Femtosecond Pulse Microscopy

Abstract

A system and method for femtosecond pulsed microscopy, preferably nonlinear microscopy, is disclosed. The microscope system includes a laser source for providing a pulse train including a series of femtosecond pulses; and a pulse picker for selecting one or more bursts of pulses from the pulse train, each of the one or more bursts including a plurality of consecutive pulses. The one or more bursts may include less than 25%, preferably less than 15%, of the pulses in the pulse train. The microscope system further includes an optical system for illuminating a sample with the one or more bursts of pulses; and a detector for detecting a signal resulting from an interaction of the one or more bursts of pulses with the sample. The detector may be configured to generate a single signal for each of the one or more bursts.

Description

[Technical field]

[0001] The present invention relates to nonlinear microscopy, and in particular, but not exclusively, to methods and systems for femtosecond pulsed microscopy, and computer program products enabling a computer system to carry out such methods.

[0002] During surgery and endoscopic procedures, it may be desirable to quickly examine tissue samples, such as biopsies, using a microscope. In this way, the physician may determine, for example, whether the tumor resection surface is clean or whether a larger volume of resection is necessary. In some cases, the physician may want to examine a living sample that is part of a subject (e.g., a patient). In these cases, preparation of the sample, for example by fixation, staining, or other processing common, for example, by pathology practice, is typically not possible or at least is not desirable. Moreover, it may be required that the tissue sample is not damaged during the examination, for example so that the tissue is still alive or can be used later for other examination purposes.

[0003] However, these requirements pose various challenges in obtaining good microscopic images: for example, obtaining sufficient contrast without staining is typically difficult, especially over short periods of time, and even more so if the tissue is not fixed or otherwise living.

[0004] Different options may be used to enhance contrast when staining or labeling is not an option, such as multi-photon microscopy or non-linear optical microscopy. Non-linear optical microscopy involves the detection of higher order signals, such as second and third harmonics. However, higher order signals require either high peak light intensity or long integration times (and therefore long acquisition times). High peak light intensity carries the risk of damaging the tissue, for example through non-linear damage mechanisms such as multi-photon ionization or otherwise through cavitation. When using low peak powers, the necessary increase in integration time results in a higher total light dose and the risk of (over)heating the tissue. Long acquisition times do not meet the requirements of fast image acquisition necessary for rapid bedside decision making. Moreover, long acquisition times may also result in motion artifacts, especially when the sample is not fixed.

[0005] G.-J. Bakker et al., 'Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy', eLife 11 (2022), e63776, describe the adverse effects (e.g. thermal damage) of too high single pulse energies in high-intensity infrared microscopy, which limits the possibility to only lower the average energy in the sample by reducing the repetition rate in favor of higher single pulse intensities.

[0006] US Patent Application Publication No. US2018 / 0106729A1 describes a bedside microscope for multiphoton microscopy that uses a 4 kHz resonant scanner to obtain raster images at high speed. However, to have an acceptable signal-to-noise ratio, 4-8 consecutive images acquired at intervals of 0.1 or 0.2 seconds are averaged. At these time scales, fresh biopsies and living samples typically exhibit dynamic behavior, and therefore averaging leads to reduced spatial resolution and / or the introduction of artifacts.

[0007] European Patent Application Publication No. EP 2 365 338 A1 describes time-resolved fluorescence microscopy, including time-resolved multi-photon fluorescence, for high-precision oxygen concentration measurements.

[0008] International Publication No. WO 2021 / 210768 A1 describes a multiphoton microscope that is equipped with a repetition rate tuner that reduces the repetition rate of a train of optical pulses emitted from a pulsed laser for time-gated detection.

[0009] BR Masters et al., 'Mitigating thermal mechanical damage potential during two-photon dermal imaging', Journal of Biomedical Optics 9:6 (Nov / Dec 2004) pages 1265-1270, describe two-photon excitation fluorescence microscopy and the thermal damage associated with one-photon absorption of infrared excitation light.

[0010] International Publication No. WO2014 / 205413A2 relates to multiphoton imaging using fiber lasers. Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, there is a need in the art for a high-speed microscope system having a large field of view with sub-micron resolution that reduces or at least mitigates the above-mentioned disadvantages. [Means for solving the problem]

[0012] The objective of the embodiments of the present disclosure is to obtain a large field of view (at least 0.5×0.5 mm) with submicron resolution on samples with little or no pretreatment. 2 , preferably at least 1×1 mm 2 The object of the present invention is to provide a microscopy system and method that allows high speed image acquisition (preferably dwell times of 1 μs or less per pixel) with high resolution and high resolution. The systems and methods may use non-linear signals, such as second and third harmonics, autofluorescence, and multi-photon interactions, to provide contrast. The systems and methods are preferably non-destructive, i.e., they cause no or only minimal damage to the sample.

[0013] In a first aspect, the present invention may relate to a microscope system, preferably a nonlinear microscope system. The microscope system comprises a laser source for providing a pulse train comprising a series of femtosecond pulses; and a pulse picker for selecting one or more bursts of pulses from the pulse train. Each of the one or more bursts comprises a plurality of consecutive pulses. The one or more bursts may comprise less than 25%, such as less than 15%, of the pulses in the pulse train. The microscope system further comprises optics for illuminating a sample with the one or more bursts of pulses; and a detector for detecting a signal resulting from an interaction of the one or more bursts of pulses with the sample. The detector may be configured to generate a single signal for each of the one or more bursts.

[0014] The femtosecond pulse may be a high-intensity pulse. For example, in the case of a microscope with a high numerical aperture microscope objective, the pulse may have an energy of at least 0.1 nJ per pulse, preferably at least 1 nJ per pulse, more preferably at least 2 nJ per pulse, when measured at a sample. For pulses with the same pulse length, a higher energy per pulse is associated with a higher peak light intensity, allowing stronger high-order signals and therefore a higher signal-to-noise ratio. However, the pulse energy may be limited by (nonlinear) damage to the sample. Thus, it may have an energy of 10 nJ or less per pulse, preferably 5 nJ or less per pulse.

[0015] In this disclosure, pulse energy refers to the pulse energy at the sample unless otherwise specified.

[0016] The pulse length may be 250 fs or less, preferably 100 fs or less. Acceptable and optimal values ​​may vary depending on system specifications, such as numerical aperture, focal spot size, pulse length and wavelength. In general, signal quality for nonlinear light-matter interactions in the sample correlates with peak light intensity, so longer pulses and / or less focused pulses may have higher pulse energy, and conversely, shorter pulses and / or more focused pulses may have lower pulse energy.

[0017] By using a laser with high (peak) pulse intensity, it is possible to obtain high quality high order images, while still providing sufficient contrast, eliminating the need to combine (e.g., average) several images. By selecting a burst with only a few pulses per pixel, overheating of the tissue can be avoided. In a typical embodiment, a raster scanner with a continuously moving mirror exposes multiple pixels to the laser beam in succession. By selecting only a small number of pulses per pixel (compared to the number of pulses that would expose the pixel without the pulse picker), the exposure time per pixel is limited, and motion artifacts, such as the motion artifacts due to the continuous movement of the raster scanner, such as smearing, are reduced.

[0018] Moreover, by avoiding the need to stitch together multiple images, artifacts due to dynamic behavior of the sample can be avoided. This is even more relevant for handheld systems, where high frame rates are desired and the acquisition head may move during image acquisition, making stitching together images acquired at different times more difficult. Handheld scanning devices may perform microscopy in situ, for example during surgery and / or during endoscopic procedures.

[0019] Thus, the microscope system described herein may be used as a pathology microscope, where the raster scanner and light source are configured to generate sufficient signal, and sufficient dynamic range in the signal, to be able to obtain a (pathology) image in a single raster scan, in this way the slow dynamics of unfixed tissue do not affect the microscope resolution.

[0020] The sample can be a sample that has not been pre-processed, for example during intra-operative imaging, such as a freshly taken biopsy or a living sample.

[0021] In some embodiments, the laser may have a wavelength of 1000-1200 nm, for example, about 1050 nm. Lasers in this wavelength range may be relatively economical. In addition, for the detection of the third harmonic signal, an optical element, for example, an objective lens, generally needs to be suitable (for example, transparent) for both the illumination light and the third harmonic signal having a wavelength that is one-third the wavelength of the illumination light. For example, when using a laser light source with a wavelength of 1050 nm, an optical element suitable for a wavelength of 350 nm to 1050 nm can be used. An optical element suitable for a wavelength of 350 nm or more, for example, a high numerical aperture objective lens, is typically more economical than one suitable for a shorter wavelength, for example, less than 300 nm.

[0022] Moreover, light absorption by water has a minimum near 1050 nm and increases substantially with longer wavelengths. Because biological samples typically contain a significant amount of water, minimizing energy absorption by water reduces tissue heating. At shorter wavelengths, the third harmonic signal shifts to the ultraviolet end of the spectrum, again resulting in higher absorption by the sample and therefore a weaker signal.

[0023] In other embodiments, the laser may have a wavelength of 700-900 nm, preferably 750-850 nm, more preferably about 800 nm. Laser sources with these wavelengths are particularly suitable for systems that do not measure the third harmonic signal. At about 800 nm, the light absorption by water is even lower than at 1050 nm, both the illumination light and the second harmonic signal are in the visible spectrum or at least close to the visible spectrum, and the availability of suitable optical components (including sensors) is generally good.

[0024] In one embodiment, the laser source may have a pulse repetition rate in the range of 10-100 MHz, preferably in the range of 20-40 MHz. The pulse repetition rate should be fast enough to allow fast scanning of the sample. In addition, a higher frequency results in shorter exposure times and therefore fewer or smaller motion artifacts.

[0025] In one embodiment, the pulse picker can be configured to change the number of pulses per burst.In this way, the illumination of the sample can be adjusted to optimize the signal, for example, based on tissue type or scanning depth.Adjusting the number of pulses per burst affects the intensity of several signal types, such as second harmonic signal, third harmonic signal and autofluorescence signal, equally in a linear manner.

[0026] In one embodiment, the microscope system can be configured to adjust the peak intensity per pulse. Adjusting the peak intensity per pulse also affects the energy per pulse. In this way, the illumination of the sample can be adjusted to optimize the signal, for example, based on tissue type or scanning depth. Adjusting the peak intensity per pulse affects different signal types, such as second harmonic signals, third harmonic signals and autofluorescence signals, in different ways. Therefore, by adjusting the peak intensity per pulse, one type of signal can be enhanced relative to a different type of signal.

[0027] In some embodiments, the peak intensity per pulse can be adjusted by a pulse picker, which reduces the need for additional components, such as filtering components, simplifying the system and reducing costs.

[0028] In one embodiment, the pulse picker comprises an acousto-optic modulator. This is an efficient and economical way to implement a pulse picker that can switch at high speed (typically with a rise time of about 10 ns) and with sufficient efficiency (typically with an efficiency of more than 80%). The acousto-optic modulator can be easily controlled to select a single pulse from the pulse train generated by the femtosecond oscillator, or can be used to select multiple consecutive pulses. Moreover, the acousto-optic modulator can also be used to reduce the pulse energy from 0% to more than 80% of the original pulse energy. Thus, a single element can be used to both select a burst containing any predetermined number of pulses from the pulse train and adjust the energy of the selected pulses.

[0029] In one embodiment, the pulse picker is configured to select one or more bursts of pulses from the pulse train, where each of the one or more bursts comprises 2-20, preferably 3-10, more preferably 4-8 consecutive pulses. The bursts are separated from each other by at least one non-selected pulse from the pulse train. Typically, for a pulse train of sufficient length, the number of non-selected pulses is at least as many as the number of selected pulses. In a typical example, the laser source has a frequency of about 40 MHz or 80 MHz. Thus, for a pixel rate of 1 MHz (corresponding to a dwell time of 1 μs), a burst comprising n consecutive pulses is followed by 40-n or 80-n non-selected pulses, respectively.

[0030] In general, the fewer the number of pulses per burst, the higher the spatial resolution of the microscope system can be. Because the fast axis of the scanner is typically implemented as a resonant scanner or as a galvo mirror or MEMS mirror operating in continuous oscillating mode, every light pulse excites a slightly different spot in the sample. This smearing effect can be minimized with short bursts, because the signals generated by successive pulses in the burst are usually indistinguishable from each other (due to the detector properties). On the other hand, a higher number of pulses increases the illumination and therefore causes the signal intensity to change in a linear manner.

[0031] Moreover, typical photodetectors, such as photomultipliers, may give more reliable signals when multiple pulses are used. This is due to the fact that the second and third harmonic signals have the same order of duration as the illumination pulse, typically 1 ns or less. As a result, peaked space charge in the photomultiplier may saturate the gain, reducing the dynamic range and / or introducing non-linear effects. This in turn reduces the possibility of quantitative evaluation of the microscopic image.

[0032] In one embodiment, the at least one detector is configured to detect second harmonic signals, third harmonic signals, autofluorescence signals, and / or multiphoton signals. In this way, high-contrast pathological images can be obtained without sample preparation, in particular without the need to stain the sample.

[0033] In one embodiment, the pulse picker is configured to repeatedly select bursts of pulses. This allows different parts of the sample to be repeatedly observed, in some cases. In such an embodiment, the optical system may include a raster scanner for scanning a target area of ​​the sample. The raster scanner may define a two-dimensional raster of pixels of the target area, where each pixel is illuminated, preferably by a single burst of pulses. In this way, a two-dimensional image of the sample may be acquired with high spatial resolution and low acquisition time. To this end, the microscope system may include a controller configured to receive a signal from at least one detector for each pixel in the target area.

[0034] In one embodiment, the pulse picker is configured to select a pulse burst having a frequency of at least 0.1 MHz, preferably at least 0.5 MHz, more preferably about 1-5 MHz. This frequency may be referred to as a burst frequency. The selection frequency of the pulse picker is preferably selected such that each pixel defined by the raster scanner is illuminated with a single burst. Thus, the period of the pulse picker preferably corresponds to the dwell time per pixel of the scanner.

[0035] The detector may be configured to have a bandwidth substantially smaller than the pulse repetition rate and larger than the burst frequency (or scan rate). For example, if the pulse repetition rate is 80 MHz and the burst frequency is 1 MHz, the detector bandwidth may be in the range of 1.5-20 MHz. Preferably, the detector bandwidth is not much higher than the burst frequency, such as about 1.5-4 times higher, such as about 2 times higher. This suppresses noise.

[0036] In one embodiment, the raster scanner is a galvo-scanner system or a MEMS mirror scanner.These types of raster scanners are fast enough to allow high image acquisition speed.In addition, they can be easy to control and implement into the system.

[0037] In one embodiment, the microscope system may further include a controller, preferably a field-programmable gate array (FPGA), for controlling the pulse picker and preferably the raster scanner and / or data acquisition from the detector. By using a single controller to control multiple components, these components can be easily synchronized. In one embodiment, the controller is synchronized with the laser light source. In this way, all related components are essentially synchronized.

[0038] In a further aspect, the invention may relate to a method for femtosecond pulsed microscopy. The method may include selecting one or more bursts of pulses from a pulse train including femtosecond laser pulses. Each of the one or more bursts includes a plurality of consecutive pulses. The one or more bursts include less than 25%, e.g., less than 15%, of the pulses in the pulse train. The method further includes illuminating a sample with the one or more bursts of pulses and detecting a signal resulting from an interaction of the one or more bursts of pulses with the sample. The signal corresponds to one of the one or more bursts.

[0039] In one embodiment, the selecting comprises repeatedly selecting bursts of pulses. In such an embodiment, the method may further comprise moving a focal spot to different positions on the sample. Preferably, the positions on the sample define a raster of sample pixels in a target area of ​​the sample, where a sample pixel is illuminated by one burst of pulses.

[0040] In one embodiment, the method may further include determining an image comprising a plurality of image pixels, where each image pixel corresponds to a sample pixel and where each image pixel has a pixel value based on the detected signal resulting from the interaction of the burst of pulses with the corresponding sample pixel.

[0041] One or more of these method steps may be performed by a microscope system as described above. Consequently, the microscope system described above may be configured to perform one or more of these method steps.

[0042] One aspect of the present disclosure relates to a control module, e.g., a field programmable gate array (FPGA), for a microscope system as described above. The control module may be configured to perform or cause the microscope system to perform one or more of the method steps described above. In some embodiments, the control module may comprise a computer-readable storage medium having computer-readable program code embedded therein, and a processor, preferably a microprocessor, coupled to the computer-readable storage medium, where in response to execution of the computer-readable program code, the processor is configured to perform any of the methods described herein.

[0043] One aspect of the present disclosure relates to a computer program, or computer program suite, comprising at least one software code portion, or a computer program product storing at least one software code portion, where the software code portion is configured to perform any of the methods described herein when executed on a computer system, e.g. a control module, as described above.

[0044] One aspect of the present disclosure relates to a non-transitory computer-readable storage medium storing at least one software code portion, where the software code portion is configured to perform any of the methods described herein when executed or processed by a computer.

[0045] As will be appreciated by those skilled in the art, aspects of the invention may be embodied as a system, method, or computer program product. Accordingly, aspects of the invention may take the form of an entirely hardware embodiment, an entirely software embodiment (including firmware, resident software, microcode, etc.), or an embodiment combining software and hardware aspects, all of which may be referred to generally herein as a "circuit," "module," or "system." Functions described in this disclosure may be implemented as an algorithm executed by a microprocessor of a computer. Moreover, aspects of the invention may take the form of a computer program product embodied in one or more computer-readable medium(s) having computer-readable program code embodied thereon, e.g., stored thereon.

[0046] Any combination of one or more computer readable media may be utilized. The computer readable medium may be a computer readable signal medium or a computer readable storage medium. The computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination thereof. More specific examples (non-exhaustive list) of the computer readable storage medium include an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM, or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the context of this specification, a computer-readable storage medium may be any tangible medium that can contain or store a program for use by or in connection with an instruction execution system, apparatus or device.

[0047] A computer-readable signal medium may include a propagated data signal having computer-readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated signal may take any of a variety of forms, including but not limited to, electromagnetic, optical, or any suitable combination thereof. A computer-readable signal medium may be any computer-readable medium, including but not limited to a computer-readable storage medium, and which is capable of communicating, propagating, or transmitting a program for use by or in connection with an instruction execution system, apparatus, or device.

[0048] Program code embodied on a computer readable medium may be transmitted using any suitable medium, including, but not limited to, wireless, wired, fiber optic, cable, RF, etc., or any suitable combination of the foregoing. Computer program code for performing operations for aspects of the present invention may be written in any combination of one or more programming languages, including functional or object-oriented programming languages ​​(e.g., Java, Smalltalk, C++, etc.), and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may run entirely on the user's computer, partly on the user's computer as a stand-alone software package, partly on the user's computer and partly on a remote computer, or entirely on a remote computer, server, or virtualized server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including any of the types of networks listed above, such as a local area network (LAN) or a wide area network (WAN), or may be connected to an external computer (e.g., through the Internet using an Internet Service Provider).

[0049] Aspects of the present invention are described below with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the present invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor, in particular a microprocessor or central processing unit (CPU) or graphics processing unit (GPU), of a general-purpose computer, special-purpose computer, or other programmable data processing device to generate a machine such that the instructions executed by the processor of the computer, other programmable data processing device, or other device create means for implementing the function / act specified in one or more blocks of the flowchart illustrations and / or block diagrams.

[0050] These computer program instructions may also be stored in a computer-readable medium, the computer program instructions may direct the computer, other programmable data processing device, or other apparatus to function in a particular manner, such that the instructions stored in the computer-readable medium produce an article of manufacture including instructions that implement a function / act specified in one or more blocks of the flowchart illustrations and / or block diagrams.

[0051] The computer program instructions may also be loaded onto a computer, other programmable data processing device, or other device to cause the computer, other programmable processing device, or other device to perform a series of operating steps, creating a computer-implemented process, such that the instructions executing on the computer or other programmable processing device provide a process for implementing a function / act specified in one or more blocks of the flowchart illustrations and / or block diagrams.

[0052] The flowcharts and block diagrams in the figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart diagrams or block diagrams may represent a module, segment, or portion of code, including one or more executable instructions for implementing one or more specified logical functions. It should also be noted that in some alternative implementations, the functions described in the blocks may occur out of the order described in the figures. For example, two blocks shown in succession may in fact be executed substantially simultaneously, or the blocks may sometimes be executed in the reverse order, depending on the functionality involved. It should also be noted that each block of the block diagrams and / or flowchart diagrams, and combinations of blocks in the block diagrams and / or flowchart diagrams, may be implemented by a special purpose hardware-based system that performs the specified functions or acts, or a combination of special purpose hardware and computer instructions.

[0053] Moreover, there is provided a computer program for carrying out the methods described herein, as well as a non-transitory computer readable storage medium storing the computer program, which may for example be downloaded (or updated) into existing data processing systems or stored upon manufacture of these systems.

[0054] Elements and aspects discussed with respect to or in connection with a particular embodiment may be suitably combined with elements and aspects of other embodiments, unless expressly stated otherwise. The embodiments of the present invention will be further described with reference to the accompanying drawings, which show, in a schematic manner, embodiments according to the present invention. It will be understood that the present invention is in no way limited to these particular embodiments.

[0055] Aspects of the present invention will now be explained in more detail with reference to exemplary embodiments thereof as illustrated in the accompanying drawings. [Brief description of the drawings]

[0056] [Figure 1] FIG. 1 illustrates a schematic diagram of a microscope system according to one embodiment. [Diagram 2] 2A-2D illustrate generally the selection of a burst that includes one or more consecutive pulses. [Diagram 3] FIG. 3 illustrates the effect of the pulse picker on the illumination of the sample. [Figure 4] FIG. 4 illustrates the control signals of the pulse picker, which is synchronized to both the laser source and the raster scanner. [Diagram 5] FIG. 5 illustrates the effect of the pulse picker on the signal generated by the detector. [Figure 6] FIG. 6 illustrates the relationship between pulse energy and image quality. [Figure 7] FIG. 7 illustrates a method for femtosecond pulsed microscopy according to one embodiment. [Figure 8] FIG. 8 is a block diagram illustrating an exemplary data processing system that may be used to implement the methods and software products described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] [Detailed Description] The embodiments in the present disclosure describe a method for obtaining high-speed images of unprepared samples, such as fresh biopsies or living tissue, using nonlinear optics for contrast. The nonlinear optics may include, for example, second harmonic, third harmonic, autofluorescence, and multiphoton interactions. Thus, the embodiments allow for obtaining high-resolution (pathological) images during surgery and / or endoscopy, allowing surgical and / or endoscopic procedures to be adjusted based on the pathological information. Although the examples given herein are primarily directed to pathological applications, other applications, such as industrial quality control, are not excluded.

[0058] 1 illustrates a schematic diagram of a microscope system according to one embodiment. The microscope system 100 includes a light source 102 comprising a femtosecond laser, for example a mode-locked Ti:sapphire laser or a Yb fibre laser. Currently suitable laser sources typically generate a pulse train 126 with a repetition rate of 10-100 MHz, for example around 80 MHz. As will be explained below, a higher repetition rate may provide a finer selection of the illumination intensity of the sample.

[0059] The selection of a suitable laser source may involve some trade-offs. In general, the higher the peak intensity of each pulse, the stronger the nonlinear optical signal. For example, the second harmonic scales with the square of the peak intensity, and the third harmonic scales with the cube of the peak intensity. However, too high a peak intensity may result in so-called non-linear damage to the sample 110. On the other hand, if the peak intensity is too low, a longer illumination time may be required to obtain a sufficient image quality, which may be obtained by longer pulses (hence higher energy per pulse) and / or by a higher number of pulses per pixel. This in turn may result in thermal damage due to overheating of the tissue. The energy at which tissue damage occurs may depend, among other things, on the tissue type, pulse intensity, pulse duration, wavelength, numerical aperture, and focal spot size. Typically, the energy per pulse should be at least 0.1 nJ, preferably in the range of about 1-5 nJ (measured on the sample). The pulse duration is less than 250 fs, preferably less than 100 fs. However, in other applications different parameters may be used. A much wider parameter range may be considered to optimize image quality, especially in applications where thermal damage is not an issue. In a typical example of imaging biological samples, a system with a numerical aperture of 1 and a laser source with a wavelength of 1050 nm may be used with a pulse time of less than 100 fs and an energy of about 1 nJ per pulse.

[0060] Similarly, the wavelength of the laser light source may be selected based on the envisaged use of the microscope system. Biological samples (including pathological samples) typically have a high water content. Therefore, to increase the maximum penetration depth and to reduce the energy absorption by the sample, it may be beneficial to select an illumination wavelength with low water absorption, for example around 800 nm or 1000-1200 nm, preferably around 1050 nm. The absorption spectrum of water has a minimum at approximately 800 nm and a local minimum at approximately 1050 nm.

[0061] The second harmonic signal has a wavelength that is half the illumination wavelength, and the third harmonic signal has a wavelength that is one third of the illumination wavelength. Thus, for an illumination wavelength of 800 nm, the wavelength of the second harmonic signal is 400 nm, and the wavelength of the third harmonic signal is 267 nm. The latter is in the (mid) ultraviolet part of the electromagnetic spectrum and may have high absorption by the sample, resulting in a weak signal. In addition, standard optical components may not be suitable for ultraviolet light, and suitable components may be more expensive than those optimized for visible light.

[0062] Thus, in a microscope system not configured to measure third harmonic signals, a laser light source having a wavelength of about 800 nm may be used. In a microscope system configured to measure third harmonic signals, the laser light source may generate light having a wavelength of approximately 1050 nm. In that case, the second harmonic has a wavelength of 525 nm, and the third harmonic has a wavelength of 350 nm. These wavelengths have relatively low absorption by typical biological samples. Moreover, optical components that are transparent in the range of 350-1050 nm may be used, which tend to be more readily available and / or more economical than optical components for use in the (deeper) infrared or ultraviolet ranges.

[0063] A pulse picker 104 downstream of the laser source selects a burst 122 comprising one or more consecutive laser pulses from a pulse train 126. In general, the optimal number of pulses per burst depends on the sample, imaging depth, repetition rate, and energy per pulse, among others. A burst may comprise 1-20, preferably 2-10, more preferably 4-8 consecutive pulses. The bursts are separated from each other by at least one non-selected pulse from the pulse train.

[0064] Typically, for a pulse train of sufficient length, the number of unselected pulses will be at least as large as the number of selected pulses. Preferably, the burst is relatively short, i.e., the proportion of selected pulses is relatively low compared to the total number of pulses, e.g., less than 30%, preferably less than 20%, more preferably less than 15% of the total number of pulses.

[0065] The pulse picker may include an acousto-optic modulator (AOM) 140. The acousto-optic modulator may be driven by an acoustic source 142, such as a piezo element. A lens 144 may focus the laser pulses within the acousto-optic modulator. Typically, a first-order diffracted signal is used to select a pulse 122 from a pulse train 126, while a zeroth-order non-diffracted signal contains unselected pulses 124.

[0066] The acousto-optic modulator can include, for example, TeO2 crystal, fused silica, or quartz. Other suitable acousto-optic modulators are known in the art. The acousto-optic modulator can have an acoustic frequency of 20 to 400 MHz, for example about 250 MHz.

[0067] Acousto-optic modulators are an efficient and economical way to implement pulse pickers that can be switched quickly (typically with a rise time of about 10 ns) and with high efficiency (typically with an efficiency of over 80% for selected pulses and essentially 0% for unselected pulses). By switching the acoustic source on and off, the acousto-optic modulator can be easily controlled to select multiple successive pulses, or a single pulse, from a pulse train generated by a femtosecond oscillator. Pockels cells can theoretically be used to quickly and precisely switch between transmitting and blocking a laser bundle, but the high drive voltages required to operate a Pockels cell make it less suitable for switching at MHz frequencies. Optical MEMS switches, e.g., optical MEMS switches based on mirrors, are currently too slow for switching at MHz frequencies. However, they could be used in slower systems. If faster switches were available, they could also be used.

[0068] Because femtosecond pulses necessarily have a significant spectral width, and because acousto-optic modulators are essentially diffraction gratings (with the grating period being the wavelength of the acoustic wave), the diffracted light beam used as the "selected" beam can be distorted. This distortion is due to differences in the diffraction angles for different wavelengths in the femtosecond pulse, resulting in dispersion of the laser bundle. This distortion can affect the shape and focusing properties of the laser beam.

[0069] The dispersion can be at least partially compensated for using a dispersing prism 146. When a TeO2 acousto-optic modulator with an acoustic frequency of 250 MHz is used, an equilateral triangular dispersing prism made of SF11 glass can be used for dispersion compensation. The dispersing prism can be placed upstream or downstream of the acousto-optic modulator. However, it has been found that the efficiency of the acousto-optic modulator is improved when the dispersing prism is placed upstream of the acousto-optic modulator. A fused silica or quartz based acousto-optic modulator provides a smaller dispersion in the laser beam than a TeO2 based acousto-optic modulator, reducing or eliminating the need for dispersion compensation. On the other hand, a TeO2 based acousto-optic modulator provides a larger diffraction angle of the selected pulse, making it easier to separate the selected pulse from the unselected pulse.

[0070] The system may further comprise an energy filter for adjusting, preferably dynamically adjusting, the peak intensity per pulse and thus the energy per pulse. In this way, the peak intensity or energy can be optimized based on, for example, the sample type and imaging depth. The pulse picker and the energy filter can be the same component (or group of components), for example an acousto-optic modulator-based pulse picker as described above.

[0071] Thus, the acousto-optic modulator can simultaneously function as a dynamically adjustable pulse picker and as a dynamically adjustable filter. This allows for varying both the number of pulses per burst and the peak intensity or energy per pulse in a relatively simple and easy-to-operate manner. A conventional femtosecond laser microscope system may include a means to turn off the illumination of the sample during the backsweep, for example using a Pockels cell. However, as mentioned above, the Pockels cell cannot be switched at a sufficiently high rate to function as a pulse picker. Compared to, for example, a Pockels cell, the acousto-optic modulator can be switched on and off at a much higher rate.

[0072] Nevertheless, other embodiments may use separate devices for pulse picking and energy conditioning.

[0073] The illustrated system further includes a two-dimensional raster scanner 106, typically downstream of the pulse picker. The raster scanner may include a galvo scanner including a pair of galvo mirrors. Other embodiments may use different raster scanners, for example, a raster scanner based on MEMS mirrors, or a raster scanner based on resonant mirrors.

[0074] The system further comprises an objective lens 108 for focusing the pulse onto a sample 110. The objective lens preferably has a high numerical aperture to enable a small focal spot, e.g., a numerical aperture greater than or equal to 1. A large numerical aperture results in a smaller focal spot size, and therefore higher spatial resolution, and a higher peak light intensity, and therefore higher signal strength.

[0075] The system further includes one or more detectors 112, 114, and 116 for detecting light reflected by and / or transmitted through the sample. The light may be split using, for example, a partially reflective mirror 113 and / or a dichroic mirror 115. For example, the system may include a detector 112 for detecting a transmitted signal at the sample opposite the objective lens.

[0076] The system may include a detector 114 for detecting the second harmonic. The second harmonic light has a wavelength that is half the wavelength of the incident light. Thus, if a laser light source with a wavelength of 1050 nm is used, the second harmonic light has a wavelength of 525 nm. The second harmonic light can be selected using a dichroic mirror 115. The second harmonic light can also be selected using a color filter, or other means known in the art.

[0077] The system may include a detector 116 for detecting the third harmonic. The third harmonic light has a wavelength that is one third of the wavelength of the incident light. Thus, if a laser light source with a wavelength of 1050 nm is used, the third harmonic light has a wavelength of 350 nm. The third harmonic light can be selected using a dichroic mirror. The third harmonic light can also be selected using a color filter, or other means known in the art.

[0078] The system may include one or more further detectors (not shown) for detecting other signals, for example a detector for detecting autofluorescent light.

[0079] One or more of the detectors may comprise a photomultiplier tube. In other embodiments, other types of detectors, for example based on charge-coupled devices (CCDs), may be used. The photomultiplier tubes typically have very high sensitivity and can measure single photons. Thus, they are suitable for detecting often weak nonlinear signals, such as second and third harmonic signals, at relatively low and therefore non-damaging peak light intensities.

[0080] However, when pulse bursts with a single pulse are used, such that each pixel is illuminated by only a single pulse (the "single-shot approach"), the signal-to-noise ratio may be limited by the inherent characteristics of the photomultiplier tube, as explained below.

[0081] Although the (dark) noise of the detector is typically negligible, typically much less than one photoelectron per pixel at a pixel rate of 1 MHz, this can limit the magnitude of the signal that can be detected. The signal-to-noise ratio varies as the square root of the number of photons detected. For example, to obtain a signal-to-noise ratio of 100, at least 10,000 photons per pixel need to be detected.

[0082] However, the response from a single femtosecond pulse to detect the second and third harmonics is very short, typically in the nanosecond range or even shorter, even after multiple scattering in the sample (this is also true to some extent for multiphoton fluorescence signals, which are broadened by the lifetimes of the excited states involved), which leads to space charge effects in the photomultiplier tube, and linear amplification in the dynode chain is not always possible.

[0083] As a result, when such detectors detect a response from a single light pulse, the detected signal is found to have a relatively high non-linearity, whereas the detector response to a burst containing multiple pulses is relatively much more linear. For many applications, such as quantitative analysis of microscopy images, especially AI-based analysis, a single burst containing multiple pulses may be preferred since a linear response is typically preferred.

[0084] By using a low number of pulses per pixel, greater than 2, for example 5-10 pulses, the arrival time of the signal is spread out, space charge effects are avoided, and signal linearity can be improved.

[0085] The system further comprises a controller 118, e.g., a field programmable gate array (FPGA). Preferably, a single controller controls the pulse picker, the raster scanner, and the data acquisition system. In this way, real-time control and optimal synchronization can be achieved. By performing relatively short bursts with good synchronization with the raster scanner, the analog bandwidth of the detector does not result in a loss in resolution. The measurement may be considered as "stroboscopic".

[0086] The controller may be connectable to a host computer via a data connection 130. System parameters, such as scan parameters, e.g., number of pulses per burst and energy per pulse, can be loaded into the controller from the host computer, and acquired data can be streamed from the controller to the host computer.

[0087] 2A-2D illustrate the selection of a burst containing one or more consecutive pulses. When an acousto-optic modulator is used as a pulse picker, the number of pulses in a burst and the amplitude (energy) of the pulses can be adjusted by adjusting the control signal provided to the acousto-optic modulator. In practice, the amplitude of the selected pulse can be varied from 0% to about 80% of the input.

[0088] In these examples, the laser source generates a pulse train 200 with a frequency of 80 MHz (hence the period between two pulses is 12.5 ns), the width of the femtosecond pulses is exaggerated to improve visibility.

[0089] 2A illustrates a control signal 202 for a pulse picker synchronized with a laser source. The control signal has a frequency of 4 MHz, a pulse width of about 37.5 ns, and a maximum amplitude. This results in a burst 204 containing 3 consecutive pulses out of every 20 pulses from the pulse train. The relative burst length is therefore 3 / 20=15%. These pulses in the burst of selected pulses have an amplitude of about 80% of the input pulse.

[0090] 2B illustrates a control signal 212 for a pulse picker, synchronized with the laser source. The control signal has a frequency of 4 MHz, a pulse width of about 37.5 ns, and an amplitude of half the maximum amplitude. This results in a burst 214 containing three consecutive pulses out of every 20 pulses selected from the pulse train. These pulses in the burst of selected pulses have an amplitude of about 40% of the input pulse. This means that the detection of the second harmonic is reduced by about a factor of four and the detection of the third harmonic by about a factor of eight, compared to the previous example.

[0091] FIG. 2C illustrates a control signal 222 for a pulse picker, synchronized with the laser source. The control signal has a frequency of 4 MHz, a pulse width of about 62.5 ns, and a maximum amplitude. This results in a burst 224 containing 5 consecutive pulses out of every 20 pulses from the pulse train (relative burst length 25%). These pulses in the burst of selected pulses have an amplitude of about 80% of the input pulse. Compared to the situation illustrated in FIG. 2A, this results in an approximately 40% increase in illumination and signal for all detectors. However, the spatial resolution is potentially lower because the illumination is more spatially spread out.

[0092] FIG. 2D illustrates a control signal 232 for a pulse picker that is not synchronized with the laser source. The control signal has a frequency of 3.95 MHz, a pulse width of about 37.5 ns, and a maximum amplitude. This results in a burst 234 containing 2-3 consecutive pulses being selected from the pulse train every 20.25 pulses. In the illustrated example, the first burst contains three pulses with an amplitude of about 80% of the input pulse. The second burst contains two pulses with an amplitude of about 80% of the input pulse and one pulse that passed through the pulse picker during the falling edge of the control signal with an amplitude of about 40% of the input pulse. The third burst contains only two pulses with an amplitude of about 80% of the input pulse.

[0093] This can affect image quality and result in the formation of banding. The effect can be different for different signal types. Generally, the effect is less noticeable when each burst contains a larger number of pulses and may become almost invisible for bursts with, for example, more than 5 or more than 8 pulses.

[0094] FIG. 3 illustrates the effect of a pulse picker on the illumination of a sample. Typically, each selected pulse produces an illuminated spot on the sample, the size of which is limited by the diffraction limit and therefore depends on the wavelength. For a 1050 nm laser pulse, the size of the focal spot is about 0.5 micrometers (given by the diffraction limit, the effective spot size is smaller due to nonlinear effects). For a raster scanner operating with a line frequency of 1 kHz and a field of view of 0.5 mm (typically limited by the collection optics) and an 80 MHz laser (the most common type), subsequent spots shift by about 12.5 nm. This is a small fraction of the spot size of about 500 nanometers, and therefore the multiple spots associated with subsequent pulses overlap. In the figure, the distance between spots is only approximate (and greatly exaggerated) in order to preserve the visibility of the individual spots.

[0095] In a first example, the pulse picker selects all pulses from the pulse train 302. The pulse picker may also function as a variable attenuator. In this case, as explained above, there is substantial overlap between the spots 304 illuminated by subsequent pulses. In the given example, each portion of the sample is illuminated by 40 subsequent pulses, which may significantly heat the sample and damage it. Moreover, it is usually not easy to separate signals from individual pulses in time, which reduces spatial resolution.

[0096] In a second example, the pulse picker selects only a single pulse 306 per pixel. Theoretically, this may result in the highest spatial resolution and the lowest sample heating. In this case, the spots 308 associated with subsequent pulses do not overlap, or overlap only a negligible amount. However, this mode may have two practical drawbacks. First, if the peak pulse intensity is high enough to obtain a good signal, there is a high risk of nonlinear damage to the sample. For biological samples, nonlinear damage typically starts to occur at around 5 nJ per pulse (measured on samples; actual value depends on wavelength and pulse length). Second, as explained above, the detector may show a (strongly) nonlinear response to a single very short pulse.

[0097] The third example shows a burst 310 with a relatively small number of consecutive pulses, greater than one, in this case three pulses per burst. Each pixel is illuminated by three spots 312, and thus the energy deposition is only 3 / 40=7.5% of that in the first example above. At the same time, the peak power can be less than in example 2 above, reducing or preventing damage to the nonlinear sample. In addition, the pulses within a burst are separated in time in a manner sufficient to avoid space charge effects in the detector, and the bursts are separated in time sufficiently to easily associate each signal with a pixel.

[0098] 4 illustrates the control signals of a pulse picker that is synchronized to both a laser source and a raster scanner. In the example shown, the raster scanner includes a slow mirror and a fast mirror. The fast mirror follows a sine pattern 408. As a result, the mirror focuses on pixels 410 that are near the center of the field of view. i Above, pixels 4101 and 4102 are moving relatively fast and are close to the edge of the field of view. n As a result, pixels near the edges will be overexposed compared to pixels near the center when constant lighting is used.

[0099] In some microscope systems, the microscope system can be compensated by adjusting the detector signal, but this does not prevent overheating of the sample. Additionally or alternatively, some systems mitigate this effect by effectively using only the central part of the mirror motion and switching the light source near the turning point. In some systems, only about one third of the mirror range is used. This limits the field of view and increases acquisition time.

[0100] This can be addressed by controlling the pulse picker with a control signal 404 based on the speed of the raster scanner. In particular, the speed of the pulse picker can be approximately proportional to the speed of the raster scanner, in particular the speed of the high speed mirror of the raster scanner.

[0101] As shown in the figure, a fixed number of pulses near the center of each pixel are selected. Similar to FIG. 2, the laser source generates a pulse train 402 at a repetition rate of 80 MHz. Because the pulse picker is synchronized to the laser source, each burst 406 contains the same number of pulses, and therefore the illumination of each pixel is the same. In this example, the mirror oscillates at a frequency of 0.47 MHz, resulting in a line frequency of 0.96 MHz. In this example, the mirror is not synchronized to the laser source, and as a result, the bursts may not line up exactly with the centers of the pixels.

[0102] In some embodiments, a resonant mirror can be used as the fast mirror. In such cases, it may not be possible to synchronize the laser source and the fast mirror. A resonant mirror is faster than a non-resonant mirror and can reduce acquisition time. However, if the mirror moves too fast, this may introduce motion artifacts or reduce the signal-to-noise ratio of the obtained image. In some embodiments, this can be at least partially corrected using known software.

[0103] In other embodiments, such as the embodiment illustrated in Figure 1, the fast mirror may be driven by a control signal, in which case the movement of the mirror may be synchronized to the laser source.

[0104] In some embodiments, a more linear control signal, such as a triangular signal, may be used to drive the mirror. In such embodiments, the mirror velocity may be essentially constant over a large range of mirror motion. In these embodiments, there may be little or no need to use a variable pulse picker rate based on the fast mirror velocity. The pulse picker may be used to effectively turn off illumination during the nonlinear portion of the mirror motion.

[0105] Synchronization of the raster scanner and the detector is described in more detail below with reference to FIG.

[0106] For pictorial clarity, the example shown includes only seven pixels on each line, although a more realistic example may have hundreds of pixels per line or more.

[0107] 5 illustrates the effect of the pulse picker on the signal generated by the detector. In the embodiment shown, a pixel rate of 1 MHz is selected. A control signal 504 is provided to the pulse picker to select a burst of 5 pulses 506 from a 35 MHz pulse train 502. This results in a detector signal 508.

[0108] Here, the detector signal represents a rough impression of the signal from the photomultiplier tube after amplification by a trans-impedance amplifier with a bandwidth of about 2 MHz. These amplifiers result in low noise, but the bandwidth is insufficient to separate the signals from the five individual pulses per bunch. An amplifier with a higher bandwidth would allow the signals corresponding to each of the five pulses to be registered separately, but this comes at the cost of higher noise.

[0109] As discussed above with reference to FIG. 3, when the bursts are relatively short, corresponding light pulses will strike approximately the same spot on the sample, so there is no need to distinguish between individual pulses. However, proper synchronization between the data collection system and the raster scanner is required to assign each detection to the correct pixel. If the raster scanner has a substantially non-uniform speed, the data acquisition rate can be adjusted accordingly. Alternatively, the data acquisition rate can be substantially higher than the pixel rate, resulting in oversampling. The data point closest to the center of each pixel can then be selected.

[0110] By measuring the burst as a single unity, relatively slow transimpedance amplifiers can be used, such as those with 2 MHz bandwidth and high gain (300 KV / A), simplifying the detection electronics, reducing noise, and reducing cost.

[0111] Figure 6 illustrates the relationship between pulse energy and signal intensity for the third harmonic signal. All images were obtained with the same microscope system, which is similar to the system illustrated in Figure 1. The images were obtained with a Yb fiber laser with a pulse repetition rate of 13 MHz. An acousto-optic modulator was used as the pulse picker. The pulse picker, raster scanner and detector were controlled by a single FPGA and therefore synchronized with each other. All five images were recorded in burst mode with 3 pulses per pixel. The energy of the pulses in the burst of selected pulses was allowed to vary from 0 to 80% of the input energy. The percentages in the figure represent the percentage of the maximum output energy that can be delivered by the pulse picker.

[0112] The image shows a calibration grid (Ibidi) with a 50 micrometer grid. The signal measured and shown is the third harmonic signal at the grid surface. The cubic power dependence of the third harmonic signal is demonstrated by varying the pulse power going from left to right from about 0.4 nJ, to about 0.7 nJ, to about 2.2 nJ, to about 3.5 nJ, to about 5 nJ per pulse delivered to the sample. In the top row, all signals are scaled the same, where the scaling is based on 100% image. The bottom row shows the same data as the top row, but contrast scaling has been used to enhance the image.

[0113] Obviously, this is 2000 times lower than when the signal is at 100% ((1 / 0.08) 3 ) Even images recorded at a low 8% power are significantly above the noise, demonstrating that the scan has a dynamic range of about 4000. This equates to 12 bits per channel, or up to 36 bits of data when using 3 channels. This poses the luxury problem of more information than can be represented on a 24-bit RGB display.

[0114] It is important to note that when adjusting the power on the sample by varying the number of pulses per burst, this has the same effect on all channels: whether it is a 3rd harmonic, 2nd harmonic, or even a linear signal, all channels are scaled proportionally to the number of pulses per burst.

[0115] When adjusting the light intensity by varying the pulse energy, the third harmonic signal scales with the cube of the pulse power, and the second harmonic signal scales with the square of the peak power, therefore varying the burst width is typically the best way to adjust the power on the sample.

[0116] FIG. 7 illustrates a method for femtosecond pulsed microscopy according to one embodiment. One or more of these method steps may be performed by a microscope system such as described above with reference to FIG. 1. In a first step 702, the method includes selecting a burst of pulses from a pulse train including femtosecond laser pulses. The burst may include one or more consecutive pulses. The burst may be bounded on either side by one or more unselected pulses.

[0117] In step 704, a sample, e.g., a biological sample, e.g., living tissue or a fresh biopsy, is illuminated with a selected burst of pulses. This is typically done using an optical system that focuses the selected burst of pulses at a location on the sample. Preferably, a high numerical aperture objective is used, e.g., NA≧1. In step 706, a signal resulting from the interaction of the burst of pulses with the sample is detected.

[0118] In a typical embodiment, steps 702-706 are repeated several times. Between two repetitions, in step 708, the focal spot may be moved to different positions on the sample. Preferably, the positions on the sample define a raster of sample pixels in a target area of ​​the sample. This may be achieved by using a raster scanner as described above. Each sample pixel may be illuminated by at least one, preferably exactly one, burst of pulses. This may be achieved by synchronizing the control of a pulse picker performing step 702 and a raster scanner performing step 708.

[0119] In optional step 710, the method may further include determining an image comprising a plurality of image pixels, where each image pixel corresponds to a sample pixel and each image pixel has a pixel value based on the detected signal resulting from the interaction of the burst of pulses with the corresponding sample pixel. The resulting image may be displayed and / or subjected to further analysis. This step may be performed by a data processing system such as that described below with reference to FIG. 8.

[0120] 8 is a block diagram illustrating an exemplary data processing system as described in this disclosure. Data processing system 800 may include at least one processor 802 coupled to memory elements 804 through a system bus 806. As such, the data processing system may store program code in memory elements 804. Furthermore, processor 802 may execute program code accessed from memory elements 804 via system bus 806. In one aspect, the data processing system may be implemented as a computer suitable for storing and / or executing program code. However, it should be understood that data processing system 800 may be implemented in the form of any system including a processor and memory capable of performing the functions described herein.

[0121] The memory element 804 may include one or more physical memory devices, such as a local memory 808 and one or more bulk storage devices 810. The local memory may refer to a random access memory or one or more other non-persistent memory devices typically used during the actual execution of the program code. The bulk storage device may be implemented as a hard drive or other persistent data storage device. The processing system 800 may also include one or more cache memories (not shown) that provide temporary storage of at least some of the program code to reduce the number of times the program code must be retrieved from the bulk storage device 810 during execution.

[0122] Input / output (I / O) devices, illustrated as key devices 812 and output devices 814, may optionally be connected to the data processing system. Examples of key devices may include, but are not limited to, for example, a keyboard, a pointing device (e.g., a mouse), and the like. Examples of output devices may include, but are not limited to, for example, a monitor or display, speakers, and the like. The key devices and / or output devices may be connected to the data processing system directly or through an intervening I / O controller. The network adapter 816 also allows the data processing system to be connected to other systems, computer systems, remote network devices, and / or remote storage devices through intervening private or public networks. The network adapter may include a data receiver for receiving data transmitted to the system, device, and / or network from the system, device, and / or network, and a data transmitter for transmitting data to the system, device, and / or network. A cable operation modem, a cable operation modem, and an Ethernet card are examples of different types of network adapters that may be used with the data processing system 800.

[0123] 8, memory element 804 may store application 818. It should be understood that data processing system 800 may further execute an operating system (not shown) that may facilitate execution of applications. The applications may be implemented in the form of executable program code and executed by data processing system 800, such as processor 802. In response to executing the applications, the data processing system may be configured to perform one or more operations described in further detail herein.

[0124] In one aspect, for example, data processing system 800 may represent a client data processing system, in which case application 818 may represent a client application that, when executed, configures data processing system 800 to perform various functions described herein with reference to a "client." Examples of clients may include, but are not limited to, personal computers, portable computers, mobile phones, and the like.

[0125] In another aspect, data processing system 800 may represent a server. For example, data processing system 800 may represent an (HTTP) server, in which case application 818, when executed, may configure data processing system 800 to perform (HTTP) server operations. In another aspect, data processing system may represent a module, unit, or function as referred to herein.

[0126] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the present invention. As used herein, the singular forms "a" and "an" as well as "the" are intended to encompass the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that as used herein, the words "comprises" and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0127] Corresponding structures, materials, acts, and equivalents of all means or step-plus-function elements in the appended claims are intended to include any structure, material, or act for performing a function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or to limit the invention to the disclosed form. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the invention. The embodiments have been selected and described in order to best explain the principles and practical applications of the invention and to enable those skilled in the art to understand the invention in various embodiments with various modifications suitable for the particular use contemplated.

Claims

1. 1. A method for femtosecond pulsed microscopy, comprising: selecting one or more bursts of pulses from a pulse train comprising femtosecond laser pulses, wherein each of the one or more bursts comprises a plurality of consecutive pulses, and the one or more bursts comprises less than 25% of the pulses in the pulse train; illuminating the sample with said selected one or more bursts of pulses; and detecting a signal resulting from interaction of said one or more bursts of pulses with said sample, said signal corresponding to one of said one or more bursts of pulses; The method comprising:

2. said selecting including repeatedly selecting bursts of pulses; The method comprises: moving the focal spot to different positions on the sample, where each sample pixel is illuminated by one burst of pulses; The method of claim 1 further comprising:

3. determining an image comprising a plurality of image pixels, wherein each image pixel corresponds to a sample pixel and each image pixel has a pixel value based on the detected signal resulting from an interaction of the burst of pulses with the corresponding sample pixel; The method of claim 2 further comprising:

4. A microscope system configured to perform the method according to any one of claims 1 to 3, wherein the microscope system comprises: a laser source for providing a pulse train comprising a series of femtosecond pulses; a pulse picker for selecting one or more bursts of pulses from the pulse train, wherein each of the one or more bursts of pulses comprises a plurality of consecutive pulses, and the one or more bursts comprises less than 25% of the pulses in the pulse train; an optical system for illuminating a sample with said one or more bursts of pulses; and a detector for detecting signals resulting from the interaction of the one or more bursts of pulses with the sample, wherein the detector is configured to generate a single signal for each of the one or more selected bursts; The microscope system comprises:

5. 5. The microscope system of claim 4, wherein the pulse picker can be configured to vary the number of pulses per burst.

6. The microscope system according to claim 4 , wherein the microscope system can be configured to adjust a peak intensity per pulse.

7. The microscope system of claim 4 , wherein the pulse picker comprises an acousto-optic modulator.

8. The microscope system of claim 4 , wherein the pulse picker is configured to select a burst having 2 to 20 consecutive pulses.

9. the pulses have an energy of at least 1 nJ per pulse as measured at the sample; and / or the pulses have an energy per pulse of 10 nJ or less as measured at the sample; The microscope system according to claim 4 .

10. The microscope system according to claim 4 , wherein the pulse length is 250 fs or less.

11. the laser light source has a wavelength of 1000 to 1200 nm; or The laser light source has a wavelength of 700 to 900 nm. The microscope system according to claim 4 .

12. the pulse picker is configured to repeatedly select bursts of pulses; and the optical system comprising a raster scanner for scanning a target area of ​​the sample, wherein the raster scanner defines a two-dimensional raster of pixels in the target area, each pixel being illuminated by one burst of pulses; The microscope system according to claim 4 .

13. The microscope system of claim 12 , wherein the pulse picker is configured to repeatedly select bursts having a frequency of at least 0.1 MHz.

14. The microscope system of claim 4 , wherein the at least one detector comprises a photomultiplier tube.

15. 5. The microscope system of claim 4, wherein the at least one detector is configured to detect a second harmonic signal, a third harmonic signal, an autofluorescence signal, and / or a multiphoton signal.

16. The microscope system according to claim 4 , further comprising a controller for controlling the pulse picker.

17. The microscope system of claim 16 , wherein the controller is synchronized with the laser light source.

18. A control module for a microscope system according to claim 4, wherein the control module is configured to perform the method according to any one of claims 1 to 3.

19. A computer-readable storage medium including a software code portion which, when executed by a control module described in claim 18, causes the control module to execute a method described in any one of claims 1 to 3.

Images