Optical microscope having multiple arrays of photon-counting detector elements and method thereof

Abstract

A method of operating an optical microscope includes emitting and directing illumination light (2) as multiple illumination light beams from one or more light sources (1) toward a specimen positioning location (6A) to form multiple separated illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning location (6A), and directing detection light beams (11) emanating from the illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning location (6A) to a detector (10) comprising multiple sensor arrays (31-34), each sensor array (31-34) comprising photon-counting detector elements (40), the detection light beams (11) forming multiple light spots (15) on the sensor arrays (31-34), and the detection light beams (11) from different illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning location (6A) are directed to different sensor arrays (31-34). The measurement signals from the sensor array (31-34) are analyzed to determine position information about the light spot (15) on the sensor array (31-34). In an adjustment process, the location where the light spot (15) impinges on the sensor array (31-34) is adjusted based on the position information. A corresponding optical microscope is further disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to optical microscopes and methods of operating optical microscopes. [Background technology]

[0002] Optical microscopes are used in a wide range of applications, such as in life sciences or materials testing.

[0003] Specifically, growing interest in living cells requires optical microscopes with particularly high sensitivity. The peak intensity of the excitation light illuminating the specimen should be low to avoid phototoxic effects in biological tissues caused by high light intensity. However, to collect the desired information from the specimen, the acquired data must have a sufficient signal-to-noise ratio (SNR). Therefore, noise sources other than quantum noise should be avoided.

[0004] In common laser scanning microscopes (LSMs), fluorescence is typically detected using photomultiplier tubes (PMTs), which convert the photon flux into a highly amplified current. However, the amplification process adds multiplication noise to the current signal. Thus, the SNR of the measured signal is reduced relative to the quantum limit of the photon flux in a Poisson distribution. This is even more true when reducing the photon flux to reduce phototoxicity and simultaneously increasing the PMT gain to achieve a certain dynamic range in the acquired image.

[0005] In principle, multiplication noise can be completely avoided by photon counting. In this case, the electrical signal is amplified, allowing the identification of single pulses that can be assigned to single-photon detection events. These pulses are counted, and the number of pulses is a measure of the intensity of the photon flux striking the sensor within a specific period. Amplification noise merely produces a statistical distortion of the pulse height, but does not affect the number of pulses, and therefore does not adversely affect the measurement.

[0006] However, after a pulse triggered by a photon striking the sensor is detected, the sensor cannot register another photon striking the sensor and must be reset to a photosensitive state. The period during which no additional photons are detected is called the dead time and lasts from the moment the first photon is detected until the sensor is fully reset and detects the second photon. The dead time can reach tens of nanoseconds, reaching the count rate limit of the photon-counting sensor. The count rate limit of a single detector element is typically on the order of a few megahertz.

[0007] The count rate limit can be increased by approximately a factor of 10 by distributing the detection point spread function (PSF) over an array of at least 10 photon-counting detector elements. In general, the count rate limit can be increased by N if the signal is distributed over at least N elements, depending on the spatial distribution of light intensity. Such an array may include single-photon avalanche diodes (SPADs). In this case, photons of detected light, e.g., fluorescence, impinge on the sensor array in a statistically distributed manner relative to their lateral positions on the array. Thus, the probability that a photon will strike a sensor element that has just been reset (and thus within the dead time) is greatly reduced. Therefore, the count rate limit depends on the size of the photon-counting array, i.e., the total number of photon-counting detector elements that can be illuminated, and the spatial distribution of light intensity.

[0008] Since the SNR of a Poisson counting process is proportional to the square root of the number of detected events, a 10-fold signal increase increases the SNR by approximately a factor of 3. However, an increase in the peak intensity of the illuminating light (excitation light) should be avoided, which also limits the number of illuminated detector elements.

[0009] To further increase the SNR while avoiding unreasonably high excitation intensities, the signal from a particular sample location should be averaged over an extended effective pixel dwell time. This increases the number of photons detected from that location, and because the SNR depends on the square root of the number of photons, it also improves image quality. Averaging can be performed in various ways. For example, the pixel dwell time can be directly increased by reducing the frequency of the LSM's scanning mirror. Alternatively, the same sample location can be scanned several times, and then various data values ​​are added. However, both approaches extend the image acquisition time of conventional LSMs, which can be undesirable, for example, when biological processes in living specimens are investigated.

[0010] Multiple laser spots can be scanned simultaneously across a specimen without increasing image acquisition time. Thus, a typical method of operation of an optical microscope includes emitting and directing illumination light as multiple illumination light beams from one or more light sources toward a specimen positioning location, forming multiple separated illumination light spots at the specimen positioning location, and directing detection light beams emanating from the illumination light spots at the specimen positioning location to a detector comprising multiple sensor arrays. Each sensor array comprises photon-counting detector elements, and the detection light forms multiple (detection) light spots on the sensor array, with the detection light beams from different illumination light spots at the specimen positioning location being directed to different sensor arrays.

[0011] Similarly, a typical optical microscope includes at least one light source and optical elements, such as an objective lens, a condenser lens, or other lenses or mirrors, for illuminating a specimen at a specimen positioning location with multiple illumination light beams that form multiple separated illumination light spots at the specimen positioning location. The microscope also includes a detector having multiple sensor arrays. Each sensor array includes a photon-counting detector element for measuring light spots formed on the sensor array by detection light beams emanating from the specimen. Detection light beams from different illumination light spots at the specimen positioning location are directed to different sensor arrays. The microscope also includes a controller for controlling at least one or more light sources and detectors.

[0012] However, increasing the number of light spots used simultaneously to scan across the specimen also increases the number of photon-counting detector elements required, and each of these pixel elements contributes to the overall dark noise of the detector, so increasing the number of pixel elements can also adversely affect the SNR. Summary of the Invention

[0013] It is an object of the present invention to provide an optical microscope and method that achieves particularly high image quality and measurement sensitivity without excessively long measurement periods.

[0014] The above object is achieved by a method comprising the features of claim 1 and an optical microscope as defined in claim 13.

[0015] Preferred embodiments are presented in the following description, particularly in combination with the accompanying drawings, as well as in the dependent claims.

[0016] The above-mentioned method according to the invention is characterized by at least the step of analyzing measurement signals from the sensor array to determine position information about the light spots on the sensor array and, based on the position information, performing an adjustment process to adjust the locations where the light spots impinge on the sensor array.An optical microscope of the above-mentioned kind according to the invention is characterized in that the controller is configured to analyze measurement signals from the sensor array to determine position information about the light spots on the sensor array and, based on the position information, to command an adjustment device to adjust the locations where the light spots impinge on the sensor array.

[0017] By using multiple light spots and ensuring accurate alignment of the light spots on the sensor array, a large proportion of all sensor elements can be efficiently used for actual measurements. Sensor elements that are not illuminated and contribute to background noise can generally be avoided. The light spots can have a relatively small pitch that may be suitable for scanning a predetermined sample area, and the sensor array is nevertheless properly positioned to accurately distinguish the light spots. A situation in which a light spot only partially hits the sensor array can be avoided.

[0018] Adjustment of the relative position between the detection light spot and the sensor array In the adjustment process, one or more of the following adjustments may be performed in response to the measurement signal from the photon-counting detector element:

[0019] The adjustment process includes adjustment steps that affect the positional relationship between all detection light spots and all sensor arrays, particularly uniformly. The process may further include additional individual adjustments of the positional relationship between a single detection light spot and the sensor array on which it impinges. The individual adjustments may be achieved so that the distance between the detection light spots matches the distance between the sensor arrays. The (collectively) adjustment of the positional relationship between all detection light spots and all sensor arrays may be achieved so that the array of detection light spots as a whole is properly aligned.

[0020] Generally, adjusting the location where the light spot impinges on the sensor array can be performed by relative movement of the light spot and the sensor array. For example, the optical path of the detection light can be adjusted by a movable optical element, such as a reflective or refractive element. Specifically, a common optical element can be adjusted, and all detection light beams move through this common optical element, for example, a tiltable glass plate. Alternatively or additionally, the sensor arrays can be moved as a whole or independently of each other during specimen observation, during an initial calibration procedure, and / or during microscope or detector manufacturing. The sensor arrays can be moved jointly, particularly in a plane perpendicular to the optical axis, so as to transverse the optical axis of the detection light beams impinging on the sensor array. These variations are described in more detail below.

[0021] Although the sensor array is typically arranged perpendicular to the optical axis of the detection light beam, the sensor array can also be tiltably mounted. For example, a printed circuit board (PCB) on which all the sensor arrays are arranged can be supported so that it can be tilted relative to the optical axis of the detection light beam. In this case, all the sensor arrays can be tilted jointly. The sensor array can be tilted according to the difference between the detection light beams, for example, when the axial positions of the detection light beams are different from each other. The detection light beams may also have different intensities, in which case it may be useful to vary the light spot size on the sensor array by tilting the sensor array. The controller can be configured to instruct the adjustment device to perform tilting according to the measurement signal from the photon-counting detector element and / or according to how the light source is controlled.

[0022] The adjustment may also include performing a relative rotation between the sensor array and the detection light beam. In these cases, the rotation axis is parallel to the optical axis of the detection light beam. For example, the sensor arrays may be rotated jointly (i.e., by rotating a common component, such as a PCB, on which all sensor arrays are located). Alternatively, an image rotator, such as an image-rotating prism, may be placed in the beam path of the detection light beam and adjusted to avoid rotational mismatch between the placement of the sensor array and the light spot on the sensor array.

[0023] Furthermore, an optical zoom element may be provided in the beam path of the illumination light beam and / or the beam path of the detection light beam. Because all light beams are directed through this zoom element, the pitch between the light beams, and thus between the light spots on the sensor array, can be changed by adjusting the zoom element. The zoom element may be adjusted so that the pitch of the detection light spots matches the pitch of the sensor array.

[0024] Adjusting the position of the sensor array Adjustment of the light spot impinging on the sensor array may also or additionally be performed during the manufacturing of the detector. In such cases, multiple sensor arrays are first movably positioned and operably connected on a common PCB so that the detector elements are fully functional and readable. For example, the bonding material may be heated to allow fine adjustment movement between the sensor array and the PCB. Next, illumination light is emitted to form multiple light spots on the sensor array. As described elsewhere, the illumination light may itself form light spots on the sensor array, or it may impinge on a specimen or reference object that emits detection light, such as fluorescence. The light source used here may be the same or different from the light source used for specimen observation. The light spots may form the same array used for specimen inspection, but at a lower or different intensity. A controller interprets the measurement signals of the sensor array, e.g., photon count values, and outputs positioning commands according to the movement of the sensor array. For example, the controller may determine the spot positions from the measurement signals, and the positioning commands may adjust the spot positions to align with the centers of the respective sensor arrays. Alternatively, the controller may determine a total signal for each sensor array (e.g., photon counts summed or averaged over all detector elements of a sensor array), and positioning commands are selected to maximize the total signal. An optimization routine may be used to adjust the position of the sensor array based on the measured signal. Each sensor array may be formed by a respective chip and may be moved independently of the other chips. Alternatively, some or all of the sensor arrays may be formed by one chip that is moved relative to the PCB.

[0025] Adjusting a movable optical element in front of the sensor array In a variation of the above procedure for adjusting the location where the light spot strikes the sensor array, the sensor array is not movable; instead, optical elements are movably positioned in front of the sensor array. The optical elements are positioned in the beam path to the sensor array and can move independently of each other to independently affect the position of the light spot on the sensor array. The number of optical elements may match the number of sensor arrays, or alternatively, adjacent sensor arrays may share a common optical element. Examples of optical elements are tiltable glass plates or lenses, movable transparent wedges, or reflective elements. The optical elements may be positioned directly on the sensor array, or may be spaced apart from the sensor array but coupled to the sensor array to ensure a stable positional relationship with the sensor array after adjustment. Light travels through the optical elements to the sensor array, and measurement signals from the sensor array are supplied to a controller as described above. The controller generates positioning commands based on the measurement signals, and the optical elements are moved according to the positioning commands. These steps may be repeated in an optimization routine until the light spot is centered at the sensor array.

[0026] For example, the optical element may be a tiltable transparent plate / glass plate placed in front of or above the sensor array, with each glass plate supported so that it can be tilted in two directions relative to the optical axis (e.g., towards X and Y axes perpendicular to the Z axis coinciding with the optical axis).

[0027] The above procedure may be carried out during manufacture of the detector, and the optical elements may optionally be secured with adhesive once properly tilted according to the positioning commands.

[0028] Alternatively, the above procedure may be performed as a dynamic readjustment before or during sample measurement. Specifically, in these cases, the optical element may be mounted on a motorized unit to allow automatic alignment with the sensor array.

[0029] Location-based binning The binning pattern for multiple superpixels can be variably set for the detector. Each superpixel is formed by jointly reading out several photon-counting detector elements to generate a common photon count value. Each detector element can be deactivated or assigned to one of the superpixels. The binning pattern can be set according to predetermined position information. For example, the center position of each light spot on the sensor array can be determined from the position information, and then the superpixels can be aligned with the center position. Specifically, one or more superpixels can be assigned to each light spot and arranged symmetrically with respect to the respective light spot. In the case of circular or ring-shaped superpixels, symmetrical arrangement is understood to mean that the center of the circle or ring shape coincides with the determined center position of each light spot. In addition, the number of detector elements binned into the same superpixel can be set according to the position information, particularly based on the cross-sectional shape or size of the light spot. If the light spots have different sizes, the superpixels for different light spots can also be set to have different sizes, i.e., to include different numbers of detector elements. Setting the binning pattern may form part or all of the adjustment process, or alternatively, setting the binning pattern may form a separate process in addition to the exemplary adjustment process described.

[0030] Sensor Array Design In some embodiments, each sensor array is illuminated by one of the detection light spots. The sensor arrays may be formed by different regions of the same chip or by different chips. These designs are further described below with reference to the drawings and have in common that the sensor arrays are located on a common PCB or in a common chip package. The sensor arrays shall be precisely aligned in a matrix corresponding to the matrix pattern of the light beams / light spots. Typically, the pattern of the light spots remains fixed, so the placement of the sensor arrays should be selected accordingly. The pitch (distance) between the sensor arrays should match the pitch of the light spots. Furthermore, the relative positions of the light spot array as a whole and all the sensor arrays with respect to the detector shall be adjusted to ensure proper alignment.

[0031] Using a common PCB or package has several advantages, particularly the fact that the sensor arrays are positioned in precisely defined positions relative to one another. The distance between the sensor arrays remains stable even if the detectors are subjected to shocks or temperature changes during transportation. The system can also be robust against misalignment between various optical paths. Precise timing between detector elements of different sensor arrays is more easily achieved due to short power lines and / or symmetrical signal trees, e.g., symmetrical clock trees. Because the sensor arrays are arranged adjacent to one another as modules on a manufacturing wafer, arranging multiple sensor arrays on a common PCB allows for higher chip production density, e.g., through CMOS fabrication. Such sensor arrays can be used in numerous applications depending on how many modules are combined in one package or one PCB, while the spacing between the sensor arrays is adjustable. Sensor arrays can be integrated into a single large sensor array without spacing between the individual sensor arrays.

[0032] For image scanning techniques (Airy scanning or photon relocation), the sensor arrays should be aligned with respect to each other to an accuracy of approximately one-tenth of the PSF diameter. This avoids artifacts in the final image due to misalignment. As an illustrative example, the PSF imaged on one of the sensor arrays may cover an area of ​​five SPAD pixels along the PSF diameter. Alignment should then be achieved with an accuracy of half the pixel pitch, which may be approximately 10 μm to 20 μm. Alignment with such accuracy requires an optical control mechanism when positioning the arrays on the PCB. Positioning can be achieved automatically by robotic operation, so the sensor arrays can remain fixed in place before and during soldering. Precision manufacturing methods such as pick-and-place, which are common for surface-mount devices, are applicable to positioning and soldering large chips, but with significantly smaller optically active sensor areas. A similar approach can also be applied to precisely position multiple sensor arrays within the same package before bonding.

[0033] In some variations, the sensor arrays are placed directly adjacent to each other to form a common array within a single chip or on a single printed circuit board.

[0034] There may be multiple bonding pads for each sensor array. At least some of the sensor arrays may be positioned directly adjacent to one another without any bonding pads between them. For example, the sensor arrays may be rectangular with bonding pads on only two of the four sides of the rectangular shape. The total number of bonding pads for outputting measurement photon counting signals is optionally less than the total number of photon counting detector elements, in which case the measurement photon counting signals of some photon counting detector elements are output through the same bonding pad.

[0035] Each photon-counting detector element may comprise at least a first memory element and a second memory element, and may allow readout of a measurement signal from the second memory element during an exposure time during which a photon detection event may be recorded in the first memory element of the photon-counting detector element. Each photon-counting detector element may optionally be formed by a single-photon avalanche detector with a SPAD anode forming the first memory element, the second memory element being configured to receive the measurement signal from the first memory element.

[0036] In some variations, the photon-counting detector elements of the same sensor array are arranged in columns and rows. A common readout line can then connect the photon-counting detector elements of the same column to one of the bonding pads. The row address for the photon-counting detector elements can be used to distinguish measurement signals from photon-counting detector elements of the same column.

[0037] Alternatively or additionally, to reduce the number of required bonding pads, a multi-bit counter may be provided, which counts several photon detection events of the same or different photon-counting detector elements.

[0038] Furthermore, multiple through silicon vias may additionally or alternatively be provided for each sensor array, with some of the photon-counting detector elements preferably sharing one of the through silicon vias.

[0039] General Features An illumination light spot can be formed by a light distribution of illumination light. Similarly, a detection light spot can be formed by a light distribution of detection light. The location of a light spot can be understood as the center of mass of the light distribution. A light distribution can generally have any cross-sectional intensity distribution that can form a continuous region or two or more separated regions. Specifically, a light distribution can include two or more separated regions, such as those used in twisted or double-helix PSFs and / or for 3D encoding of PSFs. A detection light spot can therefore be formed by a detection light distribution that generally includes laterally separated regions of any shape, as understood herein. Different detection light spots generally overlap on the detector and are distinguished from each other in that they are caused by different illumination light distributions.

[0040] By directing detection light beams from different illumination light spots to different sensor arrays, each detection light spot / distribution can be measured with a separate sensor array. Alternatively, some detection light beams from different illumination light spots can be directed to different sensor arrays, while some detection light beams share the same sensor array. The number of sensor arrays may be equal to the number of detection or illumination light spots. Alternatively, the number of sensor arrays may also be greater than the number of spots, in which case one or more sensor arrays do not receive detection light emanating from one of the illumination light spots. These sensor arrays may be temporarily deactivated or used for other purposes, such as calibration, normalization, or triggering. In the case of twisted PSFs or 3D encoding of PSFs, one sensor array receives two or more laterally separated light spot portions belonging to the same PSF / light distribution.

[0041] The light spots formed on the sensor array can have any shape. An illumination light beam with a circular cross section can give rise to a Gaussian distribution of the (detection) light spots. Alternatively, each illumination light beam can have an elongated cross section and give rise to a line of light spots on the sensor array. Multiple illumination lines may be preferred for scanning the specimen to increase the image acquisition speed. The light beams may have common or individual beam forming elements. The light distributions of the various beams may also be different.

[0042] An optical microscope may be defined as comprising a specimen holder providing a specimen positioning position and an objective lens, in particular an infinity-corrected objective lens, which sets the image distance to infinity. The specimen positioning position is to be understood as the position where the specimen to be examined should be placed. Depending on the current situation, a reference objective lens may be used instead of the specimen, or no objective lens may be used at all, for example for a specific calibration step. The objective lens may be arranged to direct or focus illumination light onto the specimen. Additionally or alternatively, the objective lens may be arranged to receive detection light emanating from the specimen and direct the detection light towards the sensor array. Typically, separate illumination and detection objective lenses may be used.

[0043] The adjustment device may be configured to perform any of the adjustment processes described herein. In particular, the adjustment device may include one or more motors, actuators, piezoelectric elements, or other means suitable for moving the described components for the adjustment process. These components may in particular include wedges, tiltable glass plates, optical zoom elements, other optical elements, spatial light modulators, or the detector itself. Depending on the components to be controlled, the adjustment device may also be formed by electronics without moving elements. In particular, the adjustment device may include or be formed by a processor or other computing unit configured to set the binning pattern of the detector. In some variations, the adjustment device and the controller are formed by a single unit.

[0044] An optical microscope may further include a tube with an imaging lens positioned in the beam path between the objective lens and the detector to focus the light from the objective lens onto an (intermediate) image plane. Unlike other optical systems, an optical microscope generates at least one intermediate image plane. The optical microscope may also include an illumination port to which a light source, e.g., one or more lasers, is connected. The detected light emitted from the specimen may be any type of light, e.g., fluorescence or phosphorescence, illumination light scattered, reflected, diffracted, or affected by the specimen via other mechanisms, illumination light transmitted through the specimen, e.g., linear laser excitation, multiphoton excitation, phosphorescence, CARS (coherent anti-Stokes Raman scattering), SRS (stimulated Raman scattering), or light emitted from the specimen for other reasons that may be at least partially caused by the illumination light due to incoherent or partially coherent optical excitation. Generally, the detected light may also be emitted by processes unrelated to the illumination of the specimen. Furthermore, the term detected light is also used in reference or test measurements and can refer to, for example, any light that would be measured by a detector when a (other) light source illuminates the detector and no sample is provided.

[0045] Illumination light beams are understood to be light beams directed to different locations laterally at the specimen positioning location. The illumination light beams may share some or all optical elements; for example, all illumination light beams may be directed through the same objective lens. The distinction between illumination light and detection light (beam) refers to whether the light travels toward the specimen positioning location or from the specimen positioning location toward the detector. However, these terms do not necessarily refer to different wavelengths or characteristics of the illumination light and detection light. The terms "detection light spot" and "light spot on the sensor array" are generally used synonymously. In contrast, illumination light spots refer to light spots spaced apart from one another at the specimen positioning location. In some described variations, the illumination light spots are light spots spaced apart laterally within the specimen plane at the specimen positioning location. However, these variations may also be modified so that the illumination spots are spaced apart axially from one another at the specimen positioning location. The axial distance may additionally or alternatively be calculated relative to the lateral distance between the illumination light spots. In particular, the illuminating light beam may be formed as described in WO2015 / 121188A1.

[0046] Position information about a light spot on a sensor array can be understood as any information derived from the measurement signals of detector elements that indicates or depends on the position of the light spot on the sensor array. For example, position information may refer to information describing which or how many detector elements of one sensor array measure a signal (e.g., have a photon count rate above a predetermined value). Position information may also refer to the center of the light spot relative to the corresponding sensor array. Also, photon count values ​​aggregated from one, several, or all sensor arrays may be used as position information, since the photon count value(s) depend on whether the position of the detected light spot is aligned with the sensor array.

[0047] The adjustment process described herein may comprise a single step of adjusting one of the described components, or it may comprise a routine in which a component is adjusted several times, with the measurement signal from the detector element being analyzed to determine how to adjust the component. For example, a transparent glass plate through which all detection light beams (but not the illumination light beams) pass may be tilted to different angles. The controller may determine the tilt angle at which the position of the detection light spot best matches the position of the sensor array, for example, by determining the maximum value of the total photon counts of all sensor arrays as a function of tilt angle. Each total photon count may constitute position information for the current tilt angle setting. The photon counts may be derived at any stage in the signal chain or from the respective acquisition screen. The routine may first take measurements at different tilt angles and then analyze the position information, or the routine may analyze measurements as they are acquired to determine the next tilt angle to be tested, or more generally, the next setting of the component to be adjusted. The adjustment process may also include adjustment of several components, for example, components that affect all detected light beams and one or more components that affect only one or a portion of the detected light beams.

[0048] The term "measurement signal" refers to the output of, or a signal derived from, one or more photon-counting detector elements. The term may be used synonymously with photon-counting signal. A photon count value, i.e., a number indicating the number of photons detected, may be derived from the measurement signal, e.g., the number of photons detected.

[0049] The optical microscope may further include a scanner disposed between the objective lens and the light source. The controller controls the scanner to perform a specimen scan in which the illumination light is scanned over the specimen. Optionally, the scanner may also direct the detection light coming from the objective lens toward the photon-counting detector array (non-scanning configuration). The scanner may be understood as a device configured to adjustably deflect a light beam. The scanner may include one or more movable optical elements, such as a mirror, a lens, or a prism. Alternatively, the scanner may also adjustably deflect the illumination light based on the acousto-optical effect or the electro-optical effect.

[0050] The PSF can be understood to define how illumination and detection light are guided by an optical microscope. Specifically, the PSF can be considered to consist of an illumination PSF and a detection PSF, where the illumination PSF defines how a point of the light source is imaged onto the specimen plane, and the detection PSF defines how a point located on the specimen plane is imaged onto the plane of the sensor array. The size of the detected light spot on the sensor array can correspond to the PSF size in image scanning techniques or confocal imaging. The sensor array can be positioned in or near the image plane or an intermediate image plane. However, the sensor array can also be located away from the image plane, e.g., in or near the pupil plane or a plane with a separated pupil.

[0051] The photon-counting detector element, also referred to for the sake of brevity as "detector element", may in particular be a SPAD (single photon avalanche diode) that is in particular operated in the so-called Geiger mode, in which an excessive bias voltage causes a voltage V OP is applied to the diodes of the SPAD. As a result, photon absorption can cause charge avalanches and thus countable events. Accordingly, the sensor array may be referred to as a SPAD array.

[0052] The controller may comprise electronic components such as an FPGA or processing unit that may be formed as a single unit or a distributed system. The functionality of the controller may be implemented as software and / or hardware. The controller or parts thereof may be specifically located as an on-chip unit adjacent to the sensor array. Portions of the controller may also be provided via a server or computer application that communicates with other components of the optical microscope over a network.

[0053] For clarity, the expression "light spot" is often used in this disclosure to refer to a light distribution on the specimen or sensor array(s). More generally, a "light spot" may be understood as a light distribution, for example, a ring-shaped pattern, one or more lines, or a spot or ring.

[0054] The present invention may be applied to other sensor devices that do not necessarily require a microscope, such as materials analysis, camera or surveillance systems, astronomy, or manufacturing monitoring. The detectors, light sources, and controllers described herein may be part of other light measurement devices for distance measurement, quality control, and monitoring, for example, in portable / handheld devices, medical devices, or vehicle sensors. In these contexts, the specimen may represent any type of object. The intended use of various embodiments of the optical microscope of the present invention results in variations on the methods of the present invention. Similarly, the optical microscope of the present invention may be configured to perform the above-described exemplary methods of the present invention. Specifically, the controller may be configured to control the sensor array or other components of the optical microscope to perform the method steps described herein. [Brief explanation of the drawings]

[0055] A better understanding of the present invention and various other features and advantages thereof will be readily obtained from the following description taken in conjunction with the following schematic drawings, given by way of example only and not of limitation, in which like reference numerals may refer to similar or substantially similar components. [Figure 1]1 shows a schematic representation of an embodiment of an optical microscope according to the invention; [Figure 2] 1 shows a schematic diagram of a detector of an optical microscope of the present invention; [Figure 3] 3 shows a schematic cross-sectional view of the detector and additional components of the optical microscope of FIG. 2. [Figure 4] 2 shows a schematic representation of another exemplary embodiment of a detector for an optical microscope of the present invention; [Figure 5] 2 shows a schematic representation of another exemplary embodiment of a detector for an optical microscope of the present invention; [Figure 6] 1 shows a schematic diagram of an exemplary design of a photon-counting detector element of a detector of an optical microscope of the present invention; [Figure 7] 2 shows a schematic representation of another exemplary embodiment of a detector for an optical microscope of the present invention; [Figure 8] 2 shows a schematic representation of another exemplary embodiment of a detector for an optical microscope of the present invention; [Figure 9] 2 shows a schematic representation of another exemplary embodiment of a detector for an optical microscope of the present invention; [Figure 10] 1 illustrates a schematic diagram of an exemplary embodiment of a modular detector for an optical microscope of the present invention; [Figure 11] 10 shows a schematic representation of another exemplary embodiment of a modular detector for the optical microscope of the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0056] Figure 1 Figure 1 shows a schematic representation of one embodiment of an optical microscope 100 of the present invention. The optical microscope comprises a light source 1 that emits illumination light 2 that is directed to a specimen positioning location 6A where a specimen 6 may be placed. Although merely optional in other embodiments, the example of Figure 1 shows the illumination light being directed through a scanner 3, optical elements 4, and an objective lens 5. The scanner 3 scans the illumination light 2 over the specimen 6, resulting in different beam paths 3A, 3B depending on the scanner position.

[0057] FIG. 1 shows a non-scanning mechanism in which detection light 11 emanating from a specimen 6 is directed to a beam splitter 7, which separates the detection light 11 from the illumination light 2, using an objective lens 5, optical elements 4, and a scanner 3. Other designs that do not use a non-scanning mechanism are possible. The detection light 11 is directed to a detector 10 using additional optical elements 8. If the optical microscope 100 is used as a laser scanning microscope, a pinhole or pinhole array may optionally be added, for example, at an intermediate image plane. Alternatively, the detector elements of the detector 10 may operate to provide the function of a pinhole by limiting the size of the detected light distribution. In the latter case, the detector may be placed directly at the intermediate image plane, thus replacing the pinhole itself.

[0058] The scanner 3 may be, for example, a galvanometer scanner, in particular a quasi-static or resonant galvanometer scanner, a MEMS (micro-electromechanical system), an acousto-optical scanner with an adjustable optical deflection element or an adjustable refractive index or a different type of scanner, including a DMD (digital micromirror device) and an SLM (spatial light modulator) or any other means for changing the direction of a light beam.

[0059] In the exemplary embodiment of FIG. 1, the detection light 11 may be fluorescent light, but in other embodiments, the detection light 11 may be generated in various ways, as described in more detail in the overview section.

[0060] To speed up measurements and improve image quality, multi-beam illumination is used. While the upper part of Figure 1 shows a simple beam path of illumination light 2, it should be understood that illumination light 2 is formed by multiple light beams, as shown in the enlarged inset at the bottom of Figure 1. The multiple light beams form multiple illumination light distributions or illumination light spots 2A, 2B, 2C, 2D at specimen positioning location 6A.

[0061] Returning again to the upper part of FIG. 1 , the light source 1 may comprise different light-emitting elements or a common light-emitting element for providing the light beam or the illumination light from which the light beam is formed. For example, a matrix element may be added to the illumination beam path (not shown) and may include a plurality of individual light transfer elements (such as holes, mirrors, or lenses) that are illuminated by the illumination light to form the separate light beams. Alternatively, a light splitting means having one or more partially reflective surfaces may be used to generate multiple light beams. Generating multiple light beams from a single light beam can be performed between the beam splitter 7 and the scanner 3 using an optical relay system, as described, for example, in DE 102014017003 A1 or WO 2018 / 073170 A1.

[0062] A controller 70 is provided for controlling at least the light source 1, the scanner 3, the adjustment device 75 and optionally the detector 10. As described in the general description, the controller 70 may be formed by any suitable hardware computing components and / or software executed by a computing unit.

[0063] The detector 10 comprises photon-counting detector elements such as SPADs and is configured to be particularly suitable for the challenge of multi-beam illumination.

[0064] Figures 1 and 2 An expanded view of an exemplary detector 10 is shown in FIG. 2. The detector 10 includes multiple sensor arrays 31-34, each with several photon-counting detector elements 40. In this example, each sensor array 31-34 is formed on a separate chip 20, and all chips 20 are mounted on a common PCB 19. In an alternative design, the sensor arrays 31-34 are formed on the same chip and then mounted on the PCB 19. The number of photon-counting detector elements 40 should be kept low, to the extent necessary for each detector element (pixel) 40 to contribute to the overall dark noise of the detector 10. Because the pixel pitch of such sensor arrays 31-34 is on the order of 10-50 μm, it is important to align the detector elements 40 with the location of the detection light spots as precisely as possible. Furthermore, the pitch of the detection light spots is related to the pitch of the illumination light spots within the specimen, and this pitch should be kept minimal so as not to overly limit the field of view scanned across the specimen and the flexibility for electronic scanning and zooming. Furthermore, the pitch of the light spots should be precisely matched respectively to obtain a constant displacement of the resulting image to fit an array optical element such as a microlens array or a pinhole array.

[0065] FIG. 2 schematically illustrates the detected light distribution / light spot 15 formed by the detected light impinging on the detector 10. Each light spot 15 is caused by one of the illumination light spots 2A-2D in FIG. 1 and is measured by one of the sensor arrays 31-34. When a confocal laser configuration is used, the light spots 15 remain in place on the sensor arrays 31-34 during scanning. As shown, each light spot 15 covers the area of ​​several detector elements 40, and an image scanning technique (Airy scan) is considered in which the point spread function (PSF) spreads across several detector elements 40. In the illustrated example, the detected light distribution / spot 15 has a circular shape. However, in other embodiments, the light distribution may take other forms and may include two or more laterally separated portions that belong to a common PSF.

[0066] Achieving particularly high image quality with photon-counting detector elements 40 is related to how many detector elements 40 are covered by the light spot 15 (which may depend on the light intensity, peak intensity, sensitivity, and other factors of the photon-counting detector elements 40). Furthermore, dark noise due to photon-counting detector elements 40 that are not or barely illuminated is a related concern. Background papers are provided in co-owned, co-pending applications PCT / EP2019 / 051927 and PCT / EP2019 / 058991. Further improvements beyond these disclosures can be achieved using multi-beam illumination, as used in FIG. 2 , in which case precise alignment of the array of light spots 15 with the sensor arrays 31-34 becomes increasingly important. For example, it may be advantageous for the light spots 15 to have a fairly short distance between each other for better scanning range, sharing of optical components by some or all light beams, or reduced size of optical components. This increases the demand for densely and precisely spaced sensor arrays 31-34. Avoiding excessively large sensor arrays 31-34 is also an objective per se to limit costs and power consumption or to avoid effects on efficiency due to, for example, background noise.

[0067] 2, the light spot 15 may be misaligned with respect to the sensor arrays 31-34. As a result, the light spot 15 may only partially strike the sensor arrays 31-34, or a large number of the photon-counting detector elements 40 may not be used as intended for measurement.

[0068] 1 and 2, the controller 70 is configured to align the light spot 15 with respect to the sensor arrays 31-34. To this end, measurement signals from the sensor arrays 31-34 are read out while the light spot 15 is impinging on the sensor arrays 31-34. From the measurement signals, the controller 70 determines position information characteristic of the position of the light spot 15 on the sensor arrays 31-34. The information can be, for example, the position of the center point of one of the light spots 15 or the total photon count value (e.g., the sum or average of the photon count values ​​of the detector elements 40) of each sensor array 31-34 or all sensor arrays 31-34 combined. Images acquired with different sensors can also be analyzed to derive position information.

[0069] Based on the information characteristic of the position of this light spot, the controller 70 instructs the adjustment device 75 to adjust one or more components of the optical microscope 100 to change the location where the light spot 15 strikes the sensor arrays 31-34.

[0070] This adjustment may affect the relationship between all light spots 15 jointly for all sensor arrays 31-34. Therefore, it is not necessary to evaluate the photon count values ​​of the different sensor arrays 31-34 separately; instead, a combined value of all sensor arrays 31-34 may be evaluated.

[0071] The adjustment device 75 can adjust the angle of the transparent (glass) plate 13 relative to the optical axis of the detection light 11. All light beams pass through this transparent plate 13, so tilting the transparent plate 13 affects the positions of all light spots 15 jointly.

[0072] Instead of the tiltable glass plate 13, a transparent wedge 14 may be provided in the beam path of the detection light 11 (shown by a dotted line in Figure 1). An adjustment device 75 may displace the wedge 14 to adjust the position of the detection light beam in a direction perpendicular to the optical axis. Two movable wedges 14 that rotate differently relative to the optical axis may be provided to allow adjustment of the position of the detection light beam in both directions (X and Y directions) relative to the optical axis (Z direction).

[0073] Optionally, the optical zoom element 8A may be adjusted by an adjustment device 75 to align the distance between the light spots 15 with the distance between the sensor arrays 31-34 (i.e., the distance from the center of one sensor array to the center of an adjacent sensor array).

[0074] In yet another exemplary embodiment, other optical elements are adjusted, particularly the optical element that directs all of the detection light beams towards the detector 10. The optical element may be a refractive element such as a mirror or a prism, or a diffractive element. The position or angle of such an optical element may be adjusted to displace all of the light spots 15 with respect to the sensor arrays 31-34.

[0075] Alternatively or additionally, the adjustment device 75 may move the sensor arrays 31-34 as a whole, in particular perpendicular to the optical axis, for example by moving a common PCB 19 or a common chip for all sensor arrays 31-34.

[0076] An optional Peltier cooler 21 is disposed on the PCB 19. Advantageously, by mounting the sensor arrays 31-34 on one PCB 19, a common Peltier cooler 21 can be used for some or all of the sensor arrays 31-34. Also, when disposed on the same PCB 19, additional resources can be shared by several sensor arrays 31-34.

[0077] Furthermore, the common PCB 19 can be advantageously used with a compact detection unit such as that described in WO 2018 / 073169 for spectrally selective detection of the detection light. In particular, the optical group described in this document may be placed before the detector 10 in the beam path of the detection light.

[0078] 2 shows a 1D arrangement of sensor arrays 31-34 and a 1D array of light spots 15, both aligned (parallel) to each other. This design may be modified to a 2D arrangement of sensor arrays and a 2D array of light spots. The 2D array may have a rectangular or Cartesian shape, or any other 2D arrangement, such as a hexagon.

[0079] Furthermore, the circular light spot 15 in Figure 2 is merely an example, for example, when the light spot 15 forms an Airy disk. Other light beam cross sections are possible, such as an elongated ellipse or a line. The sensor arrays 31-34 can be selected accordingly. The sensor arrays 31-34 may have different shapes to allow for light beams with different cross-sectional shapes.

[0080] The illumination light beams can be configured as light of the same or different wavelengths; therefore, the light beams forming the light spot 15 shown in FIG. 2 may have the same wavelength(s) or may differ from each other in wavelength / wavelength range. The sensor arrays 31-34 may be constructed and operated in the same way or differently from each other, depending on the expected light spectrum or illumination characteristics. For example, the light beams may have different temporal characteristics. One or more illumination beams may be configured as continuous wave illumination, while the other one or more illumination beams may form pulsed excitation. This may be useful, for example, for FLIM (Fluorescence Lifetime Imaging / Microscopy) in combination with standard imaging.

[0081] At least some of the illumination light beams may sequentially scan the same sample point. Measurement signals for the same sample point, i.e., photon counts measured with different illumination light beams for the same sample point, are combined, e.g., averaged or summed. The number of illumination light beams used may be flexibly adapted to an averaging factor set by a controller, e.g., depending on the current particular sample under observation or the desired image quality or signal-to-noise ratio (SNR).

[0082] The number of illumination light beams can be adapted, for example, by individually closing each light beam. Alternatively, the number of beams can be adapted by switching and attenuating the power of each illumination beam with a multi-channel AOTF. The number of activated sensor arrays can be adjusted to match the number of illumination light beams. The number of activated sensor arrays can be adapted, for example, by setting the operating bias of each sensor array.

[0083] Figure 3 FIG. 3 is a cross-sectional view of FIG. 2, showing the glass plate 13 in a tilted position. The tilted position compensates for misalignment of the light spots 15 shown in FIG. 2. FIG. 3 also shows individual transparent plates 23 in front of each sensor array 31-34. The transparent plates 23 can be tilted to compensate for misalignment of the sensor arrays 31-34 relative to one another. For example, due to manufacturing difficulties, the precision with which each chip 20 is positioned on the PCB 19 may be approximately half the diameter or the diameter of the photon detector 40. Such positional variations can be compensated for by individually tilting the glass plates 23 in front of each chip 20. The glass plates 23 are tilted so that the distance between the light spots 15 on the sensor arrays 31-34 matches the distance between the sensor arrays 31-34. It may be beneficial to perform this alignment step only initially and then fix them in place. For this purpose, the individual glass plates 23, once aligned, can be glued to prevent further movement. In contrast, the glass plate 13 may be motorized to allow readjustment during / between measurements or when changing components of the beam path.

[0084] A color filter 28 may optionally be provided in front of the sensor arrays 31-34 to enable spectrally selective detection. The color filter 28 may be a linear or rotary variable dichroic filter, or a tunable filter such as a VersaChrome® variable dichroic filter. Specifically, in the case of a VersaChrome® variable dichroic filter, the individual glass plates 23 may remain adaptable to compensate for the displacement introduced by the variable dichroic filter depending on the selected filter wavelength.

[0085] Figure 4 FIG. 4 schematically illustrates a detector 10 of an optical microscope 100. The detector 10 includes multiple sensor arrays 31-34, each including multiple photon-counting detector elements 40, specifically SPAD elements. In the example of FIG. 4, the sensor arrays 31-34 are formed as different regions of the same chip 20. More generally, at least some of the sensor arrays 31-34 may be formed as different regions of the same chip 40. On each sensor array 31-34, a respective detection light spot is formed during operation, enabling, for example, parallel measurement of multiple scanning light beams.

[0086] Using the same chip 20 for multiple sensor arrays 31-34 provides a particularly high precision of the position of the sensor arrays 31-34 relative to each other, which may be limited by the precision of lithography, which may be less than 350 nm.

[0087] Bonding pads 25 are provided around the periphery of chip 20 and function to output a measurement signal / photon count from photon-counting detector elements 40. Bonding pads 25 may be understood as conductive connection areas for electrically connecting chip 20 to, for example, a PCB on which chip 20 is mounted or to components mounted on the PCB. If sensor arrays 31-34 are spaced apart, as shown in FIG. 4, relatively large bonding pads 25 can be more easily arranged around the periphery of chip 20. However, with the arrangement of sensor arrays 31-34 as shown in FIG. 4, the areas of the chip between sensor arrays 31-34 may remain unused. This drawback is overcome by the design shown in FIG. 5.

[0088] Figure 5 Figure 5 shows an alternative detector 10 that differs from the detector of Figure 4 in that the sensor arrays 31-34 are immediately adjacent to one another, forming a common array within the same chip 20. Vertical dashed lines indicate the boundaries of the sensor arrays 31-34. Thus, the photon-counting detector elements 40 of adjacent sensor arrays are immediately adjacent, allowing for efficient or flexible use of the area of ​​the chip 20.

[0089] Each of the sensor arrays 31-34 is provided with a plurality of bonding pads 25, and at least some of the sensor arrays 31-34 are positioned (directly) adjacent to one another without any intervening bonding pads 25. In Figures 4 and 5, the sensor arrays 32 and 33 have bonding pads 25 only on their top and bottom sides (as viewed on the page), and the sensor arrays 32 and 33 do not have bonding pads on their right or left sides.

[0090] The reduced space available for bond pads 25 can make placement of bond pads 25 more difficult. This problem is exacerbated by the relatively large size of bond pads 25, as further evaluated below.

[0091] In a variation of the designs of Figures 4 and 5, all or at least some of the sensor arrays 31-34 are formed by individual chips located within the same package or on a common PCB. The description of Figure 5 can also be applied to designs in which the common chip 20 is replaced by an individual chip for each sensor array 31-34. The chips can be located directly adjacent to each other, with no bonding pads between them, for example, at a distance of less than 80 or 100 μm. The individual chips avoid unnecessary silicon area, while at the same time, placement within the same package or on a common PCB achieves accuracy on the order of one pixel size (often 10-20 μm). In such configurations, bonding wires can be a limiting factor for the minimum array pitch.

[0092] The number of bonding pads 25 should be kept small so that they can be located in only one dimension (above and below the sensor array, or to the left and right of the sensor array). In a variation of the illustrated embodiment, the bonding pads 25 shown in dashed lines can be omitted, and thus bonding pads 25 are provided only on two opposing sides of the sensor arrays 31-34. This allows for a modular design in which individual sensor arrays 31-34 are located adjacent to one another. In this case, the sensor arrays 31-34 are not regions of a single component, but rather individual chips or components. A different number of sensor arrays 31-34 can be selected in the production process depending on the desired application. Similarly, the embodiment of FIG. 4 can be modified to omit the left and right bonding pads and allow for the use of several identical sensor arrays 31-34 with bonding pads 25 on only two opposing sides.

[0093] Connecting each photon-counting detector element 40 to an individual bonding pad 25 would be impractical because the pad pitch is on the order of 50 μm, which is significantly larger than the size of the photon-counting detector elements 40. Instead, the total number of bonding pads 25 for outputting photon-counting numbers or signals can be less than the total number of photon-counting detector elements 40. The photon-counting numbers or signals of several photon-counting detector elements 40 may then be output through the same bonding pad.

[0094] Figure 6 A suitable example for reducing the number of bonding pads is described below with reference to FIG. 6 , which shows details of one of the photon-counting detector elements 40 of the detector 10, connected to a common readout line 48 to which other photon-counting detector elements (not shown) are also connected. Each photon-counting detector element 40 can be read out in a rolling readout fashion while still having a continuous global shutter exposure. The photon-counting detector element 40 includes a photosensitive diode 41, at least two memory elements 44 and 46, a latch 45, and a reset 42. The reset 42 can be formed, for example, using a transistor connected between the photosensitive diode 41 and a mass 43. Each photon-counting detector element 40 may include its own latch 45 and reset 42. The latches 45 and resets 42 of the entire detector 10 or some or all of the latches 45 and resets 42 of one of the sensor arrays 31-34 can be controlled collectively, i.e., jointly at the same time. The time between two collective reset events determines the exposure time. At the end of each exposure time, latch 45 is controlled to transfer data from first memory element 44 to second memory element 46. Second memory element 46 is then read out to a row readout mechanism which toggles via switch 47 to a common readout line 48. Readout occurs during the subsequent measurement interval, in parallel with the exposure time.

[0095] 6 is provided to facilitate understanding of the described content, but additional components may be added, or some of the components of FIG. 6 may be removed or replaced. More generally, each photon-counting detector element 40 may include at least a first memory element 44 and a second memory element 46, and during the exposure time of the photon-counting detector element 40, photon detection events are recorded in the first memory element 44 and a measurement signal can be read out from the second memory element 46. After the second memory element 46 is read out, the signal from the first memory element 44 is transferred to the second memory element 46, and the voltage on the diode 41 of the detector element 40 is restored to allow for the next exposure time or measurement.

[0096] In a SPAD, the SPAD anode capacitance can be used as a one-bit memory (in this case, the first memory element 44). Another capacitance can be provided as the second memory element 46. Such an architecture reduces the number of bonding pads required to output data. If memories 44 and 46 are one-bit memories, the pixel dead time is determined by the readout time of the entire array or pixels connected to the same readout line 48. If 10 rows are used, the dead time is 10 times the readout clock period (e.g., 10 ns). This allows for a trade-off between the number of rows and the pixel dead time. The dependency between pixel dead time and readout period can be resolved using a per-pixel multi-bit counter. In such a case, a multi-bit counter may be provided for each photosensitive diode 41 and connected to a common readout line 48.

[0097] More generally, a multi-bit counter may be provided to count several photon detection events of the same photon-counting detector element 40 and / or several photon detection events of different photon-counting detector elements 40. In both cases, the number of required bond pads 25 can be reduced. The multi-bit counters are included together with the detector elements 40 in the same chip 20, and thus photon count values ​​are transferred from one of the multi-bit counters out of the chip 20 via a respective bond pad 25.

[0098] The use of a common readout line 48 for several photon-counting detector elements 40 can be advantageous in reducing the number of bonding pads 25 and realizing a densely packed sensor array 31-34 as shown in Figures 4 and 5.

[0099] Figure 7 The photon-counting detector elements 40 of each sensor array 31-34 may be arranged in columns and rows. Some or all of the photon-counting detector elements 40 in the same column may be read out through a common readout line. Each readout line connects to one of the bond pads 25. The bond pads 25 in these and other exemplary embodiments may also be replaced with other electrical contacts. The row address for the photon-counting detector elements may be used to distinguish measurement signals from photon-counting detector elements 40 in the same column.

[0100] Such an architecture is shown in FIG. 7, which schematically illustrates an example of a chip 20 with one or more sensor arrays of photon-counting detector elements 40. Several of the photon-counting detector elements 40, e.g., one column, are connected to a common n-bit bus 49 that is used for row encoding. For simplicity, only one column is shown in FIG. 7, but the chip 20 may also include a respective n-bit bus 49 for each column of photon-counting detector elements 40. Along with the measurement signal, the n-bit bus 49 transmits row identification information, or identification information indicating the readout photon-counting detector element 40. This architecture can be particularly efficient when activity within the same column is sparse.

[0101] As another example, several photon-counting detector elements 40 may be binned, and a common output line may be used to output the summed photon count values ​​for all binned photon-counting detector elements 40. In this case, a multi-bit counter may be used, again reducing the number of required bonding pads 25.

[0102] Figure 8 FIG. 8 schematically illustrates a detector 10 or a modular detector unit 10′. Several of the illustrated modular detector units 10′ are arranged in a row to form the detector of FIG. 5 or FIG. 7. FIG. 8 also illustrates electrical contacts 26 and bonding wires 27 of the detector 10 or modular detector unit 10′. The electrical contacts 26 are arranged on the chip 20 adjacent to the columns and / or rows of detector elements 40. Photon counting signals from the detector elements 40 are routed to the electrical contacts 26. Bonding wires 27 connect the electrical contacts 26 to bonding pads 25 outside the chip 20. The bonding pads 25 may be located, for example, on a PCB. The bonding wires 26 may have a length of 1 to 3 mm, and the bonding pads 25 may be significantly longer than the electrical contacts 26, e.g., at least twice as long in width and / or length. When several of the illustrated modular detector units 10′ are arranged adjacent to each other to form a single detector, a staggered design in which the modular detector units 10′ overlap can be used. More specifically, the areas of the bonding pads 25 may overlap so that the detector elements 40 of different modular detector units 10' are closer to each other. In this way, pitch or gaps between adjacent detector elements of different modular detector units may be avoided. Each modular detector unit may form, inter alia, one of the sensor arrays described with respect to other embodiments.

[0103] Figure 9 To enable high-density 2D placement of the sensor arrays 31-34 without limitations due to the length of the bonding wires, the bonding pads 25 described above may be replaced by through-silicon vias (TSVs). FIG. 9 schematically illustrates an example of a detector 10 or modular detector unit 10B that differs from the previous examples in that the TSVs 50 extend through the wafer or die of the chip 20. The TSVs 50 may be routed to a ball grid array (BGA) 51 that can be directly connected to a PCB. Bonding technologies other than the BGA 51 may also be employed to connect the chip 20 to the PCB. For simplicity, only some of the photon-counting detector elements 40 and TSVs 50 are shown in FIG. 9. The TSVs 50 may replace the bonding pads 25 and the lines leading to the bonding pads 25 in other embodiments, but all other aspects may be similar to the above-described embodiments. Specifically, several photon-counting detector elements 40 may share the same TSVs 50. To this end, any of the above-described examples using a common output line for several photon-counting detector elements 40 may be applied to the embodiment of FIG. 9. Alternatively, particularly small TSVs 50 may be used, with one TSV 50 provided for each detector element 40. In a further variation of the embodiment shown in Figure 9, several sensor arrays are formed by separate chips 20 instead of different regions within the same chip 20. Compared to one large chip, multiple small arrays or chips may have advantages in terms of noise, power consumption, uniformity, and / or design flexibility.

[0104] Figure 10 FIG. 10 shows an exemplary design of a detector 10′, 10″, 10′″ comprising multiple modular detector units 10B-10D. Each modular detector unit 10B-10D may be formed similarly to the modular detector unit 10B of FIG. 9 using, for example, TSVs. Instead of TSVs, each modular detector unit 10B-10D may also use another technology that does not have bonding wires in at least one direction of the sensor array (e.g., the left-right direction in FIG. 9). FIG. 10 illustrates that multiple (specifically, identical) modular detector units 10B-10D are combined in different arrangements to form respective detectors. The detector 10′ comprises several modular detector units 10B-10D arranged directly adjacent to each other without spaces to form one common area of ​​photon-counting detector elements. The detector 10″ comprises modular detector units 10B-10D laterally separated from each other by gaps. In the case of detector 10''', the gap or pitch between modular detector units 10B-10D is larger than the gap in detector 10''. Thus, the same chip design can be used in a variety of applications where different detection areas may be required or preferred. Detectors 10', 10'', and 10''' of FIG. 10 may be used in different microscopes or other measurement devices, or alternatively, detectors 10', 10'', and 10''' may be used in the same microscope or measurement device.

[0105] Figure 11 FIG. 11 illustrates a detector 10 including different modular detector units 10B and 10C. The modular detector units 10B and 10C may differ in the number of respective photon-counting detector elements and / or their respective spectral detection efficiencies. Specifically, the wavelength-dependent sensitivity of the modular detector unit 10B may have a peak sensitivity to a different wavelength of light than the wavelength of the modular detector unit 10C. To compensate for the wavelength-dependent size of the PSF, the modular detector units 10B and 10C may have different cross-sectional sizes, with the cross-sectional size increasing as the wavelength of the peak sensitivity increases. For example, the modular detector unit 10B may have a peak sensitivity to blue light, while the modular detector unit 10C has a peak sensitivity to red light, with the cross-sectional size of the modular detector unit 10C being larger than that of the modular detector unit 10B.

[0106] The embodiments described with reference to the drawings are for illustrative purposes only. Other embodiments may result from combinations of these drawings. In particular, components of one embodiment may be added to another embodiment, and features of components described with reference to one drawing may be applied to components with the same reference numerals in another embodiment. The scope of the present invention is determined by the appended claims. [Explanation of symbols]

[0107] 1 light source 2. Irradiation light 2A, 2B, 2C, 2D Irradiation light spot 3. Scanner 3A, 3B Beam path depending on scanner position 4 Optical elements 5 Objective Lenses / Optical Elements 6 specimens 6A Specimen positioning position 7 Beam Splitter 8 Optical elements 8A Optical Zoom Element 10, 10', 10'', 10'' detector 10A, 10B, 10C, 10D Modular Detector Units 11 Detection light 13 Tiltable transparent plate 14 Movable transparent wedge 15 Light spot on detector 10 19 Printed Circuit Board (PCB) 20 chips 21 Peltier cooler 23 Tiltable transparent plate 25 Bonding Pads 26 Electrical contacts 27 Bonding Wire 28 color filters 31~34 Sensor array 40 Photon-Counting Detector Elements 41 Photosensitive diode of detector element 40 42 Reset detector element 40 43 Mass of detector element 40 44 first memory element of detector element 40 45 Latching of detector element 40 46 second memory element of detector element 40 47 Switch for detector element 40 48 common readout line 49 n-bit bus 50 Through Silicon Vias (TSV) 51 Ball Grid Array (BGA) 70 Controller 75 Regulating Device 100 Optical microscope VOP is the voltage applied to the diode 41.

Claims

1. 1. A method of operating an optical microscope, comprising: emitting and directing illumination light (2) as a plurality of illumination light beams from one or more light sources (1) toward a specimen positioning location (6A) to form a plurality of separated illumination light spots (2A, 2B, 2C, 2D) at said specimen positioning location (6A); directing a detection light beam (11) emanating from the illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning position (6A) to a detector (10) comprising a plurality of sensor arrays (31-34), each of the sensor arrays (31-34) comprising photon-counting detector elements (40), the detection light beam (11) forming a plurality of light spots (15) on the sensor arrays (31-34), the detection light beams (11) from different illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning position (6A) being directed to different of the sensor arrays (31-34); Equipped with analyzing measurement signals from the sensor array (31-34) to determine position information regarding the light spot (15) on the sensor array (31-34); an adjustment process for adjusting the locations at which the light spots (15) impinge on the sensor arrays (31-34) based on the position information, the adjustment process including an adjustment step that uniformly affects the positional relationship between all of the light spots (15) and all of the sensor arrays (31-34); During the manufacture of the detector (10), to adjust the location where the light spot (15) strikes the sensor array (31-34), an optical element (23) movably arranged in front of the sensor arrays (31-34), the optical element (23) influencing the position of each of the light spots (15) on the respective sensor arrays (31-34); Illumination light is emitted to form the light spot (15) on the sensor array (31-34); a controller (70) that interprets the measurement signals of the sensor array (31-34) and generates positioning commands; The optical element (23) is moved according to the positioning command; After the optical element (23) has been moved or tilted according to the positioning command, the optical element (23) is fixed with adhesive; A method characterized by:

2. 2. The method of claim 1, wherein in the adjustment process, the sensor arrays (31-34) are moved in unison across the optical axis of the detection light beam (11).

3. 3. The method according to claim 1 or 2, wherein in the adjustment process a common optical element (13, 14) is adjusted, and all illumination or detection light beams (11) are directed through the common optical element (13, 14), and the adjustment of the common optical element (13, 14) affects the position of the light spot (15) perpendicular to the optical axis of the detection light beam (11).

4. The method according to any one of claims 1 to 3, wherein in the adjustment process, the sensor arrays (31-34) are jointly tilted with respect to the optical axes of the detection light beams (11) according to differences between the detection light beams (11).

5. The method according to any one of claims 1 to 4, wherein in the adjustment process, the sensor arrays (31-34) are rotated together about the optical axis of the detection light beam (11).

6. 6. The method according to claim 1, wherein in the adjustment process, at least one optical zoom element (8A) provided in the beam path of the illumination or detection light beam (11) is adjusted to change the pitch between the light spots (15) on the sensor array (31-34), so that the pitch matches the pitch of the sensor array (31-34).

7. at least some of the illumination light beams are scanned over a common sample point; photon counts measured using different illumination light beams for the same sample point are combined; The method according to any one of claims 1 to 6, wherein the number of illuminating light beams used is set depending on the specimen under observation or depending on the desired image quality or signal-to-noise ratio.

8. During the manufacture of the detector (10), to adjust the location where the light spot (15) strikes the sensor array (31-34), the plurality of sensor arrays (31-34) are movably disposed on a common printed circuit board (19) and operatively connected; The illumination light is emitted to form the plurality of light spots (15) on the sensor array (31-34); a controller (70) that interprets the measurement signals of the sensor array (31-34) and generates positioning commands; The sensor array (31-34) is moved according to the positioning command. The method according to any one of claims 1 to 7.

9. A method described in any one of claims 1 to 8, wherein the number of optical elements (23) matches the number of sensor arrays (31 to 34).

10. As an optical element, a tiltable transparent plate (23) is arranged in front of the sensor array (31-34), 2. The method of claim 1, wherein after the tiltable transparent plate (23) is tilted according to the positioning command, the tiltable transparent plate (23) is fixed with adhesive.

11. a binning pattern having a plurality of superpixels, each of said superpixels being formed by jointly reading out some of said photon-counting detector elements (40) to produce a common photon count value; The method according to any one of claims 1 to 10, wherein the binning pattern is set depending on the position information.

12. The method of claim 11, wherein a center position of each of the light spots (15) on the sensor array (31-34) is determined, and the superpixels are aligned with respect to the center positions.

13. 1. An optical microscope, comprising: at least one light source (1) and optical elements (4, 5) for illuminating a specimen (6) at a specimen positioning location (6A) with a plurality of illumination light beams that form a plurality of separated illumination light spots (2A, 2B, 2C, 2D) at said specimen positioning location (6A); a detector (10) having a plurality of sensor arrays (31-34), each of the sensor arrays comprising a photon-counting detector element (40) for measuring a light spot (15) formed on the sensor array (31-34) by a detection light beam (11) emanating from the specimen (6), the detection light beams (11) from the different illumination light spots (2A, 2B, 2C, 2D) at the specimen positioning position (6A) being directed to the different sensor arrays (31-34); a controller (70) configured to control the at least one light source (1) and the detector (10); Equipped with the controller (70) is configured to analyze the measurement signals from the sensor arrays (31-34) to determine position information regarding the light spots (15) on the sensor arrays (31-34), and based on the position information, to instruct an adjustment device (75) of the optical microscope to adjust the locations at which the light spots (15) impinge on the sensor arrays (31-34), the adjustment including an adjustment step that uniformly affects the positional relationship between all the light spots (15) and all the sensor arrays (31-34); During the manufacture of the detector (10), to adjust the location where the light spot (15) strikes the sensor array (31-34), an optical element (23) movably arranged in front of the sensor arrays (31-34), the optical element (23) influencing the position of each of the light spots (15) on the respective sensor arrays (31-34); Illumination light is emitted to form the light spot (15) on the sensor array (31-34); a controller (70) that interprets the measurement signals of the sensor array (31-34) and generates positioning commands; The optical element (23) is moved according to the positioning command; After the optical element (23) has been moved or tilted in accordance with the positioning command, the optical element (23) is fixed with adhesive.

14. the sensor arrays (31-34) are arranged on a common printed circuit board (19); and / or The optical microscope of claim 13, wherein the sensor array (31-34) is formed as different areas of a single chip (20).

15. 15. The optical microscope of claim 13, wherein the sensor arrays (31-34) are arranged directly adjacent to each other to form a common array within one chip (20) or on one printed circuit board (19).

16. A plurality of bonding pads (25) are provided for each of the sensor arrays (31 to 34), 16. The optical microscope of any one of claims 13 to 15, wherein at least some of the sensor arrays (31-34) are arranged directly adjacent to each other without any bonding pads (25) therebetween.

17. An optical microscope according to any one of claims 13 to 16, wherein the total number of bonding pads (25) for outputting measurement photon counting signals is less than the total number of the photon counting detector elements (40), and measurement photon counting signals of some of the photon counting detector elements (40) are output through the same bonding pad (25).

18. the photon-counting detector elements (40) of the same sensor array (31-34) are arranged in columns and rows; 18. The optical microscope of claim 17, wherein a common readout line (48, 49) connects the photon-counting detector elements (40) of the same column to one of the bonding pads (25), and a row address for the photon-counting detector elements (40) is used to distinguish measurement signals from the photon-counting detector elements (40) of the same column.

19. 19. An optical microscope according to claim 17 or claim 18, wherein a multi-bit counter is provided for counting several photon detection events of the same or different photon-counting detector elements (40) in order to reduce the number of bonding pads (25) required.

20. 20. The optical microscope of claim 13, wherein each of the photon-counting detector elements (40) comprises at least a first memory element (44) and a second memory element (46), and allows reading out of a measurement signal from the second memory element (46) during an exposure time during which a photon detection event can be recorded in the first memory element (44) of the photon-counting detector element (40).

21. 21. The optical microscope of claim 20, wherein each of the photon counting detector elements (40) is formed by a single-photon avalanche detector having a SPAD anode forming the first memory element (44), and the second memory element (46) is configured to receive a measurement signal from the first memory element (44).

22. 22. The optical microscope of claim 13, wherein a plurality of through-silicon vias (50) are provided for each of the sensor arrays (31-34), and some of the photon-counting detector elements (40) share one of the through-silicon vias.

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