Devices and methods to reduce optical damage in multiphoton microscopy with a scan synchronized laser pulse repetition rate

Abstract

A laser system for multiphoton microscopy comprises a seeder oscillator, fast modulator, amplifier, second modulator, and optical parametric amplifier configured to produce pulses with variable pulse spacing temporally synchronized to a scanner having sinusoidal motion. The variable pulse spacing ensures each pixel receives substantially the same number of laser pulses, eliminating uneven energy distribution of fixed repetition rate systems. The scanner operates at 4-12 kHz frequencies while the seeder oscillator produces femtosecond pulses at 10-100 MHz with 10-100 ns pulse spacing. The optical parametric amplifier outputs wavelengths from 600-1900 nm, with 1300-1700 nm optimized for three-photon microscopy. Methods include synchronizing pulse timing to scanner motion and varying interpulse intervals across scan lines. For 8 kHz scanners with 512 pixels per line, the system achieves 32% energy reduction compared to constant pulse spacing while maintaining 100-150 mW average power on biological samples, preventing thermal damage and enabling video rate imaging.

Description

Docket Number: 00772-WODEVICES AND METHODS TO REDUCE OPTICAL DAMAGE IN MULTIPHOTON MICROSCOPY WITH A SCAN SYNCHRONIZED LASER PULSE REPETITION RATEBACKGROUND

[0001] Presently, laser systems are used in many multiphoton or non-linear microscopy applications to provide high resolution three-dimensional(3D) images. During multi-photon microscopy processes one or more photoreactive materials may be introduced into living tissue or cells. Thereafter, a laser configured to output a laser signal having a wavelength with sufficient energy to excite fluorophores within the photoreactive material may be directed to the tissue or substrate under investigation. As a result, the energy from the laser signal excites the fluorophores that generates an image. One or more sensors, scanners, or detectors may be used to capture the signal generated by the fluorophores to create an image.

[0002] While present multi-photon microscopy systems have proven to be useful, a number of shortcomings have been identified. For example, two photon microscopy is widely used presently. While two photon microscopy has proven useful in some applications, shortcomings associated with two photon microscopy include more shallow depth of resolution and lower contrast (signal to background) as compared with three photon microscopy. In addition, multiple scans of laser energy in close proximity may result in thermal damage to the tissue under investigation. For example, Figure 1 shows a substrate 11 having a laser signal 15 directed along a first scan path 17 on the surface 13 of the substrate 11 . As shown, during the scanning process the laser spot 25 traverses along a medial portion 27 of the scan path 17. As a result, laser fluence is applied to the surface 13 of the substrate 11. The scanning process necessitates the direction of travel of the laser spot 25 to change. In many scanning processes, the direction of the travel of the laser spot 25 is essentially reversed (e.g. raster scanning) forming a sawtooth-like scan path. As shown in Figure 1 , the laser spot 25 traversing along the first scan path 17 is reverse such that the laser spot 25’ travels along a second scan path 17’, the second scan path 17’ being in close proximity to the first scan path 17. As a result, the proximity of a portion of the first scan path 17 to a portion of the second scan path 17’ results in the first and second laser spots 25, 25’ overlapping (see reverse point 31 ) proximate to the change of direction. This overlapping may result in the deposited energy potentially exceeding the thermal loadDocket Number: 00772-WO capacity of the tissue in the scan area and damaging the tissue. Further, the thermal load of the tissue may be exceeded at every location where the laser spot 15 changes direction on the surface 13 of the substrate 11 .

[0003] In contrast, a limitation to the expansion of some multi-photon microscopy processes (e.g. microscopy processes using three photons or more) is the ability to image with high temporal resolution, which requires acquisition at video rate. For example, the adoption of three-photon microscopy imaging techniques in neuroscience and biological tissue is limited as the current state of the art utilizes lasers with low, 1 -4MHz repetition rates. As a result, these lasers do not deliver enough pulses per second to enable high speed imaging directly. Unfortunately, laser systems with sufficiently high repetition rate to enable the use of resonant scanners at video rate for three-photon microscopy would exceed the thermal loading of biological tissue, thereby potentially resulting in tissue damage.

[0004] In light of the foregoing, there is an ongoing need for devices and methods enabling high speed direct imaging of a biological tissue or other thermally sensitive substrate without resulting in damage to the tissue or substrate being illuminated.SUMMARY

[0005] The present application is directed to a laser system for use with multiphoton microscopy scanning systems that reduce optical damage while enabling highspeed imaging. The laser system comprises a seeder oscillator configured to output pulsed seeder signals, a fast modulator for pulse selection, an amplifier for signal amplification, a second modulator for selective beam blanking, and an optical parametric amplifier for output signal generation. The system is uniquely configured to produce pulses with variable pulse spacing temporally synchronized to a scanner having sinusoidal motion. In one embodiment, the laser system may be capable of variable pulse spacing configured such that each pixel in a scanned image receives substantially the same number of laser pulses, despite the sinusoidal motion characteristics of the scanner. This synchronization eliminates the uneven energy distribution that occurs with conventional fixed repetition rate systems, where pixels at scan edges receive excessive energy while center pixels receive insufficient energy.

[0006] The laser system may operate with scanners having frequencies from about 4 kHz to about 12 kHz, accommodating various imaging speed requirements. The seeder oscillator may comprise a mode-locked oscillator producing femtosecondDocket Number: 00772-WO pulses, typically operating at 10 MHz to about 100 MHz with pulse spacing of about 10 ns to about 100 ns. The fast modulator may comprise an acousto-optic modulator, while the amplifier may comprise a Yb-based amplifier producing output signals with wavelengths from about 1020 nm to about 1064 nm.

[0007] The optical parametric amplifier output signal may have wavelengths ranging from about 600 nm to about 1900 nm. In one embodiment, the wavelength ranges from about 1300 nm to about 1700 nm and may be optimized for three photon microscopy applications. The system produces pulse lengths of about 1 fs to about 150 fs with a minimum pulse separation of about 20 ns. In one embodiment, the pulse lengths may range from about 30 fs to about 70 fs. Those skilled in the art will appreciate the systems disclosed herein may be used in any variety of applications. As such, the wavelengths may range from about 900 nm to about 2200 nm, the pulse lengths may range from about 1 fs to about 250 fs, and the pulse separation raging from about 1 ns to about 100 ns.

[0008] The present application further discloses methods for reducing optical damage in multiphoton microscopy by synchronizing laser pulse timing to scanner motion, varying time intervals between subsequent pulses across scan lines, and maintaining substantially constant pulse numbers per pixel. For typical operating conditions with an 8 kHz scanner and 512 pixels per line, the method achieves approximately 32% reduction in energy delivered to samples compared to constant pulse spacing operation.

[0009] The methods disclosed herein may be applied to both three-photon and two-photon excitation fluorescence microscopy, with pixel densities ranging from about 32 to about 4096 pixels per scan line, providing flexibility across various imaging applications and resolutions.

[0010] The present application further discloses a complete multiphoton microscopy system integrating the laser system with a scanner having sinusoidal motion, delivering substantially equal numbers of pulses to each pixel position during scanning. The system may operate at scanner frequencies of about 4 kHz, 8 kHz, or 12 kHz and may include external pulse splitters and delay lines for increasing pulses per pixel while maintaining scanner synchronization.

[0011] The system may be configured for three-photon microscopy applications and maintain average power on biological samples of about 100 mW to about 150 mW, preventing thermal damage while enabling high-quality imaging, therebyDocket Number: 00772-WO providing a significant advancement in multiphoton microscopy technology, enabling video rate imaging with reduced optical damage and improved energy efficiency.

[0012] Other features and advantages of the devices and methods described herein will become more apparent from a consideration of the following detailed description.Brief Description of the Drawings

[0013] The drawings disclose illustrative embodiments and are not intended to set forth all embodiments of the devices and methods to reduce optical damage in multiphoton microscopy with a scan synchronized laser pulse repetition rate. Details that may be apparent or unnecessary may be omitted to save space or for more effective illustration. Conversely, some embodiments may be practices without all the detailed disclosed with regard to specific embodiments. When the same reference numbers appear in different drawings, the reference numbers refer to same or similar components or steps. The novel aspects of the devices and methods to reduce optical damage in multiphoton microscopy with a scan synchronized laser pulse repetition rate as disclosed herein will become more apparent by consideration of the following figures, wherein:

[0014] Figure 1 shows a substrate having a laser signal directed along a scan path, illustrating the overlapping laser scan spots at the scan reversal point which may result in thermal damage to tissue in conventional scanning systems;

[0015] Figure 2 shows a schematic diagram showing an embodiment of a laser system configured to reduce optical damage during laser-based processing application, including a seed oscillator, fast modulators, at least one amplifier, and optical parametric amplifier components;

[0016] Figure 3 shows the pulse train from the seeder oscillator through the laser output, illustrating the temporal characteristics of the pulse sequence fed into the optical parametric oscillator;

[0017] Figure 4a shows the variable time intervals between subsequent pulses across each line in an image;

[0018] Figure 4b shows the pulse spacing varies to accommodate the sinusoidal motion of the scanner;

[0019] Figure 5a shows a representation of the pixel dwell time across the scan line;Docket Number: 00772-WO

[0020] Figure 5b shows the variation in time spent at each pixel position due to the sinusoidal scanner motion;

[0021] Figure 6 shows the calculations for different repetition rates and the corresponding reduction in pulse energy to achieve maximum allowed average power on biological samples; and

[0022] Figure 7 shows a table of the reduction in overall energy deposition enabled by the laser system when operating at high repetition rates compared to conventional fixed repetition rate systems.DETAILED DESCRIPTION

[0023] The following description of the presently contemplated best mode of practicing the invention is not to be taken in a limiting sense but is made merely for the purpose of describing the general principles of the invention. The scope of the invention should be determined with reference to the claims.

[0024] The present application discloses various embodiments of devices and methods to reduce optical damage to tissue or other substrates during multiphoton microscopy processes. In one embodiment, the devices and methods disclosed herein may be useful in three photon microscopy applications, although those skilled in the art will appreciate that the various embodiments disclosed herein may be useful in any variety of multiphoton microscopy applications. For example, the various devices and methods disclosed herein may be used in two photon applications, application requiring three photons or more. Exemplary embodiments are described below with reference to the accompanying drawings. Unless otherwise expressly stated, in the drawings the sizes, positions, etc., of components, features, elements, etc., as well as any distances therebetween, are not necessarily to scale, and may be disproportionate and / or exaggerated for clarity.

[0025] The terminology used herein is for the purpose of describing exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should be recognized that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Unless otherwise specified, a range ofDocket Number: 00772-WO values, when recited, includes both the upper and lower limits of the range, as well as any sub-ranges therebetween. Unless indicated otherwise, terms such as “first,” “second,” etc., are only used to distinguish one element from another. For example, one node could be termed a “first amplifier” and similarly, another node could be termed a “second amplifier”, or vice versa.

[0026] Unless indicated otherwise, the term “about,” “thereabout,” etc., means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art.

[0027] Many different forms and embodiments are possible without deviating from the spirit and teachings of this disclosure and so this disclosure should not be construed as limited to the example embodiments set forth herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will convey the scope of the disclosure to those skilled in the art.

[0028] Figure 2 shows a schematic diagram of an embodiment of a laser system configured to reduce optical damage and / or overcome the increasing rate of overlap of scan spots along the approach to the scan reversal point when used during various laser-based processing applications wherein a laser signal is scanned across a surface, for example, a tissue surface. In one embodiment, the laser systems disclosed herein may be used in various multi-photon microscopy applications although those skilled in the art will appreciate that the devices and methods disclosed herein are not limited to multi-photon microscopy applications. As shown, the laser system 100 includes at least one seeder oscillator or device 102 configured to generate at least one seed signal 104 having a wavelength from about 800 nm to about 1500 nm. In one specific embodiment, the seed signal 104 has a wavelength of about 1000 nm to about 1080 nm. In a more specific embodiment, the seed signal 104 has a wavelength from about 1035 nm to about 1064 nm. The seeder oscillator 102 may comprise a mode-locked oscillator configured to produce femtosecond pulses, although those skilled in the art will appreciate any variety of seed oscillators 102 may be used, including, without limitations, fiber seed devices, YLMO seeders, optical parametric generators, and the like. In one embodiment, the seeder oscillator 102 comprises a 10 MHz to about a 100 MHz seeder configured to output one or more pulses having a pulse spacing of about 10 ns to about 100 ns. In a specificDocket Number: 00772-WO embodiment, the seeder oscillator 102 comprises a 50 MHz seeder configured to output at least one seeder signal 104 having a pulse spacing of about 20 ns.

[0029] Referring again to Figure 2, at least one first high speed modulator 106 may be in optical communication with the seed oscillator 102 and configured to receive and selectively modulate the seed signal 104. In one embodiment, the first high speed modulator 106 may comprise at least one acousto-optic modulator, although any variety of high speed modulators may be used, including, without limitations, Pockel cell modulators, electro-optic modulators, and the like. In one embodiment, the first high speed modulator 106 is configured to pick one or more pulses from a train of pulses in the seed signal 104 while blocking or otherwise attenuating the non-selected pulses. The first high speed modulator 106 may be configured to output at least one selected pulse or modulated signal 108 directed to at least one amplifier device or system 110. In one embodiment, the amplifier 110 is configured to receive the modulated signal 108 and output at least one amplifier output signal 112. In one embodiment, the amplifier output signal 112 has a wavelength from about 1035 nm to about 1064 nm. In another embodiment, the amplifier output signal 112 has a wavelength of about 1040 nm, 1045, nm, 1064 nm, and / or any desired output wavelength. In one embodiment, the amplifier 110 comprises a Yb-based amplifier, although those skilled in the art will appreciate that any variety of amplifier devices and systems may be used.

[0030] Referring again to Figure 2, at least one second fast modulator 114 may be used in the laser system 100. In one embodiment, the second fast modulator 114 may comprise an acousto-optic modulator and may be configured to operate at a beam blanking device. For example, the second fast modulator 114 may be configured to selectively blank or otherwise modulate propagation of the amplifier output signal 112 though the laser system 100. Optionally, any variety of devices may be used to form the second fast modulator 114. For example, the second fast modulator 114 may be configured to selectively blank the amplifier signal 112 while the scanning system incorporating and / or coupled to the laser system 100 is repositioned or rastered. During use, when permitted, the second fast modulator 114 may be configured to selectively permit the amplifier output signal 112 to be transmitted thereby forming a second modulator output signal 116. In one embodiment, various components of the laser system 100 may be incorporated into an amplifier system comprised of theDocket Number: 00772-WO seeder source 112, the first fast modulator 106, the amplifier 110 and the second fast modulator 114, or various components thereof.

[0031] As shown in Figure 2, the laser system 100 may include one or more optical parametric amplifiers (OPA) or similar devices 118 configured to receive the second fast modulator output signal 116 and output at least last one optical parametric amplifier signal 122. In one embodiment, the OPA comprises a noncollineat optical parametric oscillator (NOPA), although other OPA configurations may be used. As shown in Figure 2, the at least one harmonic conversion device or module 120 may be positioned before the OPA 118. Optionally, the harmonic conversion device 120 may be included within the OPA 118. In one embodiment, the harmonic conversion device 120 may be configured to produce a second harmonic of the second modulator output signal 116. For example, if the second modulator output signal 116 has a wavelength of about 1030 nm the harmonic conversion device 120 may be configured to convert the wavelength of the second modulator output signal 116 to a wavelength of about 515 nm. Those skilled in the art will appreciate that any variety of devices may be used as the harmonic conversion device 120 to produce any variety of harmonic signals of the input signal.

[0032] Referring again to Figure 2, the pulse train from seeder oscillator 102 to laser output is depicted in figure 3. This is the pulse train fed into the OPA 118 within the OPA 118 a portion of the second modulator output signal 116 is split to generate a white light continuum and the remainder is sent to the one or more parametric amplification stages (not shown) within the OPA 118. The parametric amplification stages split the pump light into two beams, signal and idler, whose summed photon energy is equal to the photon energy of the pump laser. The idler beam is used to generate light at an idler beam wavelength (e.g. about 1300nm), while the signal beam at about 853 nm is blocked internally. The wavelengths of either the idler and signal within the OPA 118 may be fixed at one operating wavelength or may be selectively tunable across a range of wavelengths. Optionally, harmonics could be implemented within the OPA 118 on the signal and idler beams to further extend the tuning range. Optionally, harmonic conversion of the idler and signal may be performed external of the OPA 118. In one embodiment, the OPA 118 may be configured to output at least one pulsed OPA output signal 122 having pulse lengths of about <50 fs pulses at about 1300 nm. Those skilled in the art will appreciate that the OPA output signal 122 may have any desired pulse length and wavelength. Exemplary pulse length rangesDocket Number: 00772-WO from about 1 fs to 150 fs. Similarly, the OPA output signal 122 may have a wavelength from about 600 nm to about 1900 nm. For example, when implemented within a two photon microscopy system, the OPA output wavelength may range from about 700 nm to about 1300 nm. In the alternative, when implemented in a three photon microscopy system the OPA output signal 122 may have a wavelength of about 1300 nm to about 1700 nm.

[0033] As shown in Figure 2, the OPA output signal 122 may be directed to at least one optical device or system 124. In one embodiment, the optical system 124 comprises one or more filters, attenuators, beam splitters, sensors, beam dumps, lenses, dispersion compensation devices or systems and the like configured to modify the OPA output signal 122 to produce at least one laser system output 126 which may be directed to a surface under investigation. For example, in one embodiment, the optical device 124 comprises at least one attenuator. In another embodiment, optical device 124 comprises one or more beam splitters, mirrors, lenses, and filters configured to produce multiple laser output signals having the same or different powers, pulse lengths, wavelengths, and / or the like. Optionally, the optical system 124 or device may be positioned anywhere within the laser system 100. Further, multiple optical systems 124 may be used within the laser system 100.

[0034] Typically, the laser system shown in Figure 2 is used in multi-photon microscopy applications such that the pulse train is temporally synchronized to a scanner having a sinusoidal motion. In the multi-photon microscopy application, a scanner, typically operating at a scan frequency (e.g. 8 kHz), oscillates to scan a laser system output signal 126, focused to a diffraction limited spot, scanning across a sample or surface. An image is rendered by digitizing the intensity of light emitted from each spot on the sample as the laser system output signal 126 is scanned. The laser system output signal 126 is scanned in a pre-selected pattern. For example, the laser system output signal 126 may be scanned in a raster fashion, line by line. Optionally, the laser system output 126 need not be scanned in a pre-selected pattern. The total number of pixels is determined by the number of spatial points that will represent each line (half oscillation of the scanner) and the number of lines. Typically, though not exclusively, an image is comprised of 512x512 pixels. For this image resolution, a scanner with an oscillating frequency (e.g. 8kHz) enables video rate imaging at 30 frames per second (FPS). Owing to the sinusoidal nature of the scanner, the amount of time that the laser system output signal 126 spends on each point in the line willDocket Number: 00772-WO vary. When scanned with a laser system 100 having a fixed repetition rate, such that the pixels in the middle portion of the scan, where the scan velocity is highest, will have the fewest number of pulses for each point in the scan line. Consequentially, the pixels at the edges of the image will have more pulses for each point. The image rendering software ensures that each pixel in the image represents the same lateral displacement of the laser beam for each point across the line. Additionally, when the image is rendered, the software will compensate for the different amount of signal generated at each point in the image. The laser system discloses herein seeks to take advantage of the repeatable nature of the scan frequency and adjust the pulse repetition rate of the laser such that each point in the image will have the same number of pulses. To accomplish this, the time interval between subsequent pulses will vary across each line in the image as illustrated in Figure 4.

[0035] By synchronizing the pulse repetition rate from the laser system 100 with the scan frequency of the scanning system and normalizing the number of pulses in each pixel, it is possible to reduce the thermal load placed on the sample by eliminating unnecessary pulses. One of the primary causes of damage to the sample occurs at the maximum and minimum of the scan waveform, where the scanner comes to a stop and turns around to change direction and scan the next line (See Figure 1 , reverse point 31 ). In laser systems with a fixed pulse repetition rate, damage to the sample can begin to occur in this region of the scan due to repeated laser pulses delivered in close proximity to one another. The solution in the prior art is to use a high speed attenuator (EOM or AOM) to rapidly modulate the laser within the scan line to blank the laser beam near the edges of the scan line. In general, 30% of the scan period is blanked and 70% of the scan period is digitized to form the image. As an example, in one embodiment, for example using an 8kHz scanner, yields 45.1 ps where the sample will be illuminated and 18.2 ps where the laser may be blanked. If we break down the 45.1 ps time per line into 512 pixels, we have an average pixel dwell time of 88ns. The minimum pixel dwell time to 72 ns and the maximum is 165 ns. Figure 5 shows a representation of the pixel dwell time across the scan.

[0036] The laser system 100 disclosed herein may be used with any variety of scanning systems and is not limited to scanners operating at 8kHz with 512 points per line. For example, the laser system 100 may be used with scanner systems having a sinusoidal oscillating frequency of about 4kHz to about 12kHz or more. Further, in another embodiment, the number of points per line could be as few as 32 or up toDocket Number: 00772-WO4096 or more. In the present case, the pulse to pulse spacing delivered from the laser system 100 may be selectively tailored to the timing requirements of the pixel up to the minimum pulse separation of 20 ns, although those skilled in the art will appreciate that the pulse separation may be less than about 1 ns and may exceed 20 ns, although those skilled in the art will appreciate that any desired pulse spacing may be used.

[0037] In one embodiment, the laser system 100 disclosed herein may be adapted for use in three-photon excitation fluorescence microscopy (3P microscopy) applications. In 3P microscopy, there is a cubic dependence of generated fluorescence signal on laser pulse energy. It is advantageous to use lasers having a lower repetition rate and higher pulse energy to achieve sufficient fluorescence signal with minimal average power delivered to the sample. Based on the parameters described previously, having an 8 kHz resonant scanner and 512x512 pixels in the image, to achieve a laser repetition rate delivering at least 1 pulse per pixel, a laser with 70 ns pulse spacing would be required. The prior art laser systems for 3P microscopy used lasers having a repetition rate of up to 4 MHz or 250 ns pulse spacing, which is insufficient to produce at least one pulse per pixel when using an 8 kHz resonant scanner. A laser with the requisite 70 ns pulse separation corresponds to a repetition rate of 14.29 MHz and would deliver 645 pulses per line with a free running laser. The laser system 100 disclosed herein has a minimum pulse separation of 20 ns, and with required integer time differences, a minimum pulse separation would be about 60 ns, corresponding to 16.67 MHz repetition rate and 752 pulses per line with a free running laser. The laser system 100 may be configured to emit pulses with non-constant pulse spacing to achieve 512 pulses per line, thereby yielding a 32% reduction in delivered energy to the sample. This reduction is significant as most biological samples can withstand a sustained average power of about 100-150 mW for an extended period of time at about 1300 nm. In this paradigm, delivering 512 pulses per line, the maximum pulse energy that can be delivered per pulse and maintain the necessary low average power is about 19 nJ. Figure 6 shows the calculations for each of the repetition rates discussed (512, 645, and 752 pulses per line) and the reduction in pulse energy to achieve the maximum allowed average power on the sample or surface.

[0038] Due to the cubic dependence on pulse energy, the pulse energy of the fully free running laser would have to be reduced by 32% to maintain the same average power on the sample as the laser in the laser system 100 disclosed herein. TheDocket Number: 00772-WO reduction in pulse energy for the freely running laser equates to a 68% reduction in the amount of fluorescence signal generated per pulse. Similarly, in two-photon excitation fluorescence microscopy (2P), the laser system 100 disclosed herein may be adapted to have a repetition rate of about 50 MHz to about 80 MHz or more to maintain a constant number of pulses within each pixel equal to the minimum number of pulses per pixel at the center of the scan. Figure 7 shows a table of the reduction in overall energy deposition enabled by an embodiment of the laser system 100 disclosed herein when running at a high repetition rate. Given the periodic and repeatable nature of the scanning mechanism, the laser system 100 may be configured to build up additional pulse energy equivalent to the average pulse repetition rate of the sequence of pulses emitted from the laser instead of the minimum pulse energy determined by the pulse separation.

[0039] In another embodiment, the laser system 100 may be configured to increase the number of pulses per pixel through an external pulse splitter and delay line should a researcher be interested in having a higher number of pulses per pixel, while maintaining the timing of the sub pulses to be synchronous with the scanner. In this methodology, the number of pulses per pixel may be maintained as constant.

[0040] The embodiments disclosed herein are illustrative of the principles of the invention. Other modifications may be employed which are within the scope of the invention. Accordingly, the devices disclosed in the present application are not limited to that precisely as shown and described herein.

Claims

Docket Number: 00772-WOClaims1. A laser system for use with multi-photon microscopy scanning systems, comprising: a seeder oscillator configured to output at least one pulsed seeder signal; a fast modulator configured to receive the at least one pulsed seeder signal and select at least one pulse from the at least one pulsed seeder signal, the fast modulator outputting at least one modulated output signal; at least one amplifier configured to receive and amplify the at least one modulated output signal and output at least one amplifier output signal; at least a second modulator configured to receive the at least one amplifier output signal and output at least one modulated amplifier output signal; and at least one optical parametric amplifier configured to receive the modulated amplifier output signal and output at least one optical parametric amplifier output signal; wherein the laser system is configured to produce pulses with variable pulse spacing temporally synchronized to a scanner having sinusoidal motion.

2. The laser system of claim 1 , wherein the variable pulse spacing is configured such that each pixel in a scanned image receives substantially the same number of laser pulses.

3. The laser system of claim 1 , wherein the scanner operates at a frequency of about 4 kHz to about 12 kHz.

4. The laser system of claim 1 , wherein the seeder oscillator comprises a mode- locked oscillator configured to produce femtosecond pulses.

5. The laser system of claim 1 , wherein the seeder oscillator comprises a 10 MHz to about 100 MHz seeder configured to output one or more pulses having a pulse spacing of about 10 ns to about 100 ns.

6. The laser system of claim 1 , wherein the fast modulator comprises at least one acousto-optic modulator.

7. The laser system of claim 1 , wherein the amplifier comprises a Yb-based amplifier.

8. The laser system of claim 1 , wherein the amplifier output signal has a wavelength from about 1035 nm to about 1064 nm.

9. The laser system of claim 1 , wherein the second modulator is configured to selectively blank the amplifier output signal.Docket Number: 00772-WO10. The laser system of claim 1 , further comprising at least one harmonic conversion device configured to produce a second harmonic of the modulated amplifier output signal.

11. The laser system of claim 1 , wherein the optical parametric amplifier output signal has a wavelength from about 600 nm to about 1900 nm.

12. The laser system of claim 1 , wherein the optical parametric amplifier output signal has a wavelength from about 1300 nm to about 1700 nm for three photon microscopy applications.

13. The laser system of claim 1 , wherein the optical parametric amplifier output signal has pulse lengths of about 1 fs to about 150 fs.

14. The laser system of claim 1 , wherein the laser system has a minimum pulse separation of about 20 ns.

15. A method for reducing optical damage in multiphoton microscopy comprising: providing a laser system having variable pulse spacing; synchronizing laser pulse timing to a scanner having sinusoidal motion; varying time intervals between subsequent laser pulses across each scan line; and maintaining substantially constant numbers of pulses per pixel across a scanned image.

16. The method of claim 15, wherein the scanner operates at about 8 kHz and scans about 512 pixels per line.

17. The method of claim 15, further comprising reducing energy delivered to a sample by about 32% compared to constant pulse spacing operation.

18. The method of claim 15, wherein the method is applied to three-photon excitation fluorescence microscopy.

19. The method of claim 15, wherein the method is applied to two-photon excitation fluorescence microscopy.

20. The method of claim 15, wherein the number of pixels per scan line ranges from about 32 to about 4096.21 . A multiphoton microscopy system comprising: a scanner having sinusoidal motion; a laser system configured to produce pulses with variable pulse spacing synchronized to the scanner motion, the laser system comprising: a seeder oscillator configured to output at least one pulsed seeder signal; a fast modulator configured to select pulses from the pulsed seeder signal;Docket Number: 00772-WO an amplifier configured to amplify selected pulses; a second modulator configured to selectively modulate amplified pulses; and an optical parametric amplifier configured to generate an output signal; wherein the laser system delivers substantially equal numbers of pulses to each pixel position during scanning.

22. The system of claim 21 , wherein the scanner operates at frequencies selected from about 4 kHz, about 8 kHz, or about 12 kHz.

23. The system of claim 21 , further comprising an external pulse splitter and delay line for increasing pulses per pixel while maintaining synchronization with the scanner.

24. The system of claim 21 , wherein the system is configured for three-photon microscopy applications.

25. The system of claim 21 , wherein the system is configured to maintain average power on biological samples of about 100 mW to about 150 mW.

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