Optical system for microscope and method for operating microscope

Abstract

To provide an optical system that enables efficient generation of high-resolution detection data, in particular for intensity images, of fluorescent samples.SOLUTION: An optical system (100) for a microscope comprises: an excitation light source (102) that generates excitation light pulses; an objective lens (104) that directs excitation light into a sample space (106) and receives detection light from the sample space; and a detection unit (110) that includes a detector array and receives the detection light. A scanning unit (114) is arranged between the excitation light source and the objective lens, and selectively directs the excitation light into different regions of the sample space via the objective lens. The optical system also comprises a beam splitter (112) that directs the excitation light from the excitation light source to the objective lens, in particular via the scanning unit, and directs the detection light from the objective lens to the detection unit. The detector array is time-gated with respect to the excitation light pulses.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an optical system for a microscope and a corresponding microscope.In another aspect, a method for operating a microscope is provided. [Background technology]

[0002] The fluorescence microscope known from US Pat. No. 1,422,348 typically shares the beam path of the excitation light with the beam path of the respective detection light. Between the sample space and the excitation light source and detector, a beam splitter is provided to combine and separate the excitation and detection light. Furthermore, spectral filters can reduce the amount of (stray) excitation light spilling onto the detector. However, these measures are technically complex, and undesired leakage of excitation light into the detector can occur due to insufficient filtering of the light path between the excitation source and the detector. Since the intensity of the excitation light is usually several orders of magnitude higher than the intensity of the corresponding detection light, even a small leakage can introduce a large amount of undesired light into the detection unit.

[0003] Stray excitation light can overwhelm the faint fluorescence emitted from the sample, reducing the signal-to-noise ratio. This can make it difficult to detect weak fluorescent species and obscure fine details necessary for accurate interpretation and analysis. This not only affects the quality of the image data, but can also lead to misinterpretation of experimental results, especially in sensitive applications such as fluorescence microscopy in medical diagnostics or cell biology research. Summary of the Invention [Problem to be solved by the invention]

[0004] The objective here is to provide an optical system that can efficiently generate high resolution detection data, especially for intensity images of fluorescent samples. [Means for solving the problem]

[0005] The above object is achieved by the subject matter of the respective independent claims. Advantageous embodiments are defined in the respective dependent claims and in the following description.

[0006] In a first aspect, an optical system for a microscope is provided. The optical system comprises an excitation light source configured to generate excitation light pulses and an objective lens configured to direct the excitation light into a sample space and receive detection light from the sample space, particularly from the sample space. The optical system further comprises a detection unit including a detector array configured to receive the detection light. A scanning unit is disposed between the excitation light source and the objective lens and configured to selectively direct the excitation light via the objective lens to different regions of the sample space. Similarly, the scanning unit can be configured to receive detection light from the different regions and direct the (descanned) detection light to the detector array. Thus, the optical system can be particularly for an (imaging) scanning microscope. The optical system further comprises a beam splitter configured to direct excitation light from the excitation light source to the objective lens, particularly via the scanning unit, and to direct detection light from the objective lens to the detection unit, particularly via the scanning unit. The detector array is time-gated with respect to the excitation light pulses.

[0007] The time-gating detector array makes it possible to reduce or completely avoid crosstalk between the excitation light and the detection light. In other words, the above-described optical system makes it possible to reduce or completely avoid the generation of detection signals from pulsed excitation light leaking or being reflected onto the detector array. In particular, by using a time-gating detector, signals responsive to the detection light can be generated and / or recorded only before or after the excitation light pulse. This can increase the signal-to-noise ratio of the generated detection signals. In particular, the optical system can include a gating unit configured to time-gate the detector array with respect to the excitation light pulse.

[0008] In particular, the detector array can be gated over at least the duration of the excitation light pulse. Thus, detected light is preferably detected and / or recorded only when the excitation light is off or not illuminating the sample space. Similarly, light is preferably not detected and / or recorded during the excitation light pulse.

[0009] The pulse shape of the excitation light pulse is taken into consideration for such gating purposes. Typically, the excitation light pulse can be modeled. For example, the intensity of the excitation light pulse with respect to time can be modeled by a Gaussian function. Therefore, the duration or pulse width of the excitation light pulse can be described by its respective full width at half maximum. In particular, if the intensity falls below a threshold value set for, for example, the full width at half maximum, the time gating can preferably be switched off.

[0010] Because the excitation light pulses are repetitive and can act as or synchronize the system clock, delays in detector element signal generation can be accounted for by shifting the gating trigger, particularly by adjusting the phase between the two repetitive excitation light clocks and the time gating clock. Any potential delay between detection signal generation and detector element switching can be considered jitter and can be addressed, for example, by extending the gating off time by a proportional adjustment.

[0011] For example, the excitation light source can include a laser light source, particularly a white-light laser. Furthermore, the excitation light pulses can be generated at a frequency preferably in the range of 10 MHz to 100 MHz, more preferably at a frequency of 80 MHz. The excitation light, particularly the generated excitation light pulses, can be sequentially directed to different regions of the sample space using a scanning unit, often referred to as scanning. The excitation light can selectively excite fluorophores in each region of the sample space. In response, the excited fluorophores can emit detection light. The fluorescence lifetime of the excited fluorophores is typically in the range of 2 ns to 6 ns. For example, a pulse rate of 80 MHz / 12.5 ns typically allows most, if not all, of the emitted photons to be captured before the next excitation pulse arrives.

[0012] In particular, the detector array may be a two-dimensional detector array. The detector array may preferably include individual detector elements, such as photodiodes, arranged in a two-dimensional array. In particular, each of the individual detector elements is configured to generate an (electrical) signal in response to incident detected light. In a particular example, the detector element may be a single-photon avalanche diode. The signals generated by each of the individual detector elements may be read out or processed individually by a control unit.

[0013] Preferably, the optical system comprises a control unit configured to receive the detection signals from the detector array. Providing the control unit in the optical system allows for efficient reception and processing of the detection signals from the detector array. In particular, the control unit may be an integrated part of the optical system rather than an external component, thereby providing a more compact structure. For example, the control unit may comprise an integrated circuit such as a field programmable gate array. The control unit may further be configured to control other units or functions of the optical system, for example a scanning unit.

[0014] Preferably, the control unit is configured to integrate the detection signals received from the detector array, thereby enabling efficient processing of the detection signals. For example, the detection signals can be integrated over a pixel dwell time or over a predetermined time interval for each pixel. The predetermined time interval may vary if the scanning speed is not linear, for example, during a line scan. In particular, transmission of detection signal data, including time-resolved data, to an external processing system, such as a computer, of the optical system is often limited by the bandwidth of the connection to the external processing system. This may reduce the maximum image acquisition rate. In particular, if time-resolved data is not required, the control unit integrates time-tagged photon count data for each pixel, thereby reducing the amount of data transmitted to the external processing system and data processing efficiency.

[0015] In a particularly preferred embodiment, the control unit comprises a system clock, which allows precise timing control of the time-gated detector array. Furthermore, the system clock can provide timing signals for the excitation light pulse and the time-gated detector array. Thus, the system clock can be configured to synchronize the excitation light pulse and the time-gated detector array.

[0016] Alternatively or additionally, the excitation light pulses can act as a system clock and trigger the time gating, or the system clock is synchronized to the excitation light pulses, particularly in the latter case, a photodetector configured to detect (at least part of) the emitted light pulses can be provided, and the detection signal of said photodetector is used as the system clock.

[0017] Preferably, the control unit includes a gating unit configured to time-gate the detection signals relative to the excitation light pulse. The gating unit may in particular be an integrated circuit such as gating logic or a field programmable gate array. The gating unit of the control unit enables the detection signals received from the detector array to be separated relative to the excitation light pulse. In particular, the gating unit can provide the detection signals only before or after the excitation light pulse for processing by the control unit. By providing a control unit with a gating unit, the detector array can be time-gated relative to the excitation light pulse without modifying the detector array itself.

[0018] Preferably, the detection unit includes a gating unit configured to time gate the detector array relative to the excitation light pulse, thereby enabling gating of the detected signals in the detector unit in the vicinity of the source of the detected signals.

[0019] In a particularly preferred embodiment, each detector element, e.g., a photodiode, of the detector array of the detection unit comprises a gating unit configured to time-gate the respective detector element with respect to the excitation light pulse. The gating unit may be, for example, an integrated circuit such as gating logic or a field programmable gate array. In particular, the gating unit is capable of closing and switching off the respective detector element and / or discarding the detection signal generated at the respective detector element.

[0020] It is particularly preferred to provide a gating unit such that each detector element is switched off. In this case, incident excitation light or detection light does not cause the detector element to generate a corresponding detection signal. Similarly, incident photons of excitation light or detection light do not trigger the dead time of the detector element. Therefore, when a detector element is switched off by its respective gating unit, each detector element cannot generate a detection signal. Such a specific embodiment can be realized, for example, by providing a detector array with detector elements each having a dedicated gating transistor for a bias voltage. Such dedicated gating transistors can also be used, for example, in active quenching circuits for Geiger-mode operation.

[0021] Preferably, the excitation light source is configured to generate excitation light pulses having a pulse width of each pulse in the range of 5 to 100 ps, ​​preferably in the range of 10 to 50 ps, ​​more preferably in the range of 10 to 20 ps, ​​and in particular, the detector array is gated at least at that time.

[0022] Preferably, the optical system comprises a pinhole, which allows discarding out-of-focus detection light and thus increases the spatial resolution of the optical system and improves image quality. For example, the pinhole can be arranged in the beam path of the detection light ahead of the detection unit, in particular in the conjugate image plane. Alternatively, the pinhole can be arranged between the beam splitter and the scanning unit.

[0023] Preferably, the detection unit includes at least one detection beam splitter configured to direct a first portion of the detection light to a detector array and a second portion of the detection light to another detector array. The detection unit may include a dispersive element that spectrally separates the detection light or separates the first and second portions of the detection light. This provides improved imaging resolution and an improved signal-to-noise ratio. This further allows for the reconstruction of spectral information about the detection light without significant loss of the detection light. The first and second detector arrays may be different regions of the same detector array or may be separate detector arrays.

[0024] In another aspect, a microscope, particularly a scanning microscope or an image scanning microscope, is provided that includes the optical system described above. For example, the microscope may include a stage on which a sample space can be positioned. In particular, the microscope is configured to generate and / or integrate a time-gated detection signal of the detector array over a pixel dwell time. The pixel dwell time may be the duration during which an excitation light pulse is directed at a particular one of the sample regions.

[0025] The microscope may be part of a microscope system that further includes a processing system, e.g., a computer external to the microscope or separate from the microscope, that processes the detection signal data generated by the detector array of the optical system. In particular, the microscope system may include a control unit configured to transfer the detection signal data, in particular the integrated detection signal data, to the processing system. The processing system may be configured to generate image data from the pixel-by-pixel integrated detection signal data generated by the optical system of the microscope as the sample is descanned. This allows for fast and efficient processing of the detection signal data and generation of image data. In particular, this is due to a reduced bandwidth required between the microscope and the processing system compared to transferring time-tagged photon arrival data as the detection signal data.

[0026] In another aspect, a method for operating a microscope with an optical system is provided, the method including generating an excitation light pulse, scanning a sample with the excitation light pulse, descanning the detection light onto a detector array, and gating the detector array for at least the duration of the excitation light pulse.

[0027] Preferably, the time-gated detection signal of the detector array is integrated over a pixel dwell time, which may be the duration that an excitation light pulse is directed at a particular one of the sample regions, thereby efficiently generating high-resolution imaging data.

[0028] The microscope and the method have the same advantages as the optical system. Furthermore, the microscope and the method can be complemented by the features of the optical system described herein, in particular the features of the respective dependent claims of the optical system.

[0029] Specific embodiments will now be described with reference to the drawings. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram illustrating an optical system. [Figure 2] 1 is a flowchart illustrating a method for operating an optical system. [Figure 3] FIG. 2 is a schematic diagram illustrating a first timing control of time gating of the optical system. [Figure 4] FIG. 10 is a schematic diagram illustrating a second timing control of time gating of the optical system. DETAILED DESCRIPTION OF THE INVENTION

[0031] 1 is a schematic diagram of an optical system 100 for a microscope, such as a scanning microscope, an image scanning microscope, or a confocal microscope. The optical system 100 includes an excitation light source 102, e.g., a laser configured to emit pulsed laser light, which is configured to generate excitation light pulses. In particular, the excitation light source 102 generates a focused beam of excitation light pulses.

[0032] 1, the beam paths of the excitation light and / or the beam paths of the detection light are represented schematically by solid lines between elements of the optical system 100. Electrical connections are represented by dashed lines.

[0033] The optical system 100 further includes an objective lens 104 configured to direct the excitation light generated by the light source 102 into a sample space 106, where a sample to be examined by the optical system 100 can be placed on a sample carrier 108. The sample can be, for example, a biological sample. In particular, the sample can have been previously stained with one or more fluorescent substances. The excitation light directed into the sample space 106 can be configured to excite fluorophores in the sample.

[0034] The objective lens 104 is further configured to receive detection light from the sample space 106. In particular, the detection light may be fluorescence light generated when fluorophores in the sample are excited by the excitation light. Similarly, excitation light reflected by, for example, a cover glass in the sample space 106 may also be regularly received by the objective lens 104.

[0035] Furthermore, the optical system 100 includes a detection unit 110 with a detector array configured to receive the detection light. In particular, the detector array is a two-dimensional array of individual detector elements, such as photodiodes. The detection unit 110 is therefore capable of detecting the detection light, in particular the fluorescence light, generated in the sample space 106.

[0036] The optical system 100 includes a beam splitter 112 that directs excitation light from the excitation light source 102 to the objective lens 104 and, in particular, to the sample space 106, and directs detected light from the objective lens 104 and, in particular, to the sample space 106, to a detection unit 110. The beam splitter 112 can be, for example, a dichroic optical element or an acousto-optical element.

[0037] The optical system 100 further includes a scanning unit 114 configured to selectively direct the excitation light to different regions or volumes of the sample space 106. Thus, the scanning unit 114 can scan the sample in the sample space 106 with the excitation light, for example, according to a meander-shaped scanning pattern. In particular, the scanning unit 114 is disposed along the beam path of the excitation light and / or the detection light between the objective lens 104 and the excitation light source 102, preferably between the objective lens 104 and the beam splitter 112. By way of example, the scanning unit can include one or more tiltable mirrors, for example, a galvanometric mirror scanner. Similar to the excitation light, the scanning unit 114 can descan the detection light generated from the specific regions of the sample space 106 to which the excitation light is selectively directed.

[0038] In certain embodiments, the optical system 100 can further comprise a pinhole, preferably arranged in the beam path of the detection light, for example between the beam splitter 112 and the detection unit 110. Furthermore, a further pinhole can also be arranged in the beam path of the excitation light, preferably between the beam splitter 112 and the excitation light source 102. Alternatively, the excitation light source 102 can comprise a further pinhole. Thus, in the case of a confocal arrangement of pinholes, both the excitation light and the detection light can share the same focal point in the sample space 106.

[0039] The optical system 100 may further include a control unit 116 configured to control functions and elements of the optical system. In particular, the control unit 116 may control the scanning unit 114 such that excitation light is selectively directed to different regions of the sample space 106. Furthermore, the control unit 116 may be configured to instruct the excitation light source 102 to generate excitation light pulses at specific frequencies.

[0040] In certain embodiments, the control unit 116 may include a system clock configured to, among other things, provide timing signals for the optical system 100. The timing signals may be used to synchronize various elements and functions of the optical system 100 with one another. For example, the excitation light source 102 may receive a timing signal to adjust the frequency and / or duration of excitation light pulses. Alternatively, the excitation light source 102 may generate the timing signal, and the time gating function may be based on the timing signal.

[0041] The timing signal may also be received by the scanning unit 114, particularly to coordinate the scanning of the sample in the sample space 106 by the excitation light. For example, the timing signal may control or determine the time that the excitation light is directed by the scanning unit 114 to each of one or more different specific regions of the sample space 106. This time may be referred to as a pixel dwell time. The pixel dwell time is typically 1 μs.

[0042] The control unit 116 can further be configured to receive detection signals from the detector array, for example, the detection signals can include time-tagged photon arrival data from each detector element of the detector array.

[0043] The detector array of the optical system 100 is time-gated relative to the excitation light pulse. In particular, the optical system 100 may include a gating unit configured to time-gate the detector array relative to the excitation light pulse. The timing signals of the control unit 116 may also be used, for example, to time-gate the detector array relative to the excitation light pulse. The gating unit may be an integrated circuit, for example, a field programmable gate array.

[0044] In certain embodiments, the control unit 116 includes a gating unit, in which case the detection signals received by the control unit 116 from the detector array can be time-gated with respect to the excitation light pulse. For example, the control unit 116 can include a gating unit that operates to cause the control unit 116 to receive the detection signals from the detector array only when the excitation light pulse is not illuminating the sample space 106. The gating unit can be operated such that the control unit 116 does not receive the detection signals when the excitation light pulse illuminates the sample space 106.

[0045] Alternatively or additionally, the detector array of the detection unit 110 may be time-gated directly by the gating unit so that detection signals are not intermittently received by the control unit 116 or so that detection signals are not intermittently generated by the detector array in the first place.

[0046] Preferably, the detection unit 110, particularly the detector array, can include a gating unit if the gating unit prevents the generation of a detection signal. In this case, each detector element of the detector array can include one of the gating units. For example, the gating unit for each detector element can be a gating transistor for biasing the detector element. When the gating unit is closed, the detector element does not generate a detection signal. The closing and opening of the gating unit can be triggered in response to an excitation light pulse such that the detector element does not generate a signal from the incident light even when the sample space 106 is illuminated by the excitation light pulse. This prevents incident light, particularly stray excitation light reflected from the sample space 106, from generating a corresponding detection signal and triggering the dead time of the detector element. In particular, the gating unit can be closed in response to a timing signal from a system clock, which can also trigger an excitation light pulse.

[0047] Additionally, a processing system 118 external to the optical system 100 may be provided. The processing system 118 may be configured to receive detection signal data generated by the control unit 116 of the optical system 100. The control unit 116 may be configured to generate the detection signal data by integrating the detection signals received from the detector array of the detection unit 110. The processing system 118 may then generate image data from the detection signal data to provide a user with an image of the sample in the sample space 106. Integrating the detection signals by the control unit 116 prior to transfer to the external processing system 118 may reduce the amount of data transferred and reduce the bandwidth requirements for transfer of the detection signal data. This allows for fast and efficient processing of the detection signal data and generation of image data.

[0048] The optical system 100 may be part of, for example, a fluorescence microscope, in which case the processing system 118 may be external to the microscope that includes the optical system 100.

[0049] In yet another embodiment, the optical system 100, and in particular its detection unit 110, can optionally include at least one further beam splitter configured to direct a first portion of the detection light to a detector array and a second portion of the detection light to another detector array. The detection light or the first and second portions of the detection light can be dispersed by a dispersive element of the detection unit 110.

[0050] 2 is a flowchart of a method for operating optical system 100. The method begins in step S200.

[0051] In the following step S202, an excitation light pulse is generated by the excitation light source 102, and a specific region of the sample space 106, in particular one sample region within the sample space 106, is illuminated by the excitation light pulse. For example, the excitation light source can be triggered to generate the excitation light pulse by a timing signal generated by the control unit 116. The detector array of the detection unit is gated during step S202, at least for the duration of the excitation light pulse. For example, the timing signal generated by the control unit 116 can close the gating unit to gate the detector array for a predetermined time, in particular for the duration of the excitation light pulse.

[0052] In step S204, after the duration of the excitation light pulse, the gating unit is opened again and the detected light generated in a particular region of the sample space 106 is descanned onto the detector array of the detection unit 110. The detector array generates a respective detection signal.

[0053] Steps S202 and S204 can be repeated to illuminate specific (one-dimensional, two-dimensional, or three-dimensional) regions of the sample volume 106 with different excitation light pulses to generate respective detection signals.

[0054] In step S206, the scanning unit 114 is adjusted to direct another excitation light pulse to another region of the sample volume 106.

[0055] Steps S202, S204 and S206 may be repeated iteratively to continuously scan the sample area within sample space 106.

[0056] The method ends in step S208.

[0057] 3 is a schematic diagram illustrating a first timing control of the time gating of the optical system 100. The graph shows four excitation light pulses 300 over time. A pixel clock 302 indicates the scanning of several pixels or regions of the sample space 106 by the excitation light pulses 300. The excitation light pulses 300 generate corresponding detection light 304, specifically the fluorescence emission of the sample in the sample space 106.

[0058] The gating state 306 indicates whether the detector array is gated. In the embodiment according to Fig. 3, the detector array is gated or the gating state is closed (e.g., has a low value) for the duration of the excitation light pulse 300. Therefore, for the duration of the excitation light pulse 300, the detector array does not generate a detection signal or no detection signal is received by the control unit 116. When the gating state 306 is open (e.g., has a high value), the detection light 304 received by the detector array generates an integrable detection signal for a particular pixel.

[0059] 4 is a schematic diagram illustrating a second timing for time gating of optical system 100. In this case, gating state 400 is controlled such that the detector array is gated from before a particular excitation light pulse 300 until the end of that particular excitation light pulse 300. This timing control is preferred when the minimum off time of the gating unit is longer than the duration of the excitation light pulse 300, especially when the gating unit switches the detector elements to the off state. This timing control maximizes the average detection probability of detected light 304, since the probability of detected light 304 or fluorescence emission is highest immediately after the excitation light pulse 300.

[0060] Elements having the same or similar function are designated by the same reference numerals throughout the figures. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0061] While some aspects have been described in the context of an apparatus, it will be apparent that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a step or feature of a step, and similarly, aspects described in the context of a step also represent a description of a corresponding block or item or feature of a corresponding apparatus. [Explanation of symbols]

[0062] 100 Optical System 102 Excitation light source 104 Objective Lens 106 Sample Space 108 Sample Carrier 110 Detection Unit 112 Beam Splitter 114 Scanning Unit 116 Control Unit 118 Processing System 300 excitation light pulses 302 Pixel Clock 304 Detection light 306,400 Gating States

Claims

1. An optical system (100) for a microscope, said optical system (100) comprising: an excitation light source (102) configured to generate excitation light pulses (300); an objective lens (104) configured to direct excitation light into a sample space (106) and to receive detection light (304) from the sample space (106); a detection unit (110) including a detector array configured to receive the detection light (304); a scanning unit (114) disposed between the excitation light source (102) and the objective lens (104) and configured to selectively direct excitation light through the objective lens (104) to different regions of the sample space (106); a beam splitter (112) configured to direct excitation light (300) from the excitation light source (102) to the objective lens (104) and direct detection light (304) from the objective lens (104) to the detection unit (110); Equipped with the detector array is time gated with respect to the excitation light pulse (300); An optical system (100).

2. The optical system includes a control unit (116) configured to receive detection signals from the detector array.

10. The optical system of claim 1.

3. the control unit (116) is configured to integrate detection signals received from the detector array; 3. The optical system of claim 2.

4. The control unit (116) has a system clock.

4. An optical system according to claim 2 or 3.

5. the control unit (116) includes a gating unit configured to time-gate the detection signal relative to the excitation light pulse (300); 5. An optical system according to any one of claims 2 to 4.

6. the detection unit (110) includes a gating unit configured to time-gate the detector array with respect to an excitation light pulse (300); 6. An optical system according to any one of claims 1 to 5.

7. each detector element of the detector array of the detection unit (110) includes a gating unit configured to time-gate the respective detector element with respect to the excitation light pulse (300); 7. The optical system of claim 6.

8. The excitation light source (102) is configured to generate excitation light pulses (300) having a pulse width in the range of 5 ps to 100 ps.

8. An optical system according to any one of claims 1 to 7.

9. the optical system includes a pinhole.

9. An optical system according to any one of claims 1 to 8.

10. the detection unit (110) includes at least one detection beam splitter configured to direct a first portion of the detection light (304) to the detector array and direct a second portion of the detection light (304) to another detector array; 10. The optical system according to any one of claims 1 to 9.

11. A microscope equipped with an optical system (100) according to any one of claims 1 to 10.

12. the microscope is configured to generate and / or integrate a time-gated detection signal of the detector array over a pixel dwell time; The microscope according to claim 11.

13. 13. A method for operating a microscope according to claim 11 or 12, said method comprising: generating an excitation light pulse (300); scanning the sample with the excitation light pulse (300); descanning the detected light (304) onto a detector array; gating the detector array for at least the duration of the excitation light pulse (300); A method comprising:

14. the time-gated detection signals of the detector array are integrated over a pixel dwell time; 14. The method of claim 13.

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