Abstract:
A method for imaging a sample is described. The sample is characterized by a limit on incident optical energy absorbed over a given time period. The method includes providing at least one input optical wave that includes pulses that each have a full-width half-maximum time duration of more than 100 picoseconds and a pulse energy sufficiently large such that a sufficient number of consecutive pulses absorbed by the sample would exceed the limit. The method also includes directing the input optical wave to focus on a first portion of the sample; detecting energy from an output optical wave generated from a nonlinear optical interaction in the first portion of the sample with the input optical wave; and generating a representation of the first portion of the sample based on the detected energy from the output optical wave.

Description:
BACKGROUND 
   The invention relates to controlling pulses in optical microscopy. 
   Various techniques for optical microscopy can be used to construct an image of a portion of a tissue sample. Some techniques for optical microscopy use nonlinear optical interactions in the tissue being imaged to provide an optical signal that can be measured to construct the image of the tissue. Nonlinear optical techniques facilitate acquisition of images deep within a sample to form, for example, three-dimensional images of biomedical samples hidden underneath non-transparent tissues, or images of defects and impurity contents situated inside light absorbing materials. 
   Nonlinear optical microscopy techniques include, for example, multiphoton excitation techniques such as two-photon-excited fluorescence laser scanning microscopy (2PLSM), techniques based on three-wave mixing such as second-harmonic generation, and techniques based on four-wave mixing such as third-harmonic generation and coherent anti-Stokes Raman scattering (CARS). Multiphoton excitation of a sample by a laser can be combined with any of a variety of optical detection techniques including fluorescence emission, harmonic generation, Raman or Brillouin scattering, or with non-optical thermal or electronic detection techniques. 
   In linear optical microscopy, the photons of an optical wave incident on a tissue sample are either scattered by the tissue or excite target molecules (e.g., fluorescent dyes) to provide signal photons that are collected by an imaging system to generate a detected signal. The detected signal depends linearly on the incident optical wave intensity. In nonlinear optical microscopy, the detected signal depends nonlinearly on the incident optical wave intensity, or in some cases, depends on the intensities of multiple interacting optical waves. However, the efficiency of nonlinear interactions are typically weak and therefore call for optical waves with high peak intensities. Ultrafast modelocked lasers are used to provide pulses with high peak intensity and short time duration (full-width half-maximum (FWHM) time duration). Modelocking provides regularly spaced pulses having a well-defined shape (e.g., approximately Gaussian). 
   SUMMARY 
   In one aspect, in general, the invention features a method for imaging a sample. The sample is characterized by a limit on incident optical energy absorbed over a given time period. The method includes providing at least one input optical wave that includes pulses that each have a full-width half-maximum time duration of more than 100 picoseconds and a pulse energy sufficiently large such that a sufficient number of consecutive pulses absorbed by the sample would exceed the limit. The method also includes directing the input optical wave to focus on a first portion of the sample; detecting energy from an output optical wave generated from a nonlinear optical interaction in the first portion of the sample with the input optical wave; and generating a representation of the first portion of the sample based on the detected energy from the output optical wave. 
   Aspects of the invention can include one or more of the following features. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 500 picoseconds. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 1 nanosecond. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 10 nanoseconds. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 100 nanoseconds. 
   The method further includes temporarily preventing the input optical wave from reaching the sample. 
   Temporarily preventing the input optical wave from reaching the sample includes periodically preventing the input optical wave from reaching the sample. 
   Temporarily preventing the input optical wave from reaching the sample includes preventing the optical wave from reaching the sample before a number of consecutive pulses are absorbed by the sample would exceed the limit. 
   The nonlinear optical interaction comprises multi-photon absorption. 
   The nonlinear optical interaction comprises two-photon absorption. 
   The output optical wave comprises a fluorescence emission from the sample. 
   The fluorescence emission from the sample comprises emission from a fluorescent molecule in the sample. 
   The nonlinear optical interaction comprises wave mixing. 
   The nonlinear optical interaction comprises four-wave mixing. 
   The nonlinear optical interaction comprises coherent anti-Stokes Raman scattering. 
   Providing at least one input optical wave, directing the input optical wave, and detecting energy from the output optical wave comprise: providing at least two input optical waves that include pulses that each have a full-width half-maximum time duration of more than 100 picoseconds; directing the input optical waves to focus on a first portion of the sample; and detecting energy from an output optical wave generated from a wave mixing interaction in the first portion of the sample with each of the input optical waves. 
   Providing at least two input optical waves comprises providing signal and idler optical waves generated from parametric downconversion of a pump optical wave that includes pulses that each have a full-width half-maximum time duration of more than 100 picoseconds. 
   The method further includes moving the input optical wave relative to the sample to focus on a second portion of the sample; detecting energy from an output optical wave generated from a nonlinear optical interaction in the second portion of the sample with the input optical wave; generating a representation of the second portion of the sample based on the detected energy from the output optical wave; and generating an image of the sample that includes the representation of the first portion of the sample and the representation of the second portion of the sample. 
   In another aspect, in general, the invention features a method for imaging a sample including providing at least one input optical wave that includes pulses that are approximately uniformly spaced by a time delay; directing the input optical wave to focus on a first portion of the sample; detecting energy from an output optical wave generated from a nonlinear optical interaction in the first portion of the sample with the optical wave during a first time period that is about equal to or longer than the time delay; temporarily preventing the input optical wave from reaching the sample during a second time period that is long enough for most of an amount of heat built up in the sample by the input optical wave during the first time period to be dissipated; and generating a representation of the first portion of the sample based on the detected energy from the output optical wave. 
   Aspects of the invention can include one or more of the following features. 
   The first time period is short enough to prevent damage to the sample caused by the amount of heat built up in the sample by the input optical wave during the first time period. 
   The first time period is shorter than 500 milliseconds. 
   The first time period is shorter than 100 milliseconds. 
   The first time period is shorter than 10 milliseconds. 
   The first time period is shorter than 1 millisecond. 
   The first time period is longer than twice the time delay. 
   Detecting the energy from the output optical wave and temporarily preventing the input optical wave from reaching the sample are repeated approximately periodically during multiple respective first and second time periods with the input optical wave directed to focus on different portions of the sample. 
   Detecting energy from the output optical wave during the first time period comprises processing detected energy during respective time windows that are shorter than the time delay and rejecting detected energy from the sample outside of the time windows. 
   The time windows are synchronized to respective pulses in the input optical wave. 
   Rejecting detected energy from the sample outside of the time windows comprises preventing detection of energy from the sample outside of the time windows. 
   Rejecting detected energy from the sample outside of the time windows comprises preventing processing of portions of a stored signal that correspond to energy detected outside of the time windows. 
   The method further includes moving the input optical wave relative to the sample to focus on a second portion of the sample. 
   The input optical wave is moved relative to the sample during the second time period. 
   Moving the input optical wave relative to the sample comprises moving the input optical wave without moving the sample. 
   Moving the input optical wave relative to the sample comprises moving the sample without moving the input optical wave. 
   The method further includes detecting energy from an output optical wave generated from a nonlinear optical interaction in the second portion of the sample with the input optical wave; generating a representation of the second portion of the sample based on the detected energy from the output optical wave; and generating an image of the sample that includes the representation of the first portion of the sample and the representation of the second portion of the sample. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 100 picoseconds. 
   The input optical wave includes pulses that each have a full-width half-maximum time duration of more than 1 nanosecond. 
   The time delay is between about 1 microsecond and about 1 millisecond. 
   The time delay is between about 10 microseconds and about 100 microseconds. 
   The nonlinear optical interaction comprises multi-photon absorption. 
   The nonlinear optical interaction comprises two-photon absorption. 
   The output optical wave comprises a fluorescence emission from the sample. 
   The fluorescence emission from the sample comprises emission from a fluorescent molecule in the sample. 
   The nonlinear optical interaction comprises wave mixing. 
   The nonlinear optical interaction comprises four-wave mixing. 
   The nonlinear optical interaction comprises coherent anti-Stokes Raman scattering. 
   Providing at least one input optical wave, directing the input optical wave, and detecting energy from the output optical wave comprise: providing at least two input optical waves that include pulses; directing the input optical waves to focus on a first portion of the sample; and detecting energy from an output optical wave generated from a wave mixing interaction in the first portion of the sample with each of the input optical waves. 
   Providing at least two input optical waves comprises providing signal and idler optical waves generated from parametric downconversion of a pump optical wave that includes pulses. 
   In another aspect, in general, the invention features a system for imaging a sample characterized by a limit on incident optical energy absorbed over a given time period. The system includes a source of at least one input optical wave that includes pulses that each have a full-width half-maximum time duration of more than 100 picoseconds and a pulse energy sufficiently large such that a sufficient number of consecutive pulses absorbed by the sample would exceed the limit; a microscope configured to direct the input optical wave to focus on a first portion of the sample; and a detection sub-system configured to detect energy from an output optical wave generated from a nonlinear optical interaction in the first portion of the sample with the input optical wave, and generate a representation of the first portion of the sample based on the detected energy from the output optical wave. 
   Aspects of the invention can include one or more of the following features. 
   The source is configured to temporarily prevent the input optical wave from reaching the sample. 
   Temporarily preventing the input optical wave from reaching the sample includes periodically preventing the input optical wave from reaching the sample. 
   Temporarily preventing the input optical wave from reaching the sample includes preventing the optical wave from reaching the sample before a number of consecutive pulses are absorbed by the sample would exceed the limit. 
   The system further includes excitation optics between the source and the microscope configured to periodically prevent the input optical wave from reaching the sample. 
   The detection sub-system is configured to process energy that has been detected within time windows corresponding to the pulses. 
   The detection sub-system is configured to prevent detection of energy outside of the time windows. 
   The detection sub-system is configured to remove portions of a signal corresponding to energy detected outside of the time windows. 
   Aspects of the invention can have one or more of the following advantages. 
   Techniques described herein for managing the timing of an excitation optical wave and the associated signal collection may reduce potential for damage due to heat, and increase quality of the signal (e.g., signal-to-noise ratio). Since the pulses are relatively long (e.g., &gt;100 ps) and the pulse repetition rate is relatively low (e.g., &lt;1 MHz), time gated signal processing can be performed at speeds that the detection electronics can handle. Without a need for an ultrafast laser, the system for nonlinear optical microscopy applications will be compact, reliable, and simple to operate. The fixed and the tunable near infrared wavelengths of some of the laser sources are able to penetrate biological tissues or materials such as silicon to reveal images embedded deep inside these samples. In wavelength scanning applications as in mode-selective molecular or biological imaging, the wavelength of lasers with relatively longer pulses can be tuned or scanned relatively easily and rapidly compared to short pulse tunable lasers. 
   Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict with publications, patent applications, patents, and other references mentioned incorporated herein by reference, the present specification, including definitions, will control. 
   Other features and advantages of the invention will become apparent from the following description, and from the claims. 

   
     DESCRIPTION OF DRAWINGS 
       FIG. 1  is a diagram of a nonlinear optical microscopy system. 
       FIG. 2A  is a plot of intensity of an input optical wave. 
       FIG. 2B  is a plot of the position of the input optical wave of  FIG. 2A  relative to a sample. 
       FIG. 2C  is a plot of intensity of an output optical wave. 
       FIG. 3  is a diagraph of a system for two-photon-excited fluorescence laser scanning microscopy (2PLSM). 
       FIG. 4A  is an AFM image of the photoresist film sample. 
       FIG. 4B  is a 2PLSM image of a photoresist film sample. 
       FIGS. 5A and 6A  are two-dimensional 2PLSM images of fluorosphere samples. 
       FIGS. 5B and 6B  are three-dimensional 2PLSM images of fluorosphere samples. 
       FIG. 7A  is a 2PLSM image of an onion sample. 
       FIG. 7B  is a white-light confocal microscopy image of the onion sample. 
   

   DESCRIPTION 
   Some techniques for obtaining high-quality images of a sample using nonlinear optical microscopy include the use of an input optical wave comprising a series of optical pulses each having a high peak electric field intensity. There are factors that limit the highest intensity that can be tolerated in a sample (e.g., a biological tissue sample). Two limiting factors are optical damage and thermal damage of the sample. Optical damage includes damage of the sample caused by a large electric field intensity, which places a limit on the peak power density (W/cm 2 ) of pulses. Thermal damage includes damage of the sample caused by heating of the sample to above a tolerable temperature due to absorption of the optical energy over time, which places a limit on the average power density of a stream of pulses, or on the energy density (J/cm 2 ) or “fluence” of each pulse and the pulse repetition rate. 
   The limits on characteristics of an optical wave associated with limiting optical and thermal damage depend on the sample. For many biological samples (e.g., cells or viruses), a typical optical damage limit on peak power density is approximately 10-100 GW/cm 2 , and a typical thermal damage limit on average power density is approximately 10 MW/cm 2 . In many cases, thermal damage is more limiting than optical damage. Typically, short pulse lasers with pulse durations in the picosecond and sub-picosecond regime are used to provide pulses with relatively high peak power density for high-quality imaging, but relatively low average power density (due to low pulse energy) to reduce the potential for thermal damage to the sample. Short pulse lasers can be, in general, relatively complex, difficult to operate, and expensive compared to lasers with longer pulse durations. 
   Various classes of lasers can become less complex, easier to operate, and less expensive as the pulse durations become longer (e.g., &gt;100 ps, &gt;500 ps, &gt;1 ns, &gt;10 ns, or &gt;100 ns). Another benefit of longer pulse durations is the associated reduction in minimum linewidth of the spectrum of the optical wave. Some spectroscopic techniques, such as CARS, scan the peak wavelength of a source&#39;s spectral line to probe the spectral properties of a sample. Not only is such wavelength scanning more complex in a short pulse source, but the spectral resolution attainable is lower due to the larger minimum linewidth. When optical wave from a short pulse source is coupled into an optical fiber (e.g., to improve delivery and alignment of the optical wave into a microscope), group velocity dispersion may result in chirping and temporal distortion of the pulses. Nonlinear processes may also occur in the fiber, altering the spectral characteristics of the optical wave. 
   Coherent optical sources that provide long pulse duration optical waves (e.g., &gt;100 ps) have been deemed by some as not suitable for nonlinear optical microscopy due to potential for damage to a sample and the lower image quality resulting from a reduced peak power density. However, by controlling the timing characteristics and delivery of an optical wave to a sample as described herein, it is possible to achieve high quality (e.g., low noise) images in nonlinear optical microscopy of the sample using longer pulse sources while satisfying damage constraints. Compact solid state pulsed lasers such as diode-laser-pumped Q-switched Nd ion doped YAG, YVO4, and YLF lasers, and Yb:doped fiber lasers and amplifiers can provide intense pulses with a time duration from a few nanoseconds to tens of nanoseconds and with excellent beam quality. The pulse repetition rates for these sources can be on the order of 1 kHz to 1 MHz. Such sources are compact, simple, and relatively inexpensive compared to short pulse lasers. The peak power of these lasers can be in the 1-10 kW range, about 10 times less than typical short pulse lasers with pulse durations in the picosecond and sub-picosecond regime. The typical pulse energy of these diode-laser pumped solid state lasers is correspondingly higher than that of short pulse lasers by about 100 to 500 times. 
   By managing the timing of an excitation optical wave and the associated signal collection, it is possible to avoid thermal damage while producing high-resolution microscopic images of good quality. Thermal damage can result from a rapid increase in temperature following accumulation of absorbed energy. If the excitation optical wave is temporarily prevented from reaching (or being absorbed in) the sample before the temperature reaches a destructive value, then accumulated heat has time to dissipate away from the sample so that damage does not occur. 
   1 System Overview 
   Referring to  FIG. 1 , in a nonlinear optical microscopy system  100 , a source  102  provides an input optical wave  104  that includes pulses that are approximately uniformly spaced by an inter-pulse time delay τ. The source  102  is a long-pulse source such that each pulse has a full-width half-maximum time duration of more than about 100 picoseconds. Excitation optics  106  direct the input optical wave  104  to a microscope  108  that focuses the wave  104  onto a portion of a sample  110  to be imaged. The input optical wave  104  serves as input for a nonlinear optical interaction in that portion of the sample  110 . The excitation optics  106  can direct the optical wave  104  to the microscope  108  over free space or guided within an optical fiber, for example. For free space delivery, the excitation optics  106  can include a lens followed by a pinhole followed by another lens to clean up the transverse mode of the beam from the source  102  and to adapt the diameter of the beam to the diameter of an entrance window of a microscope objective lens  120  to provide adequate spatial resolution. The excitation optics  106  can also include attenuation elements such as neutral density filters or a polarization rotator and polarizer pair for controlling the intensity of the input optical wave  104 . 
   The nonlinear optical interaction in the sample  110  can generate an output optical wave  112 A that travels in a forward direction relative to the input optical wave  104  to be detected in a “trans-collection” mode, and/or an output optical wave  112 B that travels in a backward direction relative to the input optical wave  104  to be detected in an “epi-collection” mode. The output optical waves  112 A and  112 B are directed by mirrors  115 A and  115 B, respectively, to a detection sub-system  114  to generate an image of the sample  110 . The output optical waves  112 A and  112 B can represent a fluorescence signal, or an optical harmonic signal, or a Raman shifted or anti-Stokes shifted signal of the input optical wave  104 , for example. Due to the nature of nonlinear optical processes, generation of these signals is typically substantially localized to the focus of the input optical wave  104 , where the intensity is greatest. This transverse and longitudinal localization of the interaction at the beam focus enables three-dimensional discrimination of the imaged location. 
   The output optical wave  112 A is detected in a trans-collection mode at a first detector  116 A, and the output optical wave  112 B is detected in an epi-collection mode at a second detector  116 B. In trans-collection mode, the forward propagating output optical wave  112 A can be collected after propagating through the sample  110  to the detector  116 A. In epi-collection mode, the backward propagating output optical wave  112 B can be propagate back through the microscope objective lens  120  and through a dichroic beam splitter (DBS)  122  that reflects most of the input optical wave  104  and transmits most of the output optical wave  112 B. In either mode, the output optical waves  112 A and  112 B can be filtered by one or more narrowband filters to discriminate from the intense input optical wave  104  and focused by one or more lenses before being detected. The detection sub-system  114  can include any of a variety of detector types including a photodiode, a charge-coupled device, or photon counters such as a photomultiplier or an avalanche photodiode. 
   The nonlinear optical microscopy system  100  can include components such as galvanometric mirrors to move the input optical wave  104  (e.g., within the excitation optics  106 ), a motorized/piezoelectric stage to move the sample  110 , or both, to scan over different portions of the sample  110  and build up an image (over x and y dimensions). Three-dimensional images can be obtained by further moving the microscope objective focus (e.g., by a piezoelectric transducer) to take image “slices” in different planes (along a z dimension). The image can be represented, for example, as a collection of image elements such as “pixels” of a two-dimensional image, or a “voxels” of a three-dimensional image that represent a property of the sample  110  based on the energy detected from the output optical waves  112 A and/or  112 B. For example, a property of the sample  110  may be related to an intensity of fluorescence emission resulting from 2PLSM, and the energy in the output optical waves  112 A and/or  112 B collected in response to one of the pulses in the input optical wave  104  can quantify that property at a sample location. Each image element can be represented as scalar data or vector data (e.g., representing spectral properties of a portion of the sample  110 ) stored on a memory storage medium. For example, the detection sub-system  114  can include a computer system  124  having a memory storage medium such as a hard drive. 
   Some implementations of the system  100  use more than one input optical wave from the source  102 . For example, an implementation of the system  100  for CARS microscopy uses two input optical waves having different frequencies. The difference of the frequencies corresponds to the Raman transition frequency of the sample  110 . The two optical waves mix in a nonlinear interaction in the sample  110  to generate a CARS output optical wave that is detected. 
   A tunable optical parametric oscillator (OPO) pumped by a pulsed laser can be used as the source  102  of two substantially collinear output optical waves: a signal wave and an idler wave. If the OPO is pumped with a pulsed wave, the signal and idler waves will include pulses that are automatically overlapped in time. The sum of the frequencies of the signal wave and the idler wave equals the frequency of the pump wave. Techniques for tuning the OPO include, for example, tuning the pump frequency and/or tuning the phase matching condition among the pump, signal, and idler. The difference of the signal and idler frequencies (and wavelengths) then can be scanned appropriately to excite the Raman transitions of the chemical bonds of the sample species that is to be imaged. An example of a tunable OPO that can be used as a source is described in U.S. application Ser. No. 11/318,234, filed on Dec. 23, 2005, incorporated herein by reference. 
   An advantage of using an OPO is that the signal, idler and output CARS signal wavelengths fall in the transmission window of most tissues so that absorption and scattering loss are kept to a minimum, allowing for penetration of the light deep inside the tissue sample to make images that cannot be observed by visible microscopy or short pulse scanning microscopy at 790 nm, a commonly used wavelength in short pulse microscopes. The output spectral bandwidth of about 1 cm −1  of nanosecond OPOs can be much narrower than that of most femtosecond or picosecond OPOs, thus the system  100  will be able to obtain a CARS signal spectrum with better spectral resolution and better species selectivity. 
   Other types of sources can be used for nonlinear interactions with multiple input optical waves. For example, the source  102  can include an OPO pumped by a second harmonic of a laser and various combinations of the fundamental, SHG-pump, signal, and idler waves can be used as input optical waves. 
   2 System Operation 
   Referring to  FIGS. 2A-2C , the system  100  is configured to collect an output optical wave  112 A and/or  112 B generated from the nonlinear optical interaction in the sample  110  during a collection time period t coll , and to temporarily prevent the optical wave  104  from reaching the sample  110  during a cooling time period t cool . The cooling time period is long enough for most of an amount of heat built up in the sample  110  by the optical wave  104  during the collection time period to be dissipated. For example, the source  102  can include a switching mechanism that periodically stops pumping the source  102  (e.g., by turning off a power supply) to quench a lasing process that generates the optical wave  104 . Alternatively, instead of quenching or otherwise shutting off the source  102 , the system  100  can block or divert the optical wave  104 , for example, using an optical device (e.g., an acousto-optic modulator) or a mechanical device (e.g., a electromechanical shutter or a piezoelectrically controlled mirror) within the excitation optics  106 . 
   For typical diode-laser-pumped solid state lasers, to provide an average power density below the thermal damage limit, the collection time period t coll  can be in the range of about 1 msec to 500 msec for a given portion of the sample that is being imaged with a beam spot size at the focal plane of the optical wave  104  of about 0.25 μm 2  to 1 μm 2 . Since no signal is detected during the cooling time period t cool , this cooling time can be used to move the optical wave  104  relative to the sample  110  to focus on a different portion of the sample to be imaged (or “imaging spot”). Thus, the speed of scanning across imaging spots of the sample  110  does not need to be faster than the distance between neighboring spot locations divided by t cool . The collection time period t coll  is large enough to ensure sufficient signal is collected. In some implementations t coll  is large compared to the inter-pulse time delay τ to encompass multiple pulses (i.e., t coll &gt;2τ), and is typically large enough to encompass tens or hundreds of pulses or more to excite the sample  110  during a collection time period at an imaging spot. For a collection time period of 500 ms, an optical wave  104  with a pulse repetition rate of 100 kHz would have 50,000 consecutive pulses absorbed by the sample  110  at each imaging spot. Alternatively, in some implementations, a single pulse could be sufficient to provide adequate signal. 
     FIG. 2A  shows a plot of intensity I(t) of the input optical wave  104  at the sample  110  over a time span of a periodic waveform that shows two collection time periods separated by one cooling time period. In this example, to facilitate visualization, only three pulses occur within the collection time period.  FIG. 2B  shows a plot of a position x(t) of the optical wave  104  relative to the sample  110  in a linear scan across the sample. In this example, the cooling and scanning are synchronized such that the optical wave  104  is substantially stationary during each collection time period, and moves during each cooling time period. In other examples, the optical wave  104  may be stationary over multiple collection and cooling time periods. 
   To provide high quality images, sufficient signal energy (e.g., photon counts) from the output optical waves  112 A and/or  112 B should be collected during the collection time period to provide a high signal-to-noise ratio (SNR). The SNR can be increased by using time gated signal processing to process signal energy from the output optical waves  112 A and/or  112 B within limited detection time windows t det  corresponding to each pulse. A controller (e.g., a computer) can use an electronic trigger pulse (e.g., from a driver that controls the pulsing of the source  102 ) to time the gating of the detection sub-system  114  to process energy that has been detected within the window t det . 
     FIG. 2C  shows a plot of intensity I(t) of the output optical waves  112 A and/or  112 B generated from the nonlinear optical interaction in the sample  110 . The shape of the “signal pulses” in the wave (along with other characteristics such as spectrum, spatial mode, and propagation direction) depends on the specific interaction being utilized. The detection sub-system  114  is gated to process light detected within the detection time window t det , but not to process light that is detected or would have been detected outside of this window. In this way, the detection sub-system  114  is likely to receive signal information based on the energy in the signal pulses that arrive at the detection sub-system  114  at a predictable time based on the regular excitation pulses, but to reject the noise that occurs outside of the window. Thus, potential noise from optical, electronic, and/or mechanical sources, for example, instead of from the nonlinear optical interaction is rejected. This increases the SNR and the resulting quality of the image. 
   Any of a variety of techniques can be used to perform the time gated signal processing. Gating can occur on-line, for example, by using electronic trigger pulses to gate a power supply within the detection sub-system  114  or to gate electronic amplifiers or photon counters within the detection sub-system  114 . Alternatively, the gating can occur off-line by storing a detected signal into a computer and digitally processing the signal according to timing information. Thus, in an on-online approach, the detection sub-system  114  is configured to prevent detection of energy outside of the detection time windows, and in an off-line approach, the detection sub-system  114  is configured to remove portions of a signal corresponding to energy detected outside of the detection time windows. 
   Various techniques can be used in combination with the time gated signal processing. A technique that uses a lock-in amplifier or a boxcar amplifier for phase-sensitive signal detection is described in U.S. Pat. No. 6,356,088, incorporated herein by reference. 
   3 Working Example 
   The following is a working example of two-photon-excited fluorescence laser scanning microscopy (2PLSM) using an implementation of the nonlinear optical microscopy system  100 . 
   Referring to  FIG. 3 , a system  300  was used for 2PLSM to obtain fluorescence images of different sample species. After repeated scans, no damage to the samples were observed. The spatial resolution of the images obtained was less than 0.5 μm. A Q-switched Nd:YAG laser was used as a source  302  of an input optical wave  304  with a 1064 nm wavelength, a beam diameter of about 9 mm, a 10 kHz pulse repetition rate, and a 19 ns FWHM pulse width. Mirrors  306  directed the optical wave  304  into a microscope  308 . A spatial filter  310  cleaned the spatial mode of the optical wave  304  and a lens  312  modematched the spatial mode into the microscope  308 . The microscope  308  included a dichroic beam splitter (DBS)  314  to direct the optical wave  304  into a 40× objective lens  316  with a 0.8 numerical aperture (NA) in air. Each sample  318  was deposited on a glass slide placed on an x-y translation stage  320  equipped with a piezoelectric nanopositioner with a full scan range of 100 μm in each of the x and y directions. Fine adjustment in the z direction was performed with a one-dimensional piezoelectric transducer  322  attached to the objective lens  316 . 
   A fluorescence optical wave  324  was collected by a lens  326  with a 0.68 NA, passed through a set  328  of narrowband bandpass filters, and detected by an avalanche photodiode single-photon counting module  330  in trans-collection mode. The photon counts were processed by a computer  332  to generate digital image to be stored and displayed. The fluorescence optical wave  324  was also collected back through the microscope  308  by a lens  336 , passed through a set  338  of narrowband bandpass filters, and detected by an avalanche photodiode single-photon counting module  340  in epi-collection mode. A CCD detector  344  also collected a non-fluorescence image of the sample  318  from excitation light that leaked through the DBS  314  was reflected by a DBS  342 . 
   Gated signal detection synchronized to the pulses of the input optical wave  304  was used to reduce any background signal to below the electronic noise limit of a few counts per second. The average power in the input optical wave  304  was typically around 16.5 mW, corresponding to a peak power of 90 W, a peak intensity of about 5.5 GW/cm 2 , and a single pulse fluence of about 105 J/cm 2 . A computer controlled the gating of the laser source  302  to start and stop the pulsed output from the laser so that the pulses were applied while the flurescence optical wave  324  was being collected and stopped while the sample  318  was being scanned. 
   The collection time period t coll  was selected to limit the total fluence absorbed during t coll  to below the damage threshold of the sample  318 . This collection time period was experimentally determined for each sample species and was between about 50-100 ms. With these settings, the photon count per collection time period ranged from a few tens to several hundreds with a background count of less then five in each case. Under these conditions, the time it took to scan an image of 30 μm×30 μm was about 10-30 minutes. For other samples and/or excitation wavelengths, the thermal damage limits may allow scan times to be decreased by increasing the pulse repetition rate and reducing t coll . 
   3.1 Sample 1: Photoresist Film 
   One sample imaged was an approximately 1 μm thick film of photoresist deposited on a glass slide and patterned into a two-dimensional (tetragonal) array of round holes (with diameter of ˜3.2 μm and a depth of ˜1.2 μm) using photolithography.  FIG. 4B  shows a 2PLSM image of the photoresist film sample. The photoresist polymer of which the film was composed yields a reasonable fluorescence signal at around 650 nm from two-photon excitation of ˜10 mW of 1064 nm radiation, even though its fluorescence quantum yield is small compared to typical fluorescent dyes. The image shows clearly the openings etched into the photoresist by the photolithographic process, including a grid of fine lines that possibly resulted from optical interference due to the photolithographic process used to make the pattern. The spacing between the lines of the grid is about 1 μm. These features also appear in an atomic force microscope (AFM) image (with resolution&lt;10 nm) of the same photoresist film shown in  FIG. 4A . 
   3.2 Sample 2: Fluorospheres A 
   Another sample imaged was an emulsion of ˜1.0 μm diameter fluorescent polystyrene microspheres (also called “molecular probes” or “fluorospheres”) deposited into the patterned ˜6.0 μm diameter holes of a photoresist film similar to that of Sample 1.  FIG. 5A  shows a two-dimensional 2PLSM image of the fluorosphere sample, and  FIG. 5B  shows a three-dimensional 2PLSM image of the fluorosphere sample. Various structural features of the deposition of the fluorospheres can be discerned from both images, including: (A) clusters of multiple fluorospheres in respective holes, (B) three distinguishable fluorospheres deposited into a hole, (C) two distinguishable fluorospheres deposited into a hole, and (D) a single fluorosphere deposited into a hole. In this example, the number of fluorospheres can be discerned by the size of the detected fluorescent portions of the image. 
   3.3 Sample 3: Fluoroshperes B 
   Another sample imaged was a dilute emulsion of ˜0.5 μm diameter fluorescent polystyrene microspheres (also called “molecular probes” or “fluorospheres”) deposited into the patterned ˜2.9 μm diameter holes of a photoresist film.  FIG. 6A , shows a two-dimensional 2PLSM image of the fluorosphere sample, and  FIG. 6B  shows a three-dimensional 2PLSM image of the fluorosphere sample. These images provide an estimate of the spatial resolution achieved in the 2PLSM imaging process using the system  300 . From the slope of the three-dimensional image of a single bead, the spatial resolution achieved was &lt;0.5 μm, close to the theoretical limit of ˜0.35 μm due to diffraction of the 1064 nm laser wavelength within the system  300  in a two-photon process. 
   3.4 Sample 4: Onion 
   Another sample imaged was a ˜50 μm thin slice of fresh onion skin that had been soaked in a Rhodamine 6G in water solution (10-3 molar) for six hours, as an example of 2PLSM applied to biological imaging.  FIG. 7A  shows a 2PLSM image of the onion sample, and  FIG. 7B  shows an image of the onion sample obtained by conventional white-light confocal microscopy. The structure of onion cell wall can be clearly identified. For this sample, the average power of the input optical wave  304  was about 22.3 mW, and no damage to the onion sample was observed through repeated scans. 
   Other embodiments are within the scope of the following claims.