Patent Description:
Molecular excitation by the simultaneous absorption of two photons (or multiple photons) provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. Two-photon microscopy and multi-photon microscopy have been used extensively to measure dynamic processes, such as calcium dynamics, in populations of neurons in the intact brain, even during animal behavior.

Photodetectors that detect light down to the single photon level, such as photomultiplier tubes (PMTs) and silicon photomultipliers (SiPMs), are commonly used in two-photon microscopy systems, due to their low cost, high sensitivity and wide coverage of wavelengths. In fluorescence microscopy, upon excitation, some structures of a sample can fluoresce more photons and other structures. For example, when imaging neuron activities, the tissue body is a lot brighter than the activities in the neural networks. The ability of imaging a structure of a sample depends on the number of photons detected by a detector, as well as the background signal. In order to detect a faint structure in the sample, the gain of the detector needs to be increased, or the integration time increased. However, the gain or integration time increase is limited by the brightest object in the sample, as the photons emitted by the brightest object would saturate the detection system, including detector, amplifier and digitizer, etc., if the gain or integration time is adjusted too high. Thus, the photodetectors, amplifiers and digitizers used in fluorescence microscopy have a limited detection dynamic range, as it is bounded by the maximum and minimum intensities that can be simultaneously detected within a field of view. Current imaging techniques can provide up to about <NUM>-bit of dynamic range. However, a high dynamic range of <NUM> to <NUM>-bit is desired in some imaging applications, such as optical imaging of neural activities and fine neural structures. Therefore, there is a need for a method to overcome the above limitation, sothat high dynamic range imaging of a sample can be performed.

<CIT> discloses a tissue imaging system includes a laser module for outputting a laser pulse, an optical delay module configured to split a laser pulse received from the laser module into a plurality of time-delayed sub-pulses, a telescope for delivering the sub-pulses from the optical delay module to a target volume and a photodetector configured to collect photons generated within the target volume in response to excitation of the target volume by the first and second sub-pulses. The system may further include a spatial multiplexing module configured to receive the temporally multiplexed laser pulse from the optical delay module and splitting the temporally multiplexed laser pulse into a plurality of sub-beams including a first sub-beam and a second sub-beam, wherein the first sub-beam and the second sub-beam are spatially separated with respect to a first image plane formed at a first depth within the target volume and with respect to a second image plane formed at a second depth within the target volume.

<CIT> discloses system(s) and method(s) to probe electromagnetic fields at the surface of a solid-state material. The technique combines ultrafast (e.g., less than <NUM> fs) optical excitation and electron microscopy to generate electronic excitations and image the ensuing electromagnetic fields with nanometer-scale spatial resolution and
femtosecond time-scale resolution. In addition, time-of-flight energy analysis facilitates imaging of relaxation a generated electronic excitation. The dynamics of the electromagnetic fields can be probed interferometrically through generation of multi-frame imaging, with inter-frame frequency of the order of a few hundreds of attoseconds, of interference patterns among an electric field associated with an excitation in a sample or device and the electromagnetic field of a probe pulse coherent with an excitation pulse. Quality assurance of nanoscopic devices based on plasmonic, photonic, electronic, spintronic operation can be analyzed with spectroscopy provided in the subject innovation.

<CIT> discloses that a multiphoton excitation type observation device is equipped with a pulse laser light source for emitting an ultra-short pulse laser light; the observation device body for irradiating the sample with the ultra-short pulse laser light emitted from the pulse laser light source, and observing fluorescence emitted from the sample; and an acoustic optical modulation filter arranged between the pulse laser light source and the observation device body, for modulating the ultra-short pulse laser light emitted from the pulse laser light source.

<CIT> discloses a time-resolved fluorescence imaging (TRFI) system that images a target medium without lifetime fitting. Instead of extracting the lifetime precisely, the system images the fluorophore distribution to allow for a simple and accurate method to obtain the fluorescence image without lifetime-extraction for time-resolved fluorescence imaging. An illumination source circuit for TRFI is also disclosed that shapes the excitation pulse. In one embodiment, the illumination source comprises an LED and stub line configured for generating a linear decay profile.

<CIT> discloses non-linear optical microscopy and micro-spectroscopy imaging systems employing efficient dual frequency laser sources, in particular for coherent anti-Stokes Raman scattering (CARS) microscopy using two pulsed laser excitation beams (pump and Stokes beams).

An embodiment of the present invention provides an imaging system including: a light source configured to generate successive light pulses having a pulse interval; and a microscopy system configured to image a sample and to process signals detected from the sample; characterized in that the generated successive light pulses are of diminishing intensity and in that the signal processing is based on intensities of the successive light pulses, wherein the successive light pulses are more than two and the pulse interval is constant.

Further with an imaging system, an embodiment of the present invention provides a light source configured to generate a series of reducing intensity laser pulses, including: a pulse laser configured to generate light pulses having a repetition interval; a first beam splitter configured to receive a light pulse and to direct a first percentage of the light pulse onto a delay loop and output a second percentage of the light pulse; wherein the delay loop is configured to direct the first percentage of light pulse back to the first beam splitter with a time delay; and wherein the system is configured to, by having continued looping of a light pulse in the delay loop, output successive light pulses of diminishing intensity with a pulse interval being equal to the time delay.

Further with an imaging system, an embodiment of the present invention provides a microscopy system including: a sample objective; a detector; and one or more optical elements configured to direct the successive light pulses to the sample objective; wherein the sample objective is configured to focus the successive light pulses at a focal plane within a sample; wherein the detector is configured to detect light emitted from the focal plane within the sample in response to the focused successive light pulses.

An embodiment of the present invention provides an imaging method including: generating, by a light source, successive light pulses having a pulse interval; and imaging a sample and processing signals detected from the sample; characterized in that the generated successive light pulses are of diminishing intensity and in that the signal processing is based on intensities of the successive light pulses, wherein the successive light pulses are more than two and the pulse interval is constant.

Further with an imaging method, an embodiment of the present invention provides a method for generating a series of reducing intensity laser pulses, including: generating, by a pulse laser, light pulses having a repetition interval; receiving, by a first beam splitter, a light pulse and directing a first percentage of the light pulse onto a delay loop and outputting a second percentage of the light pulse; directing, by the delay loop, the first percentage of light pulse back to the first beam splitter with a time delay; and by having continued looping of a light pulse in the delay loop, outputting successive light pulses of diminishing intensity with the pulse interval being equal to the time delay.

Further with an imaging method, an embodiment of the present invention includes: directing, by one or more optical elements, the successive light pulses to a sample objective; focusing, by the sample objective, the successive light pulses at a foc plane within a sample; detecting, by a detector, light emitted from the focal plane within the sample in response to the focused successive light pulses.

Relative terms such as "lower," "upper," "horizontal," "vertical," "above," "below," "up," "down," "top" and "bottom" as well as derivative thereof (e.g., "horizontally," "downwardly," "upwardly," etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion.

This disclosure describes the best mode or modes of practicing the invention as presently contemplated. This description is not intended to be understood in a limiting sense, but provides an example of the invention presented solely for illustrative purposes by reference to the accompanying drawings to advise one of ordinary skill in the art of the advantages and construction of the invention. In the various views of the drawings, like reference characters designate like or similar parts.

It is important to note that the embodiments disclosed are only examples of the many advantageous uses of the innovative teachings herein. In general, statements made in the specification of the present application do not necessarily limit any of the various claimed inventions. Moreover, some statements may apply to some inventive features but not to others. In general, unless otherwise indicated, singular elements may be in plural and vice versa with no loss of generality.

In one embodiment, an imaging system includes: a light source configured to generate successive light pulses of diminishing intensity having a pulse interval, as shown in <FIG>; and a microscopy system configured to image a sample and to process signals detected from the sample based on intensities of the successive light pulses, as shown in <FIG>. <FIG> is a diagram of a system for generating a series of reducing intensity laser pulses according to an embodiment. The system includes a pulse laser <NUM> that generates light pulses having a repetition rate. For example, the pulse laser may be a tunable femtosecond Ti:sapphire laser having a repetition rate around <NUM> to <NUM> and a tunable wavelength range of <NUM> to <NUM>. It is understood that different specifications or types of pulse lasers may be used depending on the specific application. In one embodiment, the light pulses generated by the pulse laser is directed to a polarizing beam splitter (PBS) <NUM>, and the light pulse that is in a first state of polarization passes through the PBS to reach a beam splitter <NUM>. In one embodiment, the beam splitter <NUM> is a partially reflecting mirror, for example, a <NUM>/<NUM> mirror. Note that different reflection/transmission ratios, or percentages, such as <NUM>/<NUM>, <NUM>/<NUM>, etc., are also contemplated, depending on specific requirements. The beam splitter <NUM> splits the light pulse into two paths: a first percentage to a delay loop and a second percentage to an output. In one embodiment, the output goes on to an imaging path for a microscopy system. As shown in <FIG>, in the imaging path, there are one or more optical elements for directing the light pulse to a desired location. For example, the one or more optical elements may be a lens, mirror, beam splitter, or scanner, etc., or some combinations of these elements. In one embodiment, an x-y scanner <NUM> scans the second percentage of the light pulse to cover an area within the plane of a sample <NUM>. By scanning the location within the plane, scanning microscopy can be performed. A dichroic mirror <NUM> reflects the light pulse into an objective <NUM>. It is noted that a skilled person would be able to use a configuration of one or more optical element, or the equivalents to direct the light to a location within the plane. The objective <NUM> then focuses the light pulse onto an image plane <NUM> of a desired depth into the sample <NUM>. It is noted that a skilled person would be able to use a configuration of one or more optical element, or the equivalents to direct the light to a location within the plane. In one embodiment, the wavelength of the laser is selected to cause fluorescence at a focal point in the sample. In one embodiment, the wavelength of the laser is selected to cause a two-photon excitation in the sample. Light emitted by the sample <NUM> is collected by a detector <NUM>. In one embodiment, the detector is a photomultiplier tube (PMT). In one embodiment, the detector is a silicon photomultiplier (SiPM). In one embodiment, the light emitted by the sample <NUM> passes through the objective <NUM> and through the dichroic mirror <NUM> to reach the detector <NUM> above. Note that the arrangement of the dichroic mirror and detector shown in <FIG> is an example setup only. Other optical elements and/or arrangements are possible to achieve the desired direction of light to the sample and detection of the emitted light from the sample.

In one embodiment, the delay loop includes a path traverses the beam splitter <NUM> that directs a first percentage of the light pulse to a first mirror <NUM>, which reflects the pulse to a second mirror <NUM>, which reflects the pulse through a half-wave plate <NUM> to the PBS <NUM>. The half-wave plate <NUM> changes the pulse to a second polarization state, allowing the PBS <NUM> to reflect the pulse back to the beam splitter <NUM>. Note that the half-wave plate <NUM> may be placed anywhere within the path. The arrangement shown in <FIG> is an example setup. The delay loop introduces a desired delay time to the first percentage of the light pulse relative to the second percentage of the light pulse. Note that the delay loop setup shown in <FIG> is an illustrative example. Other optical elements and/or arrangement may be used to create such a delay loop, or equivalents thereof.

The beam splitter <NUM> further splits the delayed light pulse according to its reflection/transmission ratio, and thus the intensity of the delayed light pulse transmitted by the beam splitter <NUM> is further diminished. Each time the light reflected by the beam splitter <NUM> into the delay loop would be delayed by the desired delay time as it loops around the delay loop, and the intensity of the light transmitted by the beam splitter <NUM> would be reduced according to the reflection/transmission ratio of the beam splitter. Thus, a series of delayed pulses of diminishing intensity is generated from the initial light pulse. <FIG> is a plot of intensity over time of the modulated light pulses according to an embodiment. As can be seen from <FIG>, the second light pulse is delayed relative to the first light pulse and the intensity of the second light pulse is less than that of the first light pulse. The third pulse is delayed by the same amount and with its intensity further diminished. The pulses shown in <FIG> are for illustration purposes. The number of pulses, their relative intensities, delays, etc., may vary depending on specific setup and application requirements. Note that <FIG> also shows a fourth pulse with the same intensity as the first pulse. This fourth pulse is due to a pulse from the pulse laser arriving at the next repetition interval.

Using knowledge of the pulse intensity, location on the test sample, and amount of fluorescence measured, an imaging system according to an embodiment can create an increased dynamic range of the image relative to what can be obtained in a normal two-photon imaging system.

In one embodiment, the detector includes a plurality of temporal buffers which would store signals from the sample. The signal detected by the detector for the fluorescence in response to a focused light pulse may be stored in one of the buffers. If the delay time introduced by the delay loop is selected such that when the delayed pulse arrives at the sample, the fluorescence due to the previous pulse has already substantially subsided, then detected signal due to the fluorescence in response to the focused delayed light pulse may be stored in another buffer. Thus, at each focal point in the sample, the buffers store fluorescence data at different times, each correspond to a light pulse of different intensities. Since the intensity of the fluorescence depends on the intensity squared of the excitation pulse, for some pulse intensities, the light emitted by a structure in the sample may be so high that the buffer is saturated, and for some other pulse intensities, the light emitted by a structure in the sample may be so faint that the signal is not registered. In one embodiment, the imaging system includes a processor <NUM> configured to select a buffer among the plurality of temporal buffers, where the selected buffer is not saturated by the brightest object. Thus, for each spot, the processor has a choice among different intensity-modulated pulses and can select the excitation intensity that corresponds to the most appropriate dynamic range for that spot. In one embodiment, the processor may select the data in a buffer that corresponds to the highest intensity pulse among those pulses that do not cause buffer saturation.

The choice of the appropriate buffer may be performed in real time as the sample is being scanned by the system. In one embodiment, the processor may include a field programmable gate array (FPGA), which allows programmable logic to be incorporated with high speed and flexibility on the processor.

In another embodiment, the creation of a series of pulses of reducing amplitude is realized by a synchronous electro-optic modulator (SEOM) with a delay loop as shown in <FIG> shows an example setup of the SEOM according to an embodiment. A quarter wave plate <NUM> and a first PBS or polarizer <NUM> configured to receive an input a pulsed laser to provide circularly polarized laser pulses into the electro-optic modulator EOM (such as a Pockels cell) <NUM>. If the laser pulses are reflected the PBS <NUM>, they are directed into a sink <NUM>. The electro-optic modulator <NUM> is driven by a waveform driver <NUM>. The modulated output from the electro-optic modulator is split by the second PBS <NUM> into output <NUM> and output <NUM> according to their respective polarization states.

The EOM modulation waveform is generated by the waveform driver <NUM>. The waveform drive includes a custom circuit to lock to the laser and create a phase locked signal, with the ability to step phase shift that signal, to a RF power amplifier which drives a transformer, providing the AC voltage to the EOM. In one embodiment, a sinusoidal waveform, which is representative of the EOM modulation waveform, is expressed as: <MAT> where VP is the amplitude, or "Peak Amplitude" of the waveform, f is frequency in Hz, and φ is the phase in radians.

A periodic impulse train, which is representative of pulsed lasers, is expressed as: <MAT> where Ts is the period of the pulses going around the delay loop. This means that the series of laser pulses arrive at t = <NUM>, t = Ts, t = <NUM>Ts,. , etc. Now the phase of the EOM drive sinusoid is relative to this and is defined by φ. The voltage on the EOM is relevant only at the instant in time when the laser pulse is present in the EOM material (crystal). That means the voltage on the sinusoidal waveform is relevant only at time t = <NUM>, t = Ts, t = <NUM>Ts,. , etc. The drive voltage is thus the sinusoid equation evaluated at those instants in time, and now looks like a discrete-time sampled signal: <MAT>.

When the sinusoidal waveform is synchronized with the successive delayed pulses, we have f = <NUM>/Ts = repetition rate of the series of pulses of reducing amplitude. The amplitude reduction of the series of pulses can be adjusted by changing the phase of the EOM drive φ.

The present disclosure overcomes the limits in the dynamic range of the traditional detection/digitization system by creating a series of pulses of reducing amplitude, and then using knowledge of the pulse intensity, location on the test sample, and amount of fluorescence measured to assemble a high dynamic range final image. Thus, embodiments of the present invention represent significant improvements over existing microscopic imaging technology.

Claim 1:
An imaging system comprising:
a light source configured to generate successive light pulses of diminishing intensities having a pulse interval;
and
a microscopy system configured to image a sample and to process signals detected from the sample based on intensities of the successive light pulses;
characterized in that the successive light pulses are more than two and in that the pulse interval is constant.