate with an actuator that translates the light source (and optionally the optical sensor), for example along orthogonal directions, but it is much more preferred if the actuator is configured to rotate the light source around one or more directions. For example, preferably the actuator has variable yaw and pitch to enable at least one light source to illuminate different areas across two dimensions. That is, the actuator may preferably rotate the light source around two different, preferably orthogonal, rotation axes that are substantially orthogonal to the direction in which the light is emitted. It will be appreciated that the actuator may not directly rotate the light source, but may rotate a body to which the light source (and optical sensor) is attached. This form of actuator allows the system to be mounted in a fixed position while still taking chlorophyll measurements from a relatively large area. Instead, the system may have one direction of translational motion and one direction of rotational motion of the light source to take measurements from a two-dimensional area.

[0025] In some particularly preferred embodiments, the at least one light source and the optical sensor are mounted in an enclosed housing that includes a transparent window through which the at least one light source and the optical sensor can operate. In some embodiments, the actuator can be mounted inside the housing, or alternatively, the actuator can move the housing in which the light source and the optical sensor are located. This arrangement helps to protect the system from, for example, potentially humid environments and contact by other equipment or personnel near the system. This is particularly preferred when the actuator rotates the light source, as this allows the light source to take measurements from a wide area with only a small amount of light source movement. In such embodiments, it may be preferred that the transparent window is provided by an at least partially transparent dome-shaped element of the housing. The term "partially transparent" may be understood to mean that at least a portion of the dome-shaped housing part is transparent, so that the light source and the optical sensor can operate through it. The dome-shaped housing element may be positioned such that light from the light source is transmitted through that portion of the dome-shaped housing element in a direction substantially perpendicular to the portion of the dome-shaped housing element in substantially any direction in which the light source (and the optical sensor) is pointed. This can minimize distortion of the light by the window and can also minimize distortion of the light received by the optical sensor, especially if the optical sensor uses a relatively small field of view.

[0026] As mentioned above, preferably, at least one light source includes a laser. Preferably, the laser is configured to illuminate a spot-shaped area, although a line-focused laser configured to illuminate a linear area may be used instead. Lasers are particularly useful because they generate tightly collimated light and typically illuminate a very small area, and can be selected to use a desired wavelength of light. It is therefore possible to meet the energy density threshold required for the illuminated area while using a relatively small amount of power, and also to function over a wide range of distances. Preferably, the laser is a power-adjustable laser. This allows the same laser to perform both the saturation pulse and the measurement pulse. Alternatively or additionally, this may allow the laser to adjust the power to compensate for variations in the illuminated area due to the distance to the measurement target and / or the tilt of the target relative to the tilt direction. It will be appreciated that the light source may include two or more lasers, for example, if it is desired to perform separate measurements using light of different wavelengths. If a laser is used, preferably the system further includes at least one beam-shaping optical element, such as a holographic diffuser. Such a beam shaper can be configured to achieve a desired spot size, shape and uniformity of the laser, thus improving chlorophyll fluorescence measurements.

[0027] Another preferred type of light source used instead of or in addition to a laser is one or more LEDs (light emitting diodes). LEDs typically have the advantage over lasers of being cheaper and having fewer safety concerns. As described in more detail below, some preferred embodiments may use both one or more LEDs and one or more lasers. The use of supplemental LEDs may reduce the required power of the laser, i.e., less power is required from the laser to reach the required energy density for the saturation and measurement pulses, which may make the system safer to use around workers. Nevertheless, the system may still benefit from using less power.

[0028] In practice, the at least one light source may include a divergent light source, of which an LED may be an example, and focusing optics for substantially collimating or converging the light emitted from the divergent light source. A divergent light source, such as an LED bulb, may have at least a portion of its light, initially emitted over a wide angular range, focused by a focusing optic, such as one or more lenses, to increase the energy density at the measurement target. It may be preferable for the light to be substantially collimated by the focusing optics. It will be understood that light collimated by the focusing optics is typically more loosely collimated than, for example, a laser, and may therefore have a larger variation in illuminated area size depending on the distance to the measurement target. In some cases, it may be preferable to converge and focus the light. This may be useful to illuminate a small area equivalent to a laser spot at a given distance. In other cases, the focusing optics may be adjustable, changing the focus of the light depending on the distance to the measurement target.

[0029] Regardless of the type of light source used, preferably the light source is an adjustable power light source, such that the light source is capable of performing both saturation and measurement pulses and can adjust the power based on the distance to the target.

[0030] As mentioned above, the system may include multiple light sources, each configured to emit substantially collimated or convergent focused light, and arranged to illuminate a common area. Illumination of a "common area" will be understood to mean that the two light sources overlap, at least partially, where they strike the target. It should be noted that the light sources do not necessarily have to illuminate exactly the same area. For example, it may be common for the spot from the focused LED to be larger than the spot from the laser, and still be directed to the same target, defining a common area illuminated by both light sources. It should be noted that all light sources of the system are not necessarily required to illuminate the exact same common area. For example, the system may include a first laser and one or more LEDs emitting light of a first wavelength for a first measurement type, and a second laser and / or one or more LEDs emitting light of a different second wavelength for a second measurement type, where the light from these light sources does not necessarily have to overlap, and may in fact preferably not overlap.

[0031] As alluded to above, different types of light sources have their own advantages and therefore it may be preferable to integrate different types in the same system to benefit from the strengths of each. Thus, preferably, a first of the multiple light sources includes a laser, preferably a laser with adjustable power, and a second of the multiple light sources includes a diverging light source (e.g. one or more LEDs) and focusing optics for substantially collimating or converging and focusing the light emitted from the diverging light source. As mentioned above, supplementing the laser with one or more focusing light sources of this type can reduce the laser power required to reach the energy density required for the saturation and measurement pulses, and therefore make the use of the system safer around the operator. In fact, in most cases, three or more light sources will be used, rather than only two. Preferably, a third of the multiple light sources includes a diverging light source and focusing optics for substantially collimating or converging and focusing the light emitted from the diverging light source. These preferred cases may have an array of diverging light sources and focusing optics as well as at least one laser, all of which are configured to illuminate a common area for measuring chlorophyll fluorescence. A number of light sources, in particular a number of divergent light sources (e.g. LEDs) and associated focusing optics, can be arranged in a ring array around the optical sensor. A ring array of light sources is advantageous for improving the uniformity of light on the measurement target at different illumination directions.

[0032] When the laser is used in conjunction with a divergent light source (e.g., one or more LEDs) and focusing optics to substantially collimate or convergently focus the light emitted from the divergent light source, preferably the laser is an adjustable power laser and the system further includes a controller configured to adjust the power of the laser based on the target distance and / or based on the power of the second light source and / or the beam divergence of the second light source. As mentioned above, a divergent light source with focusing optics will typically have a larger variation in the illuminated area as a function of the distance to the measurement target. By using an adjustable laser, the laser power can be changed to accommodate this. For example, if the target is close and the light from a divergent light source with focusing optics is concentrated in a small area, only a lower power laser spot is needed to reach the energy density required for the saturation pulse or measurement pulse. On the other hand, if the target is far away, the collimation may not be perfect or the target is beyond the focus of the convergent focus, so the light may spread over a larger area and therefore more power may be needed from the laser to reach the required energy. Adjustment of the laser may also be based on the position, size and / or shape of the illuminated area (by the laser or diverging light source and focusing optics) within the field of view of the optical sensor, which, as previously described, may be an indication of the distance to the target and its inclination.

[0033] As mentioned above, it may be desirable to use light of different wavelengths or wavelength ranges in measurements made using the system, and thus a system having multiple light sources may include at least two light sources of the multiple light sources that emit light of different wavelengths or wavelength ranges from each other. While this is possible, it is also possible to have a system that uses only one wavelength or wavelength range of light.

[0034] Many advantages of the present system arise from constraining the system to act on a small target area during any one measurement. Preferably, the or each light source has a resolution of less than 50 cm at a given working distance. 2Less than 25cm, preferably 2 Less than 5cm, more preferably 2 less than, and even more preferably, 1 cm 2 Less than 5 mm, most preferably 2 For example, a focused LED can illuminate a larger area than a laser, which is typically smaller than 1 cm 2 Less than 5 mm in most cases 2 It will be understood that the light source has a spot size of less than 100 nm. In general, a smaller illumination area is preferred because it allows the use of a sensor with a smaller field of view, which reduces noise and data processing requirements, as well as the energy used by the light source to reach the target energy density. The predetermined working distance may be in the range of 0.1 m to 20 m, preferably in the range of 0.1 m to 10 m, more preferably in the range of 0.1 m to 5 m, and most preferably in the range of 0.5 m to 1 m.

[0035] The size of the illumination area is determined by the collimation or convergence of the emitted light and the distance from the light source at which the light impinges on the target. Typically, the system will be configured to function over a range of distances and therefore it may be preferred that the target illumination area size is achieved over the entire working range, which may be between 0.1m and 20m, preferably between 0.1m and 10m, more preferably between 0.1m and 5m, and most preferably between 0.5m and 1m. This may be achieved simply by ensuring that a substantially collimated light source meets the target illumination area size at both ends of the working range. In one notable example, the or each light source is configured to have a collimation of at least 50cm over at least one working distance in the range of 0.1m to 20m, and preferably over the entire working distance. 2The or each light source is configured to illuminate an area of ​​less than 1 cm2 over the entire range of 0.1 to 20 m. If a laser is used, the illuminated area may be less than 1 cm2 over the entire range of 0.1 to 20 m. However, as a result of limitations to detection of fluorescence at long distances, the system is likely to have a smaller working distance and, as a result, the achievable illuminated area will be smaller. In one such example, the or each light source is configured to illuminate an area of ​​less than 25 cm2 over at least one working distance in the range of 0.1 m to 10 m, preferably over the entire range. 2 In another example, the or each light source is configured to illuminate an area of ​​less than 10 cm at at least one working distance in the range of 0.1 m to 5 m, and preferably throughout said range. 2 Individually, the lasers are configured to illuminate an area of ​​less than 1 cm over an operating range of 0.1 m to 10 m or 0.1 m to 5 m. 2 Of course, it will become apparent that the operating range and illumination area can be configured as needed for a particular installation by appropriate selection of light sources and optical sensors.

[0036] In many embodiments, a controller is also provided that is configured to: a) illuminate a first area using at least one light source; b) detect chlorophyll fluorescence from the first area using an optical sensor; c) redirect the at least one light source using an actuator; d) illuminate a second area using at least one light source; and e) detect chlorophyll fluorescence from the second area using an optical sensor. The controller will typically repeat steps c), d) and e) for additional measurement targets, i.e., by sequentially illuminating different areas using the light source and detecting chlorophyll fluorescence from said areas across a two-dimensional array of measurement locations. This allows the system to build up an image of plant growth across a crop canopy with a limited number of measurements.

[0037] The system may be connected to a controller and / or data processing and / or storage device via a network, preferably the Internet. For example, data collected by the system may be communicated via the Internet to a cloud processing and / or storage network. The system may include, for example, a WiFi or Ethernet connectivity module for connecting to such a network.

[0038] A number of systems, each as described above, may be provided to measure chlorophyll fluorescence of plants in different areas, each of which may be connected to the same network for control and / or collection of measurement data.

[0039] The measurements can be reported to an operator, e.g., a grower, through an interface, such as a display connected to one or more systems, or can be reported to an operator's device via an API. If multiple systems are provided, the measurements can be reported and attributed to each system where the detection was made.

[0040] An advantage of the system is that the same light source may be used for both saturation and measurement pulses. Preferably, the controller is configured to illuminate each of the first and second areas with multiple pulses of light of different intensity and / or duration. In particular, this may include a first measurement phase that includes illuminating each area with at least one pulse, preferably multiple measurement pulses having a lower intensity, lower frequency and / or shorter duration (compared to the second measurement phase), to provide a so-called F0 measurement. In this first measurement phase, the light will typically be modulated at an intensity having a lower average intensity (again compared to the second phase). This will typically include modulation between a providing on-state and an off-state, but may vary between two different intensities, both of which may be lower in intensity than the pulses in the second measurement phase. The second measurement phase may then include illuminating said areas with at least one saturation pulse, typically having a higher intensity, higher frequency and / or longer duration than the pulses in the first measurement phase, to provide a so-called Fm measurement. In this second measurement phase, the light will typically be modulated at an intensity having a higher average intensity than the first phase. This may involve varying the light between two different intensities, both typically higher than either of the intensities used in the first measurement phase. After performing the second measurement phase, a third phase typically follows, in which measurement pulses of lower intensity, lower frequency and / or shorter duration are resumed, e.g., the light will typically be modulated at an intensity having a lower average intensity (compared to the second measurement phase), such as between a low intensity on state and an off state.

[0041] In a particularly preferred embodiment, the controller is configured to control at least one light source to illuminate each area with light such that in a first measurement phase the light is modulated in intensity to define one or more pulses of light having a low average intensity (compared to the second measurement phase), and in a second measurement phase the light is modulated in intensity to define a plurality of discrete different pulse types that together have a high average intensity. A saturation pulse (i.e. the second measurement phase in this term) differs from measurements made with a saturation pulse in a conventional measurement process, since it typically comprises a single light pulse formed by the superposition of several different light pulses.

[0042] Preferably, the intensity of the illuminating light in the target area returns to substantially zero between each pulse type. It is preferred that the light intensity return to zero between each pulse type because this serves the linearity requirements of the light source. However, alternatively, the intensity can return to a low baseline intensity between pulses.

[0043] In this example, preferably, the second measurement phase comprises a plurality of light pulses of a first pulse type and a plurality of light pulses of a second pulse type, the light pulses of the second pulse type occurring between the light pulses of the first pulse type, preferably the first and second pulse types alternating. The first pulse type preferably comprises the light being modulated from a low value, preferably an off value, to a first high value, such that said first pulse type has a first intensity and duration before returning to a low value, and the second pulse type preferably comprises the light being modulated from the same low value, preferably an off state, to a second high value (which may be the same or different from the first high value), such that at least one, preferably both, of the intensity and duration of the second pulse type differs from that of the first pulse type in the second measurement phase. In practice, typically the duration of the pulses will differ by several orders of magnitude. The second measurement phase here is similar to a conventional saturation pulse, but in this example the target is illuminated with a saturation pulse, the saturation pulse is switched off for a short period of time, and during this period that the saturation pulse is off the target is illuminated with a measurement pulse.

[0044] The above measurement profile has several advantages: the pulse profile is less sensitive to rise or fall times and less sensitive to amplitude, i.e. intensity; it reduces the linearity requirements of the source and is less sensitive to switch-on currents; it also improves laser safety, since the two laser pulses do not overlap.

[0045] In another preferred embodiment, the controller is configured to control at least one light source to illuminate each area such that in a first measurement phase, the light is modulated in intensity to define one or more discrete pulses of light constituting a first percentage of the first measurement phase and having a low average intensity (compared to the second measurement phase), and in a second measurement phase, the light is modulated in intensity to define a plurality of discrete pulses constituting a second percentage of the second measurement phase that is greater than the first percentage and having a high average intensity. The percentage of the measurement phase refers to the amount of time the light intensity is high, e.g. the light is on, compared to the amount of time the light is low, e.g. the light is off. The second measurement phase should have a higher percentage at high intensity values ​​than the first measurement phase to provide a higher average intensity. For example, these different percentages can be provided by providing pulses in the first measurement phase that have a lower frequency than the pulses in the second measurement phase. Preferably, the pulses in the second measurement phase have a frequency of 5-1000 kHz, preferably 10-1000 kHz, more preferably 10-500 kHz, so that the biological response of the crop is not affected by the light frequency and responds only to the average intensity.

[0046] Preferably, the light pulses in the first measurement phase have the same intensity as the light pulses in the second measurement phase. Preferably, the light pulses in the first measurement phase have the same duration as the light pulses in the second measurement phase. Preferably, the light pulses in the first measurement phase are the same as the light pulses in the second measurement phase but have different frequencies to provide different average intensities.

[0047] In this embodiment, the conventional saturation pulse (equivalent to this second measurement phase) is replaced by a series of high frequency pulses. These pulses can be the same as the measurement pulses used in the F0 measurement (first measurement phase in this terminology). The pulse amplitude, duration and duty cycle during the first measurement phase can be calculated to achieve low average illumination during the first measurement phase so that photosynthesis is not triggered, and to achieve saturation of the reaction centers during the second measurement phase.

[0048] The advantage of using only one pulse type as in this embodiment is that the driving circuitry generating the pulses can be simple. The pulse profile is also less sensitive to rise or fall times and less sensitive to the amplitude of the pulse. The linearity requirements of the light source are lowered and the sensitivity to switch-on currents is reduced. This technique means that the detector can use a lock-on amplifier. Laser safety can be improved because a single amplitude can be used, which can be lower than the peak amplitude required for a typical saturation pulse. Finally, an important advantage of this measurement technique is that the high frequency pulses allow measurements to be made at high frequencies, especially with non-imaging sensors. This allows measurements of chlorophyll fluorescence kinetics such as OJIP.

[0049] The conversion of the light intensity captured by the detector system into chlorophyll fluorescence measurements (Fo, Fm) may require additional processing steps to obtain representative fluorescence intensities from plant leaves. This is due to the typical laser spot profile having a Gaussian distribution of output intensity, where only the central region will have sufficient light intensity for the measurement. Therefore, the region isolated to calculate the fluorescence intensity should be restricted to the central region. The region should be calculated to ensure a non-overlapping, symmetric normal distribution for the fluorescence intensity returned by the detector at each excitation intensity. In the case of an image sensor, computer vision techniques can be used to first detect the region of interest and then refine the specific pixels of interest to ensure accurate measurements.

[0050] In many embodiments, the or each light path between the light source and the target and the light path between the target and the optical sensor impinge on the target area at different angles. This may be a result of the light source and the optical sensor being spaced apart and aimed at the same target. In some circumstances, such an arrangement is advantageous, and indeed we have already described how this suggestion can be used to help determine the distance to the target. However, in some circumstances, it may be preferable to have a coaxial arrangement, whereby the light path is substantially the same at the target. Thus, some embodiments further include a beam splitter or optical combiner, where the light source is arranged to illuminate said area, i.e. the target area, along a first optical path through the beam splitter or optical combiner, and the optical sensor is arranged to receive the chlorophyll fluorescence emitted from said ceria along a second optical path different from the first optical path through the beam splitter or optical combiner, and the light source and the optical sensor are arranged such that the light path is substantially coaxial, i.e. the same, between the beam splitter or optical combiner and said illuminated area. In other words, the illumination direction at the target is substantially the same as the sensing direction of the sensor. Both the beam splitter and the optical coupler convert each input direction into two output directions, and the present arrangement uses one of the two outputs from the light source to receive the fluorescence as an input, and the optical sensor is placed at the unused output of this fluorescence. This allows the optical sensor to be provided effectively on-axis with the light source via the beam splitter or optical coupler. This may provide that the fluorescence from the target area always hits the optical sensor in the same plane, for example, regardless of the nature of the target, which may make the measurement faster by eliminating the need to identify the target.

[0051] In these embodiments, if an optical filter is used, the filter should be placed between the beam splitter or optical combiner and the optical sensor, i.e., the first optical path does not pass through the filter. In this way, the illumination light is not affected by the filter and only the fluorescent light directed to the optical sensor is filtered out.

[0052] One advantage of using a beam splitter is that it may further include focusing optics between the beam splitter and the area to be illuminated, the focusing optics being configured to substantially collimate or focus the light from the light source and collimate or focus the chlorophyll fluorescence from the area towards the optical sensor. Thus, a similar set of optics may be used to shape the light from the light source as is used to shape the light directed towards the sensor. This may make the system cheaper to manufacture.

[0053] One advantage of using an optical coupler is that the light source may be configured to emit light into a first end of a first optical fiber, and the optical sensor is configured to receive light from a first end of a second optical fiber, said first and second optical fibers being coupled together by an optical coupler such that the second end of either the first or second optical fiber emits light from the light source to illuminate said area, and said second end receives chlorophyll fluorescence from said area, and preferably the actuator is configured to controllably direct the light from the light source by moving said second end of either the first or second optical fiber. In particular, this arrangement means that the light source and the optical sensor can remain stationary, and the directing of the light and the aiming of the optical sensor are performed by moving the optical fiber. In such an embodiment, it may be preferable to include focusing optics for collimating or converging the emitted light from the second end of either the first or second optical fiber towards a target, as well as for focusing the chlorophyll fluorescence into the optical fiber.

[0054] The above embodiments are particularly advantageous when used in conjunction with non-imaging optical sensors, since chlorophyll fluorescence will always be incident on the optical sensor at the same plane and imaging is not required to identify target areas within the field of view. As noted above, non-imaging sensors are typically capable of higher measurement frequencies and may be useful for measuring pulse response curves.

[0055] According to a second aspect of the present invention, there is provided a method of measuring chlorophyll fluorescence, the method comprising: a) illuminating a first area with at least one light source configured to emit substantially collimated or focused light; b) detecting chlorophyll fluorescence from the first area with an optical sensor; c) moving the at least one light source with an actuator; d) illuminating a second area with at least one light source; and e) detecting chlorophyll fluorescence from the second area with an optical sensor.

[0056] The method corresponds to a method of measuring chlorophyll fluorescence using a system according to the first aspect of the invention, in which the light source is movable by an actuator to selectively illuminate different areas for testing. Each of the above-mentioned advantageous features according to the first aspect of the invention may be implemented in the method according to this aspect as well.

[0057] It will be appreciated that steps c), d) and e) may be repeated for one or more further areas. These future areas may include areas different from the first and second areas, but may also include repeating the measurements at a later time in substantially the same first and / or second areas to detect changes in chlorophyll fluorescence over time. The method may be used to provide real-time monitoring of plant photosynthetic efficiency, i.e. the conversion of photons to excited electronic states in a photochemical process, by measuring chlorophyll fluorescence over time and reporting the changes to an operator, e.g. a grower.

[0058] The method may be used to determine a common chlorophyll fluorescence metric, such as Fv / Fm, from the detected chlorophyll fluorescence. The method may also be used to monitor and report plant growth and productivity. For example, chlorophyll fluorescence metrics may be measured over time and changes may be identified and reported to an operator, e.g., a grower. From these monitored metrics, it may be possible to recommend changes to the measured plant's environment. For example, the method may include recommending changes to one or more of lighting, e.g., artificial lighting, temperature, water, humidity, CO2 or O2 concentration, soil nutrient concentration, and soil pH, based on the detected chlorophyll fluorescence or changes in the detected chlorophyll fluorescence over time.

[0059] According to a further aspect of the invention, there is provided a method of measuring chlorophyll fluorescence, the method comprising illuminating a first area with light using at least one light source, the illuminating the first area comprising illuminating the first area such that in a first measurement phase the light is modulated in intensity to define one or more pulses of light having a low average intensity, and in a second measurement phase the light is modulated in intensity to define a plurality of discrete different pulse types together having a high average intensity, preferably the intensity of the illuminating light in the target area returns to substantially zero between each pulse type in the second measurement phase, and detecting chlorophyll fluorescence from the first area using an optical sensor. Preferably, the second measurement phase comprises a plurality of light pulses of a first pulse type and a plurality of light pulses of a second pulse type, the light pulses of the second pulse type occurring between the light pulses of the first pulse type, preferably the first and second pulse types alternating. The first pulse type preferably comprises a light pulse modulated from a low value, preferably a substantially off state, to a first high value such that said first pulse type has a first intensity and duration before returning to the low value, and the second pulse type preferably comprises light modulated from the same low value, preferably a substantially off state, to a second high value (which may be the same as or different from the first high value) such that at least one, preferably both, of the intensity and duration of the second pulse type differs from that of the first pulse type in the second measurement phase.

[0060] According to another aspect of the invention, a method of measuring chlorophyll fluorescence is provided, the method comprising illuminating a first area with light using at least one light source, the illuminating the first area comprising illuminating the first area such that in a first measurement phase, the light is modulated in intensity to define one or more pulses of light constituting a first percentage of the first measurement phase and having a low average intensity, and in a second measurement phase, the light is modulated in intensity to define a plurality of discrete pulses constituting a second percentage of the second measurement phase different from the first percentage of the first measurement phase and having a high average intensity. Again, the percentage of the measurement phase refers to the amount of time the light intensity is high, e.g. the light is on, compared to the amount of time the light is low, e.g. the light is off. The second measurement phase should have a higher percentage at high intensity values ​​than the first measurement phase to provide a higher average intensity. For example, these different percentages can be provided by providing pulses in the first measurement phase having a lower frequency than the pulses in the second measurement phase. Preferably, the pulses in the second measurement phase have a frequency of 5-1000 kHz, preferably 10-1000 kHz, more preferably 10-500 kHz, so that the biological response of the plant is not affected by the frequency of the light, but only responds to the average intensity. Preferably, the light pulses in the first measurement phase have the same intensity as the light pulses in the second measurement phase. Preferably, the light pulses in the first measurement phase have the same duration as the light pulses in the second measurement phase. Preferably, the light pulses in the first measurement phase are the same as the light pulses in the second measurement phase, but with different frequencies to provide different duty cycle ratios and therefore different average intensities. In this embodiment, the conventional saturation pulses (equal to the present second measurement pulses) are replaced by a series of high frequency pulses. These pulses may be the same as the measurement pulses used in the F0 measurement (the first measurement phase in this term).

[0061] The present invention will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0062] [Figure 1] 1 illustrates diagrammatically a system according to a first embodiment of the invention; [Diagram 2] 1 shows a perspective view of a system according to a first embodiment of the invention; [Diagram 3] 1 shows a perspective view of a part of a system according to a first embodiment of the invention; [Figure 4] 1 shows a side view of a portion of a system according to a first embodiment of the invention; [Diagram 5] 3 is a flow diagram illustrating the operation of the system. [Figure 6] 13 shows measurement images taken during operation of the system. [Figure 7] 2 illustrates diagrammatically a system according to a second embodiment of the invention; [Figure 8] 5 illustrates diagrammatically a system according to a third embodiment of the invention; [Figure 9] 5 illustrates diagrammatically a system according to a fourth embodiment of the invention; [Figure 10] 5 illustrates diagrammatically a system according to a fifth embodiment of the invention; [Figure 11] 13 illustrates diagrammatically a system according to a sixth embodiment of the present invention; [Figure 12] 1 is a graph showing light intensity over time for a first method of measuring chlorophyll fluorescence. [Figure 13] 1 is a graph showing light intensity over time for a second method of measuring chlorophyll fluorescence. [Figure 14] 13 is a graph showing light intensity over time for a third method of measuring chlorophyll fluorescence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] A first embodiment of the present invention will now be described with reference to FIGS.

[0064] 1 shows a schematic of the system showing the main components and their use in making measurements. System 1 includes a dome-shaped housing 10 that encloses a laser 20 and a camera 30 that are mounted on a support 40. The support is connected to an actuator 50 that moves the support 40 within the housing 10 to change the direction in which the laser 20 and camera 30 are pointed. The housing also encloses a controller 60, which controls the operation of the actuator, the laser, and the camera.

[0065] As shown in FIG. 1, the dome-shaped housing 10 is mounted above a canopy C of growing plants in the area below, with a substantially flat base so that the dome portion of the housing projects downwards. In this arrangement, the support 40 is operated by an actuator 50 to aim the laser 20 and camera 30 at a target in the canopy C below. The laser 20 is controlled by a controller 60 to illuminate the area 21 and emit a saturation pulse or a measurement pulse depending on the type of measurement being made. The target may be 0.5-1 meter from the system 1, and the laser spot size at this distance may be about 2 mm in diameter. The camera 30 has a field of view 31 of the canopy that encompasses the area 21 that is illuminated by the laser light. The camera has a field of view of about 20 cm at a distance of 0.5-1 meter. 2 The filter 35 is positioned between the camera 30 and the target such that light emitted from the target as chlorophyll fluorescence is detected by the camera 30 and reflected light from the laser spot 21 is filtered out by the filter 35.

[0066] 2 shows the system in more detail, with the camera 30 omitted, and shows that the dome-shaped housing 10 includes a transparent dome-shaped cover 11, which is attached to a circular base 12 with a raised rim at the periphery. The cover 11 is attached to the circular base 12 by screws (not shown) that connect and fasten the cover 11 to the base 12 through threaded holes 13 in the peripheral lip of the cover 11. The remaining components of the system are then enclosed within the housing and contained between the base 12 and the cover 11, with the optical components, i.e., the laser 20 and the camera 30, still able to operate through the transparent cover 11. The entire system 1 can be mounted above a cluster by attaching the base 12 to a surface above the cluster, such as a ceiling, via threaded holes (not shown) or other mounting means in the base 12.

[0067] Within the housing 10 is the control electronics 60. This includes a controller for the actuators 50, a controller for the laser 20 and a controller for the camera 30 which work together to perform a series of measurements of different targets within a constellation C below the system. These control electronics are mounted in the base 12 of the housing 10 so as to be stationary during use.

[0068] As mentioned, within the housing 10 are the laser 20, the camera 30, the support 40 and the actuator 50, which are shown separated in Figures 3 and 4. The actuator 50 includes a ring-shaped base 51 within which is mounted a platform 52 having a circular portion and a mounting portion that is received by the base 51. Located on the mounting portion is a pan servo motor 53, which operates through a central axis through the platform and is preferably capable of rotating the platform 360°. Similarly mounted to the platform 52 via axis 54 is a tilt servo motor 55, which is preferably capable of rotating 90° relative to the platform 52 in this configuration. This type of actuator, with variable yaw and pitch, allows the laser and camera to be pointed over a wide area, thus allowing large communities to be surveyed in a small footprint. The dome-shaped housing ensures that the laser and camera are always substantially perpendicular to the area of ​​the housing between them and the target, regardless of where they are pointed.

[0069] Support 40 is a molded plastic that is attached to the housing of tilt motor 55 of actuator 50 and supports both laser 20 and camera 30 along with filter 35. Support 40 includes, among other things, a camera holder 41 and a laser holder 42, which are positioned such that both laser 20 and camera 30 are pointed along the same direction, i.e. parallel to each other, and laser 20 is spaced a few centimetres above camera 30.

[0070] The camera housing section 41 includes a back plate 41a, and the camera 30 is attached to the back plate 41a and points away from the back plate at a target. The camera holder section 41 also includes two arms 41b, which extend forward from the back plate 41a and down the camera barrel. The two arms connect to a filter assembly 33 at their front ends. The filter assembly includes a filter wheel 34 connected to a filter motor 36 attached to the end of the arm 41b. The filter motor 36 operates to rotate the filter wheel 34 so that the entire filter 35 or filter 37 in the filter wheel can be placed in front of the camera 30. The filter 35 is held in front of the camera lens and can be a long pass interference filter that filters out normally incident light having wavelengths less than 650 nm, allowing the camera 30 to measure fluorescence in the range of 650-750 nm, or it can be a band pass filter that blocks all light outside the range of 650-750 nm. Filter 37 can be a different filter, such as a 650-700 nm or 700-750 nm bandpass filter, to acquire PSI or PSII fluorescence separately. Alternatively, filter 35 can be a 650-700 nm bandpass filter and filter 37 can be a 700-750 nm bandpass filter, to enable the system to measure both PSI and PSII fluorescence independently of each other.

[0071] The laser holder 42 of the support 40 is a substantially cylindrical sleeve that is open at both ends, with a smaller opening at the front end through which the laser beam is emitted. At the open rear end, the laser holder 42 receives a substantially cylindrical laser, which points forward through the open front end.

[0072] The laser 20 itself is configured to emit collimated light having a wavelength of approximately 450 nm with a beam diameter of 3 mm, and a fluence of 8000 μmol / m. -2 s -1It should be possible to deliver a saturation pulse lasting about 1 second to the target, with an average photosynthetic photon flux density (PPFD) of 1000 nm. The laser should also be capable of delivering measurement pulses lasting 1 μs to 10 ms.

[0073] The camera 30 may be an OV9281 manufactured by OmniVision®, 4275 Burton Drive, Santa Clara, Calif. 95054, USA. The camera may be configured with a relatively small field of view of 6°×4°, which reduces noise in the signal and increases sensitivity to the area illuminated by the laser. As mentioned above, the field of view of the camera must be large enough so that the laser spot is visible over the operating range of the system. In an embodiment where the laser 20 and the camera 30 are aimed parallel to each other, the laser spot is in the center of the infinite field of view, and the closer the target is to the system, the closer it is to the top of the field of view. Indeed, as mentioned above, the position of the laser and the spot size in the field of view of the camera may be used to determine the distance to the target. In other embodiments, the laser and the camera may not be parallel, but may instead be at a small but constant angle in the direction of the pointing such that the laser spot is in the center of the field of view of the camera at approximately the center of the operating range of the system. This allows the spot to move over the entire field of view of the camera depending on the distance to the target, thus increasing the sensitivity of the measurement of the distance to the target. An angle of about 8° has been found to be suitable for typical separation distances between the camera and laser and working distances in the range of 0.5-1 m, but it will be appreciated that these may be configured as necessary depending on the particular system installation, including the working distance and the spacing between the camera and the laser. In other embodiments, the angle between the laser and the camera may be adjustable. The control system may adjust the angle between the laser 20 and the camera 30 until the spot is centered in the field of view, at which point the angle between the camera and the laser required to achieve this centering of the laser spot may determine the distance to the target.

[0074] 5 is a flow diagram showing the basic operation of the system according to this embodiment. When the measurement process is started, in step S100, the actuator 50 is used to direct the light source, i.e. laser 20, and the sensor, i.e. camera 30, to a new target in the lower cluster C. This target can be, for example, from a memory stored in the control electronics 60, and is typically one of a two-dimensional array of targets in the lower cluster. This can be a regular array of targets, for example one target every 5 cm in two dimensions in a regular square grid in a plane at a distance of 0.75 m from the system. Alternatively, the target can be a pre-programmed point of interest in the cluster.

[0075] Once the laser and camera are aimed at the target, in step S200, the laser is used to illuminate the target. Typically, the first measurement (the so-called F0 measurement) includes a series of pulses, and in one example, the series of pulses includes a 6000 μmol m -2 s -1 with amplitudes up to 100 μs, pulse widths from 0.1 μs to 100 μs and periods up to 10 s, and with a measurement interval of approximately 0.1 μmol m -2 s -1 To measure saturation (so-called Fm measurements), a saturation pulse is delivered instead. The strength of the saturation pulse is variable, but in one example this is about 8000 μmol m over an interval of about 0.8 s. -2 s -1 The saturation pulse itself is configured to provide an average PPFD of 8000-14000 μmol m -2 s -1 and may include a series of pulses from low to high amplitude having pulse widths of 0.1 μs to 10 ms and periods of 10 to 100 ms.

[0076] As described above, before executing the required illumination profile, the controller may first determine the distance to the target by illuminating the target and detecting the position of the light source within the field of view of the sensor, and then adjust the output of the light source to arrive at the correct PPFD, taking into account any known variation in spot size with distance as a result of beam divergence, as well as taking into account the tilt angle of the target relative to the beam.

[0077] In step S300, the fluorescence from the target is detected using a camera. Although this step is shown after step S200, it will be understood that these steps are performed approximately in parallel, with the camera continuously detecting the fluorescence level over time during the course of the illumination step. The camera may take a series of images of the target during this step, which may correspond to different measurement types. For example, the light source may first illuminate the target with a low energy pulse for F0 measurement, during which the camera takes a series of images, and then the light source may illuminate the target with a high energy pulse for Fm measurement, during which the camera again takes a series of measurement images.

[0078] 6 shows one measurement image M taken using a camera through a bandpass filter, as described above. In some embodiments, the controller may perform a background compensation technique on each measurement image M. In particular, this technique may be used to compensate for background interference in a target area M containing pulse-induced fluorescence. T This area is typically identified as the brightest area that conforms to the expected shape of the light source, in this case the target is expected to be a roughly circular spot. The controller then identifies the background area M in the measurement image M. B In some embodiments, the background is simply the target area M TThe background area M may be the entire area outside the target area, but in this embodiment the controller identifies only a portion of the measurement image outside the target area to serve as the background area. This may be, for example, an area of ​​similar shape and size adjacent to the target area, or may include using computer vision techniques to select the same portion of a plant or leaf as is included in the target area. To compensate for any background light not due to chlorophyll fluorescence, the controller may select the background area M B The controller then determines the average intensity of each pixel in the target area M. T This can be subtracted from each pixel in to obtain a background-adjusted measurement of chlorophyll fluorescence.

[0079] The method may also include identifying a portion of the image from an image such as that shown in FIG. 6 where chlorophyll fluorescence is to be measured. As mentioned above, the light sources used typically do not have a flat-top illumination profile, so some portions of the area illuminated by the laser light may not reach the saturation threshold. Thus, the target area M T Edge regions of the light may skew the fluorescence measurement data. Thus, from the camera image, one may isolate a central portion of the illuminated area where the required threshold is met, and chlorophyll fluorescence measurements can be detected only from this area, optionally adjusted to compensate for background light as described above. For example, the central 50% of the illuminated spot seen by the camera may be selected for measuring chlorophyll fluorescence.

[0080] In step S400, the controller checks whether there are any additional targets that need to be measured. If so, the process returns to step S100, where the controller directs the light source and sensor to the new target, and then repeats steps S200 and S300. If no targets remain, the process ends.

[0081] Figure 7 illustrates in diagrammatic form another embodiment of the present invention, which in particular illustrates the use of multiple light sources of different types and their illumination of a common target area. For simplicity, Figure 7 illustrates only the support 40, camera 30, filter 35, and multiple light sources. The figure omits the housing 10, actuator 50, and controller 60, which may be substantially as described above.

[0082] In this embodiment, the light source includes a laser 20 substantially similar to that described above in relation to the first embodiment, together with three further light sources 22a, 22b and 22c. Although only three further light sources are shown in this embodiment, it will be appreciated that a greater number of light sources may be used.

[0083] Each of these further light sources is an LED with associated focusing optics. Each LED should emit the same wavelength as laser 20, in this case about 450 nm, if they are intended to be used together in the same measurement process. The focusing optics in this case includes a collimating lens so that each light source 22a, 22b, 22c emits substantially collimated light, but could also include a focusing lens to emit converging light focused in the middle of the working range, or an adjustable lens set to adjustably focus the light to a target distance.

[0084] The further light sources are arranged in a ring array surrounding the camera 30 such that they are each equidistant from the centrally located camera 30. In some embodiments, each alternating LED may have one of two wavelengths or wavelength ranges, such that the further light sources can perform measurements using two different wavelengths of light. In such embodiments, this may be supplemented by a second laser having a second wavelength, and an adjustable filter wheel 35 may be provided to accommodate different types of measurements utilizing different wavelengths of interest.

[0085] Each light source 22a, 22b, 22c illuminates a respective area 23a, 23b, 23c in the canopy below. In most measurements, the illuminated areas only partially overlap, which is shown exaggerated in FIG. 7. The light sources may be directed parallel to one another, in which case the separation of the spots, and therefore the overlap, will depend on the spacing of the light sources and the beam divergence. Alternatively, the light sources may be directed inwards to completely overlap at a given distance from the system, in which case the light sources will only partially overlap on either side of this given distance. As a further alternative, the light sources may be adjustable so that the spots can be aligned at each measurement distance.

[0086] 7, the laser 20 is aimed so that the laser spot 21 overlaps each of the areas 23a, 23b, 23c illuminated by the light sources 22a, 22b, 22c. Thus, an area of ​​the canopy is illuminated with light from each of the light sources 20, 22a, 22b, 22c, and this common area is the measurement target where the camera will detect chlorophyll fluorescence.

[0087] The light sources 22a, 22b, 22c typically have a larger beam divergence than the laser 20. As a result, the spot size increases with increasing distance to the target, and therefore the energy density at the target will be greater than with a more tightly collimated laser, and will fall off with distance. To compensate for this, the control electronics can adjust the power of the laser so that the energy density at the target, i.e., the area of ​​the laser spot, remains at a level desired for the measurement being made. The energy output of each light source is known, and by knowing the beam divergence, it is possible to determine with each light source the size of the illuminated area at any particular distance, and thus the energy density at the target. As mentioned above, the distance to the target can be determined by the position and / or size of the illuminated area within the field of view of the camera, with the laser spot 21 being particularly preferred for distance determination. The control system can therefore increase the power of the laser to compensate for the lower energy density that typically accompanies the longer distance to the target, and thus the larger spot size of the light sources 22a, 22b, 22c. Thus, the control system can achieve a target energy density with measurements being made at any distance within the working distance.

[0088] A further embodiment will now be described with reference to Figure 8. This embodiment in particular shows the use of a beam splitter to allow for a coaxial arrangement of the light source and the optical sensor. For simplicity, Figure 8 shows only the support 40 and the elements that reside on the support. This figure omits the housing 10, the actuator 50 for moving the support 40 and the controller 60, which may be substantially as described above.

[0089] This embodiment includes a light source 20, again in the form of a laser. The laser is configured to emit collimated light having a wavelength of approximately 450 nm with a beam diameter of 3 mm, and a fluence of 16000 μmol m -2 s -1It should be possible to deliver a saturation pulse lasting about 1 second to the target, with an average photosynthetic photon flux density (PPFD) of 1000 nm. The laser should also be able to deliver a measurement pulse lasting 1 μs to 1 ms. Note that in this embodiment the laser is more powerful than the previous embodiment due to the use of a beam splitter.

[0090] A high laser power is required because the laser illuminates a beam splitter 70, which is also on the support 40. In the figure, the laser emits light along the vertical axis and the beam is incident on the mirror at an angle of 45°. The beam splitter can be, for example, a semi-transparent mirror. The beam splitter should be configured so that, as far as possible, 50% of the incident light is transmitted through the beam splitter and 50% of the incident light is reflected. In this arrangement, the light from the laser 20 that is transmitted through the beam splitter is used to illuminate the target area 21, so twice the illumination power is required for the splitter beam to reach the required energy density at the target. In this example, the light from the laser 20 that is reflected by the beam splitter to the horizontal axis is not used.

[0091] Also on the support 40 is an optical sensor mechanism including a non-imaging optical sensor 30 such as a photodiode, an optical filter 35, which may be a filter wheel substantially as described with respect to the first embodiment, and a collection lens 38. These components are located on the other side of the beam splitter, i.e. on the horizontal axis in the figure and on the side of the beam splitter 70 where none of the laser light is directed. The collection lens 38 is preferably motorized so that it is movable towards and away from the optical sensor 30 to compensate for the variable distance to the sample. This increases the working distance of the detector and improves the signal generated. A motorized collection lens can be used in any of the embodiments described herein, for example as part of the camera or spaced away from the non-imaging detector as in this embodiment. With this arrangement, chlorophyll fluorescence in the detector area 31 is incident on the beam splitter 70 on the vertical axis and light reflected by the beam splitter to the horizontal axis is directed to the optical sensor 30. This light reflected to the horizontal axis first enters the collecting lens 38, which is positioned to focus the light reflected from the beam splitter 70 towards the optical sensor 30. As the light is focused from the collecting lens 38 towards the optical sensor 30, it passes through the optical filter 35, which may filter out all light outside the range of, for example, 650 nm to 750 nm. The intensity of the light received at the optical sensor 30 will therefore indicate the level of chlorophyll fluorescence in the target area 21. It should be noted that in this embodiment, the collecting lens 38 is not essential and may be omitted if the optical sensor 30 and the filter 35 are large enough to receive sufficient light from the area 31. Furthermore, although a non-imaging sensor is used, it is also possible to use an imaging sensor such as a camera. However, the depth perception of the spot due to the suggestion is lost.

[0092] As can be seen in Fig. 8, by mounting the laser 20 and the optical sensor mechanism on a vertical axis with the beam splitter 70 in between, the emission and detection directions can be made coaxial between the beam splitter 70 and the target 21, and the area 31 from which light is detected by the sensor 30 is centered on the target 21, regardless of the distance to the target. With the laser 20, the sensor 30 together with the filter 35, the collecting lens 38 and the beam splitter 70 fixedly mounted relative to each other on the support 40, the system can be directed to different targets to be imaged by moving the support, for example by an actuator (not shown in this embodiment), and light from the target area will be incident on the sensor 30 in the same way, regardless of the distance of the target. Alternatively, the coaxial illumination and sensor directions can be directed to different targets while the support 40 remains fixed, for example by steering optics such as a scanning mirror mechanism located in the coaxial optical path.

[0093] Figure 9 shows a variation of the embodiment shown in Figure 8. Whereas in Figure 8 the light from the laser is already collimated when it enters the beam splitter 70, in this embodiment the laser is replaced by a diverging light source 22, i.e. a collecting lens 38 for collimating this light is placed downstream of the beam splitter 70 in the light path transmitted through the beam splitter. This lens 38 is therefore also used to focus the chlorophyll fluorescence from the target area 21 onto the beam splitter 37, where about half of the light is reflected through the filter 35 to the optical sensor 30. By positioning the light source at the transmission focus of the lens 38 and the optical sensor at the reflection focus of the lens 38, this lens serves the dual purpose of collimating the light from the light source and focusing the light from the target onto the optical sensor.

[0094] FIG. 10 shows another embodiment of a system that achieves on-axis illumination and detection, in this case using optical fibers 81, 82 and a fiber optic coupler 71.

[0095] This embodiment includes a laser source 20, which should again be configured to emit collimated light having a wavelength of about 450 nm with a beam diameter of 3 mm, and a resolution of at least 16000 μmol m -2 s -1 It should be possible to deliver a saturating pulse lasting about 1 second to the target, with an average photon composite photon flux density (PPFD) of 1000 Hz. Depending on the losses from the optical fiber, more power may be needed. The laser injects light into a first end 81a of an optical fiber 81. This optical fiber transmits the light to an optical fiber coupler 71, which splits this input light, as far as possible, so that 50% continues towards the second end 81b of the first fiber 81 and 50% is directed towards the second optical fiber 82, whereupon it travels towards the second end 82b of the second optical fiber 82 as soon as it is directed. The light directed towards the second end 82b of the second optical fiber 82 is not used further in this embodiment.

[0096] The second end of the first optical fiber is attached to the movable platform 40. This second end of the fiber is coupled to a fiber collimator 81c, which ensures that the light emitted from the fiber is converged into a substantially collimated light beam that can be directed to the target 21. This fiber collimator 81c can be a separate component attached to the end of the fiber or can be integrated into the second end 81b of the first optical fiber 81. Preferably, the optical fibers 81, 82 have a low numerical aperture aperture, with a diameter on the order of 100 nm. The relatively narrow optical fibers and the low numerical aperture aperture ensure that tight collimation of the output light can be more easily achieved. The movable platform can be moved by an actuator, thus moving the end of the optical fiber and the collimator to controllably direct the light to different target areas.

[0097] Chlorophyll fluorescence from the target 21 enters the fiber collimator 81c, which acts to couple this light to the second end 81b of the optical fiber, whereupon the light is transmitted to the optical fiber coupler 71. The optical fiber coupler thus splits the chlorophyll fluorescence so that, wherever possible, 50% continues towards the first end 81a of the first fiber 81 and 50% is directed into the second optical fiber 82, whereupon it travels towards the first end 82a of the second optical fiber 82.

[0098] The chlorophyll fluorescence traveling towards the first end 82a of the second optical fiber is transmitted through a fiber Bragg grating or thin film filter 35, which should be configured to transmit only light in the range of 650 nm to 750 nm to filter out the light from the laser. Finally, the filtered light is received at a coupled optical sensor 30, which detects the intensity of the chlorophyll fluorescence.

[0099] FIG. 11 shows a further variation of the embodiment shown in FIG. 8. In addition to the non-imaging optical sensor 30, this embodiment includes an imaging optical sensor 32, i.e. a camera. The non-imaging optical sensor 30 is arranged with a light splitter 70 to define a coaxial illumination and a second direction, but the camera 32 is arranged at a different angle to the illumination direction. The camera 32 in this embodiment has a wider field of view than the non-imaging optical sensor 30 and defines a field of view 33 that squares the field of view 31 of the non-imaging optical sensor 30. For example, the non-imaging optical sensor 30 may receive light from an area of ​​200 square degrees or less, while the camera 32 may receive light from an area of ​​at least 2000 square degrees. In this embodiment, the camera 32 is also mounted on the support 40 and moves together with the laser 20 and the non-imaging optical sensor 30. However, since the camera may have a wide field of view, it is also possible for the camera to be fixedly mounted such that the light source and the optical sensor are moved relative to the camera by an actuator. In this embodiment, the camera may be used for several functions. First, the camera may determine the distance to the target using the same parallax techniques as described above. Second, the camera may be used to direct a laser, for example, to ensure that the crop is illuminated and not the background, and to build up an image of the canopy to provide information about the nature of the area where chlorophyll fluorescence is being measured, for example. This embodiment thus offers the advantages of both imaging and non-imaging sensors. It will be appreciated that any of the above embodiments can be modified to use a non-imaging sensor for chlorophyll fluorescence measurement and have a separate imaging sensor, such as a camera.

[0100] 12-14 illustrate several different ways of operating the above-described embodiments, particularly with respect to driving the light source used to illuminate the crop to take chlorophyll fluorescence measurements.

[0101] Figure 12 is a graph illustrating the first measurement technique. The y-axis represents PPFD, the amplitude or intensity of the light illuminating the target area in μmol / m -2 s -1The graph is shown in units of 1000 μmol m and the x-axis shows time. As can be seen in the graph, in a first measurement phase the target is illuminated with a series of measurement pulses (ML) to perform a so-called F0 measurement. Each measurement pulse is formed by changing the light from an initial off state to an on state with an intensity specified for the F0 measurement. These measurement pulses can be repeated a series of times in the first measurement phase. Each measurement pulse has an intensity of about 6000 μmol m -2 s -1 The PPFD is set to about 0.1 s to 10 ms and has a duration of about 1 μs to 10 ms. The period between each pulse can be 0.1 s to 10 s. In a second measurement phase, the plant is irradiated with a saturation pulse. The saturation pulse is set to about 0.8 s. In the saturation pulse, the light is initially irradiated with about 8000 μmol m -2 s -1 The light is turned on with a first amplitude of about 14000 μmol m -2 s -1 Pulsed to a higher amplitude of 8000 μmol m -2 s -1 These superimposed pulses have a duration of about 1 μs to 10 ms and a period of about 10 ms to 100 ms, which corresponds to the measurement process described above.

[0102] Figure 13 is a graph illustrating a second measurement technique. Again, the y-axis represents PPFD and the x-axis represents time. The measurement process again begins with a first measurement phase measuring F0, which may be the same as that described above with respect to Figure 12, at approximately 6000 μmol m -2 s -1 The method includes one or more measurement pulses of PPFD of about 1 μs to 10 ms and a duration of about 1 μs to 10 ms. The period between each pulse can be 0.1 s to 10 s. However, the method differs in the saturation pulse, which in this case is divided into a series of discrete different pulses. In particular, the saturation pulse has a duration in the range of 0.1 ms to 100 ms and a concentration of about 2000 to 8000 μmol m -2 s -1 and a series of first pulses each having an amplitude of 0.01 μs to 100 μs, typically 10 μs duration and 8000 to 24000 μmol m-2 s -1 The amplitude, duration and spacing of these pulses are adjusted so that the average PPFD during the second measurement phase is 8000 μmol m -2 s -1 The first and second pulses alternate in a second measurement phase lasting about 0.8 s. The second pulse is the measurement pulse (ML) and occurs 100 μs after the first pulse has ended. In this embodiment, the first pulse saturates the reaction centers of the plants in the target area, and then the second pulse allows the so-called Fm measurement.

[0103] Figure 14 is a graph illustrating a third measurement technique. Again, the y-axis shows PPFD and the x-axis shows time. The measurement process again begins with a first measurement phase that measures F0. The strength and timing of the pulses are adjusted so that the average PPFD delivered to the target is less than 0.1 μmol m-2 in this first measurement phase. -2 s -1 As shown in FIG. 14, this can be achieved using measurement pulses with a first frequency, although in some cases there may be only one measurement pulse in the first measurement phase. The second measurement phase is composed of a series of pulses with the same intensity and duration as the pulses in the first measurement phase. However, here the frequency of the pulses is selected so that the average PPFD delivered to the target is about 8000 μmol m -2 s -1 The pulses in the first and second measurement phases are set to have the same intensity and duration, differing only in frequency, which simplifies the manufacture and control of the system.

Claims

1. 1. A system for measuring chlorophyll fluorescence, comprising: at least one light source configured to emit substantially collimated or focused light to illuminate an area, the at least one light source including a laser configured to illuminate a spot-shaped area; an optical sensor configured to detect chlorophyll fluorescence from each illuminated area, the optical sensor having a field of view of 400 square degrees or less; an actuator configured to controllably direct the light from the light source to enable the light source to illuminate a plurality of different areas; an actuator configured to move the optical sensor and / or move one or more optical elements relative to the optical sensor to enable the optical sensor to detect chlorophyll fluorescence from each illuminated area; Including, system.

2. the actuator configured to move the optical sensor and / or move one or more optical elements relative to the optical sensor is the same actuator configured to direct the light from the light source. The system of claim 1 .

3. the optical sensor has a field of view that is smaller than the total space accessible for illumination by the at least one light source, preferably less than 10%, more preferably less than 5%, even more preferably less than 1%, and most preferably less than 0.5% of the total space accessible for illumination by the light source; 3. The system according to claim 1 or 2.

4. the optical sensor has a field of view of 200 square degrees or less, preferably 50 square degrees or less, and most preferably 25 square degrees or less; 3. A system according to claim 1 or 2.

5. the at least one light source and optical sensor are fixedly mounted relative to one another; 3. A system according to claim 1 or 2.

6. The optical sensor includes an imaging sensor such as a camera.

3. A system according to claim 1 or 2.

7. a controller configured to distinguish, in the measurement image, a target area including the area illuminated with light from the at least one light source and at least one background area; the controller is further configured to adjust the detected intensity in the target area based on the detected intensity in the background area. The system of claim 6.

8. The controller is preferably configured to select a portion of the measurement image outside the target area as the background area based on the detected intensity across the measurement image. The system of claim 7.

9. the controller is configured to adjust the detected intensity in the target area based on the detected intensity in the background area by calculating an average background intensity over the background area and subtracting the average background intensity from the detected intensity in the target area.

8. The system of claim 7.

10. The system further includes a controller configured to identify a target area in a measurement image of the imaging sensor; the target area is the central portion of the illuminated spot-shaped area; 7. The system of claim 6.

11. the optical sensor is a non-imaging optical sensor; the system further includes an imaging optical sensor configured to capture an image of the area illuminated by the light; Preferably, the imaging optical sensor has a wider field of view than the non-imaging optical sensor.

3. A system according to claim 1 or 2.

12. an optical filter positioned to block reflected light from the at least one light source and to allow the optical sensor to detect light resulting from chlorophyll fluorescence.

3. A system according to claim 1 or 2.

13. The optical filter is an interference filter. The system of claim 12.

14. a controller configured to determine a target distance based on a position, size, and / or shape of the illuminated area within the field of view of the optical sensor.

3. A system according to claim 1 or 2.

15. The or each light source may have a working distance of 50 cm at at least one working distance in the range of 0.1 m to 20 m, preferably in the range of 0.1 m to 10 m, more preferably in the range of 0.1 m to 5 m, and most preferably in the range of 0.5 m to 1 m. ２ Less than 25 cm ２ Less than 5 cm, more preferably ２ less than, and even more preferably less than 1 cm ２ Less than 5 mm, most preferably ２ configured to illuminate an area of ​​less than 3. A system according to claim 1 or 2.

16. a) illuminating a first area with light using the at least one light source; b) detecting chlorophyll fluorescence from the first area using the optical sensor; and c) redirecting the light from the at least one light source using the actuator; and d) illuminating a second area with light using said at least one light source; e) detecting chlorophyll fluorescence from the second area using the optical sensor; and and a controller configured to:

3. A system according to claim 1 or 2.

17. a controller configured to control the at least one light source to irradiate each area with light such that in a first measurement phase, the light is modulated in intensity to define one or more pulses of light having a low average intensity, and in a second measurement phase, the light is modulated in intensity to define a plurality of discrete, different pulse types that together have a high average intensity.

3. A system according to claim 1 or 2.

18. a controller configured to control the at least one light source to irradiate each area with light such that, in a first measurement phase, the light is modulated in intensity to define one or more discrete pulses of light that constitute a first proportion of the first measurement phase and have a low average intensity, and in a second measurement phase, the light is modulated in intensity to define a second proportion of the second measurement phase that is greater than the first proportion of the first measurement phase and have a plurality of discrete pulses that have a high average intensity.

3. A system according to claim 1 or 2.

19. the pulses in the first measurement phase have a first frequency; the pulses in the second measurement phase have a second frequency higher than the first frequency; Preferably, the pulses in the first and second measurement phases have the same intensity and duration.

20. The system of claim 18.

20. 1. A method for measuring chlorophyll fluorescence, comprising: a) illuminating a first area with light using at least one light source configured to emit substantially collimated or focused light, the at least one light source including a laser configured to illuminate a spot-shaped area; b) detecting chlorophyll fluorescence from the first area using an optical sensor, the optical sensor having a field of view of 400 square degrees or less; c) using an actuator to redirect the light from the at least one light source and to move the optical sensor and / or one or more optical elements relative to the optical sensor to enable the optical sensor to detect chlorophyll fluorescence from each illuminated area; d) illuminating a second area with light using said at least one light source; e) detecting chlorophyll fluorescence from the second area using the optical sensor; and Including, method.

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