WO2019200112A1 - Frequency-shifted pulsed swept laser apparatus - Google Patents

Abstract

A frequency-shifted pulsed swept laser configured to output a broadband periodic sweep, in a wavelength centered around a frequency, to a pulse modulator and into a frequency-shifting element which shifts the pulsed output to different frequencies. The apparatus makes use of the quadratic power dependency of the second harmonic generation (SHG) process, with the sweep of a swept source laser modulated to short pulses and consecutively amplified to high instantaneous powers and broadband frequency-doubling. A broadband sweep of visible pulses is achieved which can be employed for many applications, a number of which are described.

Description

FREQUENCY-SHIFTED PULSED SWEPT LASER APPARATUS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional patent application serial number 62/656,499 filed on 04/12/2018,

incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant No.

GM 107924, awarded by the National Institutes of Health. The Government has certain rights in the invention.

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

[0003] A portion of the material in this patent document may be subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records, but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C.F.R. § 1.14.

BACKGROUND

[0004] 1. Technical Field

[0005] The technology of this disclosure pertains generally to a wavelength swept laser, and more particularly to a frequency-shifted pulsed swept laser configured for operation in the visible portion of the frequency spectrum.

[0006] 2. Background Discussion

[0007] Wavelength swept lasers usually operate at near-infrared wavelengths (e.g., greater than 800 nm), because several problems including dispersion, gain media bandwidth and availability, optical losses prevent operation at visible wavelengths. However, rapidly swept laser sources in the visible portion of the frequency spectrum would be of high commercial interest. On the other hand, frequency-doubling of near- infrared sources is well known and frequently employed. Nevertheless, the operation is restricted to narrowband operation since second harmonic generation (SHG) requires a so called phase-matching condition. This matching of the phase velocity of both the incoming and the generated light is required for high SHG efficiency. One famous application is green laser pointers which employ Potassium Titanyl Phosphate (KTP) crystals for SHG generation. However, the narrowband phase-matching condition opposes a practical application of SHG to the frequency-doubling of a broadband swept-source laser source.

[0008] Accordingly, a need exists for a frequency-shifted pulsed swept laser which operates in the visible portion of the electromagnetic spectrum. The present disclosure fulfills that need and provides additional benefits over previous swept laser technologies.

BRIEF SUMMARY

[0009] The technology described in this disclosure makes use of the

quadratic power dependency of the second harmonic generation (SHG) process. To this end, the sweep of a swept source laser is modulated to short pulses and consecutively amplified to high instantaneous powers.

The high powers provide a broadband frequency-doubling in a non phase- matched Beta Barium Borate (BBO) crystal. The angle of the BBO crystal relative to the beam path and the polarization of the light are critical to SHG efficiency. The result is a broadband sweep of visible pulses which can be employed for many applications such as the aforementioned, where peak powers can be much lower. Typically, an application in the visible spectrum requires about 1 mW to 10 mW of optical peak power, which can be achieved by non phase-matched frequency-doubling of 10 W to 100 W peak power pump light.

[0010] Further aspects of the technology described herein will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the technology without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0011] The technology described herein will be more fully understood by reference to the following drawings which are for illustrative purposes only:

[0012] FIG. 1 is a block diagram of the visible pulsed swept source laser according to an embodiment of the present disclosure.

[0013] FIG. 2 is a rendition of an SHG swept diffraction line produced by a KTP crystal according to an embodiment of the present disclosure.

[0014] FIG. 3 is a spectrum plot of the swept laser (calculated to doubled energy) and the experimentally realized broadband SHG swept laser pulses according to an embodiment of the present disclosure.

[0015] FIG. 4 is a block diagram of fast two-dimensional excitation using diffracted, swept light pulses in the visible portion of the spectrum according to an embodiment of the present disclosure.

[0016] FIG. 5 is a block diagram of a fast excitation system for imaging

applications according to an embodiment of the present disclosure.

[0017] FIG. 6A and FIG. 6B are plots of intensity showing illumination for successive pixels and detection based on high-speed lifetimes, for the setup shown in FIG. 5, according to an embodiment of the present disclosure.

[0018] FIG. 7 is a plot of a recorded time-trace of the fluorescence signal obtained according to an embodiment of the present disclosure.

[0019] FIG. 8A and FIG. 8B are plots of fitted exponential decay used for FLIM according to an embodiment of the present disclosure.

[0020] FIG. 9 is a schematic of a periodically-poled crystal (PPC) for

efficient broadband frequency doubling according to an embodiment of the present disclosure.

[0021] FIG. 10A through FIG. 10E are plots of waveforms for the

wavelength swept laser as utilized according to an embodiment of the present disclosure.

[0022] FIG. 11 are plots of optical Spectra showing the effect of the swept EOM bias compensation as obtained according to an embodiment of the present disclosure.

[0023] FIG. 12A through FIG. 12D are plots illustrating wavelength tuning performance of different buffered versions of an FDML laser according to an embodiment of the present disclosure.

[0024] FIG. 13 is a representation of subset imaging utilized according to an embodiment of the present disclosure.

[0025] FIG. 14 is a schematic of alternative pulse modulation and

amplification according to an embodiment of the present disclosure.

[0026] FIG. 15A through FIG. 15D are renditions of pulse distribution over multiple laser sweeps according to an embodiment of the present disclosure.

[0027] FIG. 16 is a schematic of a fast excitation system utilized for flow imaging according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

[0028] The disclosed technology is a pulsed swept source laser that

operates in the visible wavelength range. Traditionally, swept source lasers operate at near-infrared wavelengths (e.g., 800 nm to 1700 nm) because of the limited availability of laser gain media. However, the use of rapidly swept sources in the visible wavelength range would be advantageous in a number of fields, such as for the study of spectroscopic measurements, optical coherence tomography (OCT), stimulated Raman scattering (SRS) spectroscopy and imaging, fluorescence and phosphorescence

spectroscopy, fluorescent lifetime measurements, quantum yield

measurements, time bandwidth-limited pulse generation, ophthalmic imaging, fluorescent imaging, fluorescent lifetime imaging (FLIM) and many other applications.

[0029] One embodiment of the disclosure is a swept source laser operating in the visible wavelength range (e.g., 530 nm) by frequency-doubling the entire sweep of a near-infrared swept source laser. High instantaneous power pulses are frequency-doubled through utilizing second-harmonic generation (SHG) in a Beta Barium Borate (BBO) crystal, resulting in a pulsed, swept source laser in the visible wavelength range.

[0030] Possible advantages of this embodiment include rapid and

adjustable sweeping speeds (up to several MHz), broadband coverage, and narrow instantaneous linewidth. This narrow linewidth in swept lasers is achieved through a narrowband spectral filter, which is used for frequency sweeping (commonly a scanning Fabry-Perot filter or other optical resonator) with the line narrowing achieved through lasing operation.

Instantaneous linewidths are achieved which are less than about one hundredth of the instantaneous frequency, more preferably less than about one thousandth of the instantaneous frequency, and most preferably less than about a few hundred parts per million (ppm); and these linewidths can be tailored to the specific application. Typical linewidths are less than about one thousandth of the center wavelength, for example a length of several picometers; but even narrower swept lasers are possible with linewidths in the GHz to sub-MHz regime.

[0031] In at least one preferred embodiment, the technology may be

employed for high speed imaging, since the swept source output can be diffracted by an optical grating to produce a wavelength dependent angle scan. This interoperation between swept source and optical grating results in a rapid beam steering mechanism at the speed of the wavelength sweep, which can reach speeds above the MHz range.

[0032] In particular, the technology may be utilized for fast fluorescence imaging with simultaneous detection of fluorescence lifetime imaging (FLIM). In one embodiment, this can be achieved using a sensitive detection mechanism and preferably synchronized digitization. By way of example and not limitation, one practical application is fluorescent flow cytometry and cell sorting, in which the combination of the two imaging modalities at MHz speeds could dramatically accelerate workflows in biotechnical laboratories. The disclosed frequency swept laser has a sweep range that is adjustable, which allows it to be adjusted to fall within an absorption bandwidth of a chosen fluorophore, and can be configured with a pulse repetition rate of laser pulses which is chosen to approximately correspond to fluorescence lifetime of a sample being imaged. In addition, pulses from the frequency swept laser have a repetition rate that is adjusted to avoid bleaching/triplett state formation. Still further, the disclosed swept laser apparatus can have programmable pulse modulation patterns (which may extend over several frequency sweeps) that allow allotting denser and/or sparser sampling to regions of interest, and using pulse patterns that support interleaved sampling.

[0033] The pulse modulator can be configured to generate time varying

pulse modulation which controls amplitude, width, or shape of the pulses, or any combination of amplitude, width and shape of the pulses. These pulse patterns can be reprogrammed in real-time (“on-the-fly” during

measurements), for example as part of a feedback loop based on output parameters of an algorithm or machine learning assistant. This is a special feature of this actively generated pulse modulation that modulation patterns can be intelligently chosen, such as the pulses and/or pulse pattern and/or pulse length and/or pulse repetition rate, in real-time according to a sample dependent factor.

[0034] It should be noted that the pulse modulator (e.g., EOM) is configured to generate a modulation pattern to modify and/or flatten intensity of the input light over the frequency sweep. This flattening can circumvent, or at least reduce, different pulse heights due to the non-constant gain applied by the amplifier. Since an EOM is a non-binary modulator with adjustable amplitude, the pulse height out of the modulator can pre-corn pensate the wavelength-dependent gain curve of the amplifier, which effectively cancels out the different gain and achieves equal-power pulses out of the amplifier.

[0035] In one embodiment, the technology makes use of the quadratic power dependency of the SHG process. To this end, the sweep of a swept source laser is modulated to short pulses and consecutively amplified to high instantaneous powers. To provide this modulation, common light modulation devices and/or pulse modulators such as electro-optic modulators (EOMs), acousto-optic modulators (AOMs), semiconductor optical amplifiers (SOAs) or other semiconductor devices, mechanical shutters or electronic pulsing or other technologies and the like can be utilized. Modulation patterns can be arbitrary, yet in one preferred embodiment optical pulses of short pulse widths are modulated, with typical pulse widths ranging from one to one thousand picoseconds. For amplification, various mechanisms may be utilized, such as rare-earth doped fiber amplifiers or laser rods (or similar shaped gain media). The pulse modulator is configured to provide differing pulse durations

depending on the application. For example providing pulse durations of less than about 10 ns, or less than about 1 ns, or less than about 100 ps.

[0036] The high powers grant a broadband frequency-doubling in a non phase-matched BBO crystal. The angle of the BBO crystal relative to the beam path and the polarization of the light dictate SFIG efficiency. The result is a broadband sweep of visible pulses which can be employed for many applications like some of the aforementioned, where peak powers can be much lower. Typically, an application in the visible wavelength range requires about 1 to 10 mW of optical peak power, which can be achieved by non phase-matched frequency-doubling of 10W to 100W peak power pump light.

[0037] FIG. 1 illustrates an example embodiment 10 of a visible pulsed

swept source laser. A swept laser 12 outputs a wavelength swept light source 14 about a frequency wq . The intensity versus time is shown 15 for this swept light source. Frequency doubling is then performed on the swept light source as follows. Pulse modulation 16 is performed that outputs a swept pulsed light source 18 to amplifier 20, whose amplified output 22 is then received by a second harmonic generation circuit 24 to produce 26 a swept light source 27 at a center frequency 2 wq . Apart from frequency- doubling, the use of second-harmonic generation (SHG), or other nonlinear mechanisms can be harnessed to shift the frequency of the primary swept laser, for example using sum- or difference frequency generation (SFG, DFG), optical parametric oscillators (OPOs), step-up frequency converters, four-wave mixing (FWM), third or higher harmonic generation (TFIG, HHG), Raman shifting or stimulated Brillouin scattering (SBS).

[0038] The wavelength swept laser 12 outputs a periodic sweep in

wavelength 15. Through electronic synchronization, each sweep can be modulated to a number of n pulses 18 which effectively translates each sweep into a train of pulses, where each pulse has a different color. Thus, a frequency swept train of pulses is generated. This frequency swept pulse train is then amplified 20 to high instantaneous powers 22. The broadband sweep, centered around the optical frequency wq , is frequency-doubled via second-harmonic generation (SFIG) 24, to output a pulsed swept laser around the center frequency 2 wq 26, as seen in plot 27.

[0039] In at least one embodiment the frequency-doubled pulsed swept laser was implemented using a BBO crystal for broadband SFIG. Both the angle of the crystal relative to the beam path and the polarization were important factors in achieving high SFIG efficiency. The resultant visible light (e.g., green light) can then be sent onto a diffraction grating.

[0040] FIG. 2 shows the diffracted swept SFIG signal (shown rendered as a line drawing of outer and inner perimeters of line intensity) such as produced by a Potassium Titanyl Phosphate (KTP) crystal. In the figure, a line 32 is seen with bright center region 34. It will be noted that the signal intensity differs significantly over the sweep. It should be appreciated that the phase-matching condition favors a narrow spectral linewidth, resulting in an uneven intensity over the SFIG sweep. Use of a BBO crystal should be more suitable for broadband operation. The spectrum of the swept SFIG output was recorded and compared to the input spectrum of the swept near-infrared laser, and it was seen that the energy of the input was doubled mathematically.

[0041] FIG. 3 illustrates an example embodiment 50 of a spectrum plot for the swept laser (calculated to doubled energy) 52 and the experimentally realized broadband SHG swept laser pulse spectrum 54. This plot shows clear correspondence of the input spectrum (calculated double energy) and the spectrum measured experimentally by frequency-doubling.

[0042] FIG. 4 illustrates an example embodiment 70 of fast two-dimensional excitation using diffracted, swept light pulses in the visible wavelength region. The frequency doubled swept source laser 72, such as described for FIG. 1 , is shown by way of example and not limitation as passing through an optical fiber 74 (e.g., single mode fiber), from which is output a free air beam 76 of the visible spectra swept light source. A line scanning mechanism is formed by sending the beam of swept light source onto a diffraction grating 78, creating a group of pointwise diffracted beams 80, in which each pulse has a unique time and wavelength, which is directed to a unique temporal and spatial mapping on a sample 88. An optional secondary scanning mechanism 82 can be employed in order to scan frames (89a - 89n) of sample 88. By way of example and not limitation, the secondary scanning is performed using a galvanometric mirror 82 which can be angularly modulated 84, to redirect beams 80 along different paths 86 toward target 88.

[0043] FIG. 5 and FIG. 6A through FIG. 6B illustrate an example

embodiment 110 of an interesting application for rapid fluorescence imaging by inertia-free, diffracted beam scanning. It should be appreciate that using visible light provides the ability to perform linear fluorescence imaging (one photon absorption). A similar setup is shown as in FIG. 4 with frequency doubled swept source laser 72 through an optical fiber 74 generating swept light source beam 76 to a line scanning mechanism of a diffraction grating 78 outputting pointwise diffracted beams 80, to a secondary scanning mechanism 82 which can be angularly modulated 84 along different paths 86.

[0044] The light is reflected from a dichroic beamsplitter 112 and passed through focusing element (e.g., a lens) 114 to focus the beams to illuminate sample 116. Reflections from the sample pass back through element 114 to then pass through the dichroic beamsplitter. It will be noted that any generated fluorescence is separated from the excitation light by using this dichroic beamsplitter which reflects a specified range of wavelengths while it is transmissive to a different range of wavelengths. After passing through the dichroic beamsplitter the fluorescence light is detected by a fast photodetector 118. The photodetector may comprise any known detector, such as photomultiplier based devices (Photomultiplier Tubes (PMTs)) upon which absorption of photons results in emission of electrons in electrical signal output 120. Light detection is thus performed in the epi- direction for the weak signals received from sample 116. The electronic signal(s) 120 from photodetector 118 are then digitized, such as by an analog-to-digital converter 122, which allows for digital processing (e.g., computer processing) of the sample information.

[0045] FIG. 6A illustrates 130 a well-defined time-to-wavelength behavior of the swept light source seen in FIG. 5, which allows mapping of the time- domain fluorescence signals to the image pixels.

[0046] FIG. 6B illustrates 140 the recording of fluorescence lifetimes if a sufficiently high bandwidth detection mechanism (detector 118 and digital conversion 122) is utilized. Thus, the present disclosure can be used for the generation of fluorescence lifetime images (FLIM).

[0047] The main advantage of this technique over other fluorescence

imaging techniques is that it can be performed extremely rapidly. It will be noted that the line scanning rate corresponds to the wavelength sweep rate of the laser, which can be on the order of kHz to MHz. Thus, up to kHz frame-rates can be achieved for both fluorescence imaging and FLIM recordings.

[0048] It should also be mentioned that applications exist for directing the beam to pass through (transmissive) the sample, wherein transmission and/or absorption of the wavelengths can be registered by a lens and detector combination on either side of the sample. This measurement can be used to generate transmission and/or absorption images, which are preferably in addition to the measurement of other imaging modalities such as fluorescence imaging or fluorescence lifetime imaging (FLIM). In this case also the fundamental wavelength before frequency-shifting can be used to gather more absorption/transmission information than by just using one wavelength band. Evidently, also many frequency-shifting modalities and/or wavelength bands can be combined to achieve multiple spectral measurement bands.

[0049] For the diffraction based excitation described above, the following should be noted (a) The spectral width of each diffracted spot is preferably sufficiently small that it allows adjacent pixels to be separated. Two requirements are generally necessary toward obtaining a sufficiently small spot size: (i) the spectral width of each modulated pulse should be sufficiently narrow and (ii) the grating resolution should be high. The first requirement is fulfilled as the wavelength swept laser can provide instantaneous linewidths of 20 pm, which is further lowered due to the non- linear SFIG generation. On the grating side, the grating resolution can readily be engineered to fulfill the needed resolution (b) The absorption bands of fluorophores that are to be excited with this system should be sufficiently broadband over the excitation sweep span. This is important to ensure fluorescence excitation over a wavelength swept line. This requirement is fulfilled, as sweep spans after SFIG for the presented systems are in the range of 10 nm, which is comparably small to the absorption bandwidth of most fluorophores.

[0050] Example 1

[0051] The system as described above has been utilized in our lab setup with images of Pollen samples obtained as one-photon excited

fluorescence images of a Pollen grain, which by way of example and not limitation was excited around 530 nm using an image acquisition rate of 1.157 kFIz and a line scan rate of 118 kFIz. The line scan rate of 118 kFIz was obtained by employing a 2-times buffered Fourier domain mode locking (FDML) laser at 1060 nm with 18 nm sweep span. It will be appreciated that an FDML laser is a laser modelocking technique that creates a continuous wave with a wavelength-swept light output. An amplitude electro-optical modulator (EOM) was used to modulate pulses of approximately 130 ps at a multiple repetition rate of the wavelength swept laser.

[0052] In this case, 128 pulses per sweep were used, but it will be

recognized that the number of pulses per sweep can be readily

programmed to other values, including higher numbers of pulses per sweep. This is conveniently achieved with programmed waveforms running on an arbitrary waveform generator. The pulses were amplified by

Ytterbium doped fiber amplifiers (YDFA) (e.g., a Master Oscillator Power Amplifier (MOPA)). A fiber-based bandpass filter was used to block the amplified spontaneous emission (ASE) from the YDFA stages. In this example, the bandwidth of these ASE filters was chosen at 20 nm, according to the desired sweeping span of the swept laser. The swept pulses were frequency-doubled by focusing the light into a BBO crystal.

The crystal and the polarization were oriented to maximize performance. After the crystal, a dichroic filter was installed to filter out the 1060 nm light and transmit the green SFIG light. The green light was then sent to a diffraction grating to achieve swept line scanning. A grating with 1200 lines/mm was employed and the first order diffraction used for line scanning. Apart from this order, only the zeroth-order was present and minimized by adjusting the polarization. The galvanometric mirror was operated at a 204-times slower frequency (578.5Flz) than the wavelength swept laser, thus resulting in a frame-rate of 2 * 578.5 Flz = 1.157 kHz.

[0053] FIG. 7 illustrates an example embodiment 150 of scanned frames of the fluorescence image. Software was configured with a graphical interface to generate the image of the Pollen sample, and show the recorded time- trace of the fluorescence signal. In FIG. 7 this image of two consecutively scanned frames is seen for use with a PMT whose output voltage was negative. The time scale seen in the image is in seconds. Each frame was recorded within the aforementioned 1/1157Hz = 864 mb . Thus, the image is generated from the time-trace by extracting relevant fluorescence signals. [0054] In at least one embodiment, the time-trace is copied from the digitizer, and a sampling rate determined. In this example the time between samples is seen as approximately 1.6 ns, corresponding to a sampling rate of 625 MSamples/s. The pulse repetition rate of the laser was 128 * 118 kHz = 15.1 MHz and thus the number of samples between pulses is calculated to be 41.3735. For correct image generation, the frame start from the digitizer trigger has to be properly set. Once this first sample is set, the image pixels can be simply extracted by extracting samples in multiples of samples between pulses. The first sample for the image is set manually by choosing the number for the offset (first pulse) for any excitation pulse, then the pixel offset (setting the start of a sweep) and finally the lines offset (corresponding to the relative phase of the galvo mirror). Additionally, a number of samples can be chosen for averaging if the fluorescent signal persists for more than one sample, which can enhance signal-to-noise ratio (SNR).

[0055] Example 2

[0056] One application for the disclosed visible pulsed swept-source laser is fluorescence lifetime imaging (FLIM). The same imaging setup used for fast fluorescence imaging can be employed for FLIM by using a high detection bandwidth. In testing this mode two fluorescent beads were imaged within 864 ps for fluorescence imaging. By using a fast

photomultiplier tube (PMT) and a fast digitizer at 3.125 GSamples/s, it was also possible to record the analog fluorescence lifetime decays. By fitting the exponential decays to every pixel, a FLIM image can be generated. In our tests a FLIM image was created imaging fluorescent lifetimes between 3 ns and 8 ns with post-processing color-coded representations.

[0057] FIG. 8A and FIG. 8B illustrate plots 160, 170, comparing between the single pulse fluorescence lifetime decay and the fitted exponential curve used to extract the lifetime. The FLIM imaging capability is generated in parallel to the fluorescent imaging, at the same rapid 864 ps acquisition time. In FIG. 8A the decay curve for fitted exponential decay used for FLIM is shown depicting the fluorescence signal, and the exponential fit used to generate a FLIM image. In FIG. 8B is shown histograms of fitted lifetimes in a particular line of the FLIM image with values around 4 ns.

[0058] The fits were determined only on the falling part of the fluorescence signal, so deconvolution with an instrument response function (IRF) of the detection system was not necessary (“tail-fitting”). It should be noted that the pulse length is much shorter than the fluorescence lifetime. For more precise lifetime measurements, deconvolution can be implemented and multi-exponential fits can be performed.

[0059] The fit length can be chosen and optimized to the fluorescence

lifetime and the pulse repetition rate. Upper and lower cut-off values were chosen by highest lifetime of 8000 ps, lowest lifetime of 3000 ps and minimum fitted amplitude of 100 mV which corresponds to about one third of the maximum fluorescence signal.

[0060] The pulse repetition rate can be chosen according to the lifetimes occurring in the sample. This adaptive control can be implemented by a control loop which measures the lifetimes and then adjusts the time between subsequent pulses. In a preferred embodiment, the lifetime of a previous excitation has already decayed enough that the next excitation lifetime can be recorded and determined independently. This can be applied even to individual timing distances between pulses/pixels in an imaging application, where individual pixels might have differing fluorescent lifetimes and thus require different pulse time separations. The adaptive pulse rate of the system can also provide advantages in avoiding triplett formation and bleaching of a fluorescent sample.

[0061] The speed and data efficiency of the present disclosure can be

increased by performing intelligent sampling. As a first example this can be performed by applying intelligent driving waveforms to the swept laser as described by Eigenwillig, C.M., et al., "K-space linear Fourier domain mode locked laser and applications for optical coherence tomography", Optics Express, 16, 8916-8937, 2008] As a second example a specially engineered grating can be utilized in which the line periodicity is varied, or by using other components that provide spatial variation in diffraction behavior (such as per an article [Hu, Z. and A.M. Rollins, "Fourier domain optical coherence tomography with a linear-in-wavenumber spectrometer", Optics Letters, 32, 3525-3527, 2007]).

[0062] A warped sampling approach may be employed which results in

temporally varying density of impulses. In combination with a diffraction mapping setup, this can lead to a spatially varying density of sample points. Utilizing warped sampling can provide an optimized data throughput when performing rapid imaging. The dynamic control of the pulse modulation allows for a control loop setup to manipulate this according to the individual application. For example, in flow cytometry that is imaging various cell focusing positions or parallel flow channels a higher pixel density can be allotted in order to image the passing specimen under optimized conditions.

[0063] FIG. 9 illustrates an example embodiment 190 of performing efficient broadband frequency-doubling utilizing a custom-designed periodically- poled crystal (PPC) 198. If the different wavelengths are offset along one axis, a spatially varying PPC (SV-PPC) can be constructed so as to generate quasi phase-matching for many colors of the visible pulsed swept source. This special crystal can be constructed by spatially offsetting the different colors, with a spatially varying periodically-poled crystal (SV-PPC) 198 can be employed to efficiently frequency-double a broad range of wavelength by quasi phase-matching.

[0064] Therefore, first the colors are separated, for instance by a diffracting element, such that they emanate from the same point in space 192 at different angles 194. If a lens 196 is placed in the focal distance, all colors are refracted to a parallel pattern and are individually focused. The SV- PPC can now be constructed such that each color sees a quasi-phase- matched periodicity in the PPC 198, for instance with 200a for the reddest part of the spectrum, 200b for a slightly less red spectral component, and 200c through 200n for even longer wavelengths. The second harmonic generated light waves can then be reunited 204 using a second lens 202 in focal distance to the SV-PPC. This embodiment achieves efficient frequency-doubling for a broad range of wavelengths. It should also be appreciated, that apart from using a discrete SV-PPC, a fan-out PPC can be utilized with similar results.

[0065] It will be appreciated that a wavelength dependent angle illumination can be achieved by proper angle-polishing of a fiber output. Since every color has a different refractive index in the fiber, all colors will exit the fiber tip at different angles if the fiber tip is angle-polished. This angle can be matched to the desired application, for example by phase-matching, angle scanning and so forth.

[0066] It will be further appreciated that other rapidly swept narrowband wavelength sources can be utilized for frequency doubling. Depending on the application, and in particular for diffraction imaging, a certain

instantaneous linewidth might be desirable. Interesting candidates are MEMS based VCSEL lasers, as used for Optical Coherence Tomography (OCT), Vernier tuned semiconductor lasers, polygon based tunable lasers, or short cavity semiconductor lasers. In view of the similarity of the sources to OCT sources, they are well-suited for use on combined OCT and fluorescence platforms. It is understood, that a combination of the technology with OCT, Raman and reflection imaging is advantageous.

[0067] It should be noted that the use of an Echelle grating can enable 2D scanning, assuming a sufficiently narrowband light source is used. It should also be appreciated that a seed, generated by the wide spectrum of a pulsed source in combination with a dispersive element, is also an adequate wavelength swept seed source for the subsequent amplification and frequency-doubling chain.

[0068] It will be appreciated that there are light sources that exhibit primarily stepwise scanning. Examples of these sources include VERNIER tunable lasers, FDML or regular swept lasers with an intra-cavity etalon of fixed free-spectral range. Stepwise scanning in combination with the presented technology has the substantial benefit of enabling a very precise stepwise spatial scanning on a predefined scan pattern. Such an assembly would be the ideal system for coupling to the distal side of fiber bundles without excessive dead time. Also in cases where a sparse sampling pattern is desired, stepwise scanning sources are advantageous.

[0069] Further, the speed and the data efficiency can be increased by

intelligent sampling from the sample. Either, by applying intelligent driving waveforms to the swept laser, by using a specially engineered grating with for example varying line periodicity, or by other components showing spatial variation in diffraction behavior.

[0070] The wavelength of the frequency swept laser and/or the wavelength of the frequency shifted output can further be shifted by employing a stimulated Raman scattering (SRS) shifter or stimulated Brillouin scattering (SBS) shifter. This can be particularly efficient when operated inside an optical fiber. To this end, the frequency swept laser could act either as a seed laser in the SRS and/or SBS shifting process, in combination of a pump laser, or itself be a pump laser of this process.

[0071] For applications requiring exact timing (e.g., fluorescence lifetime measurements, SRS spectroscopy, and so forth) a time-controlled detection can be employed. To this end, a phase-locked loop (PLL) can be employed which synchronizes the modulation of the light source to secondary systems such as a detection system.

[0072] There are a number of important aspects of the disclosed frequency- swept Laser Configuration. One challenge was the extinction ratio of the employed EOM. The model used for building the prototype has a specified extinction ratio of greater than 30 dB. This value is important in order to achieve duty cycles in the same order, thus storing all the optical power in the pulses and not in the time between pulses. We found that this extinction ratio is only achieved over a small optical bandwidth ( less than about 1 nm), for these interferometer-based EOMs. Thus the extinction ratio was not optimal over the whole wavelength sweep (typically 20 nm). This issue was addressed for the present disclosure by applying a swept bias voltage to the EOM. . As the bias voltage is linear in wavelength in the EOM, the part of the sine wave that is applied to the wavelength swept laser is also applied to the bias of the EOM in order to achieve the maximum extinction ratio over the whole sweep (e.g., the maximum extinction ratio possible with an EOM device is typically 100 to 100,000). Thus, the maximum extinction ratio is achieved throughout the whole sweep by sweeping the bias voltage synchronously with the instantaneous frequency of the swept laser.

[0073] FIG. 10A through FIG. 10E illustrate example waveforms 210, 220, 230, 240, 250 for the wavelength swept laser. In FIG. 10A the waveform 210 applied to the wavelength swept laser is shown. In FIG. 10B the EOM bias as a swept bias voltage 220 is depicted. This case represents a 2- times buffered FDML laser, thus the bias voltage is two copies of a half period sine wave. The amplitude and phase of this swept bias

compensation is carefully adjusted according to the EOM voltage-to- wavelength calibration curve and the synchronous timing of the wavelength sweep time-of-arrival at the EOM, respectively

[0074] The curve of FIG. 10B shows the applied swept voltage to the EOM.

This was inserted via a custom-built Bias-Tee to the DC port of the EOM used (e.g., Photline® EOM). The internal Bias-Tee in the EOM had a cut- off frequency above 400 kHz, as was tested experimentally. Thus, a swept voltage could be successfully applied to the DC port of the EOM. The amplitude was carefully adjusted according to the voltage response of the EOM and the phase adjusted to the relative phase of the wavelength- sweep passing through the EOM.

[0075] FIG.10C shows the modulation pattern supplied to the

semiconductor optical amplifier inside the FDML swept laser showing a 50 percent duty cycle. This modulation is used to achieve 2-times buffering of the FDML output. FIG. 10D shows the digital pattern used to modulate short pulses out of the FDML output light. The pulse generator transforms each rising or falling edge of this electronic waveform into an electronic pulse which then carves out, (i.e. , actively shapes) the pulse out of the light field. In this example only a moderate pulse rate of eight times the FDML frequency is shown for the sake of clarity, however, the present disclosure can be utilized at much higher rates. FIG. 10E further shows a 50 percent duty cycle waveform which can be utilized as a trigger output for secondary devices such as a data acquisition device. Also, this waveform can be low- pass filtered and amplified to generate a sine wave which can subsequently be used to drive a scanning mirror to scan the laser beam in

synchronization to the FDML laser.

[0076] FIG. 11 illustrates an example result 270 of the optical spectra in response to the use of swept EOM bias compensation. The spectrum of the wavelength swept laser shown as the uppermost curve 276 depicts the case when the EOM bias voltage is set to maximum transmission. The middle curve 274 depicts transmission when the extinction is maximal for the center of the sweep, for example with a constant DC bias being applied. A narrow extinction condition for the interferometer-based EOM is seen and to the sides the extinction ratio is less than the specified 30dB. Once the swept bias compensation is applied as seen by the lower curve 272, the wings (left and right side portions of the plot) also drop to below 30 dB extinction ratio. Thus, the whole sweep experiences (high) maximum extinction ratio due to the applied time-dependent swept bias voltage to the pulse modulator (EOM) to synchronously match the bias voltage to the instantaneous frequency of the frequency swept laser. This was verified later by manually reproducing this curve with tuning a static bias voltage.

[0077] This measure successfully achieves the specified extinction ratio over the whole sweep. This was experimentally verified by manually tuning the static voltage over the sweep 274 which reproduced the curve 272 perfectly.

[0078] An important aspect about the possibility of buffering the FDML laser is that an almost linear (substantially linear) sweep can be achieved. A 4- times or 8-times buffered FDML laser sweep can be considered linear in wavelength and thus results in a linear scan over one line of the image.

This is preferable, as usually a sinusoidal sample pattern is achieved with galvanometric mirrors. Thus, even though the scanning line rate is very fast in the current technology, the mapping can be made linear.

[0079] The wavelength swept laser can also be operated with an electronic waveform, such that an almost linear sweep in wavelength is created. Also, a linear sweep in frequency can be obtained (k-space linear sweep).

[0080] FIG. 12A through FIG. 12D illustrate waveforms 290, 300, 310 and 320, for wavelength tuning of different buffered versions of an FDML laser. The fundamental cavity output 290 in FIG. 12A is a sinusoidal sweep in wavelength over time, as the fast Fabry-Perot Filter is driven with a sinusoidal wave. By externally buffering the central part of the sweep, a linear part of the sweep can be used and copied two, four, eight and more times. This linear wavelength sweep thus results in a linear line scan in combination with the diffraction grating, thus circumventing the usually achieved sinusoidal mapping of the image pixels.

[0081] FIG. 12B shows the waveform applied to the bias voltage of the

EOM, resulting in a swept bias voltage. This ensures maximum extinction ratio in the interferometer-based EOM over the whole bandwidth by keeping the maximum extinction ratio exactly synchronized in time with the instantaneous wavelength of the FDML laser entering the EOM. FIG. 12B shows the electronic waveform applied to the EOM for a two-times buffered FDML laser, where an analytic cosine function was programmed to match the EOM bias voltage to the instantaneous output wavelength of the FDML laser. Accordingly, FIGs 12C and 12D show the waveforms for four-times and eight-times buffered versions of an FDML laser.

[0082] The following describes a different parameter regarding the offset voltage as monitored by the feedback loop. It should be appreciated that both the swept laser offset wavelength and the EOM bias voltage have to be controlled precisely in order to ensure desired long-term imaging quality over hours or even days. Therefore, both of these voltages can be readily controlled. This can be achieved for example by observing the time function or the spectrum of the laser. Therefore, a tap fiber coupler or the zero order diffraction of the line scanning grating can be used. A feedback loop was coded in the lab that recorded the spectral shape and position of the output of the MOPA laser and controlled both the FDML and the EOM voltage on their optimal operating points.

[0083] In order to ensure uniform signal height over the image, the power of the swept MOPA laser has to be constant over the whole sweep. This is not a given specification, as both the gain medium of the swept laser (e.g., a semiconductor optical amplifier (SOA)) and the Ytterbium gain profile have an asymmetric shape. Fortunately, in our case they compensate for each other, as the gain maximum of the SOA is around 1060 to 1080 nm and the gain maximum of the YDFA is at 1030 nm. For precise adjustment, the pulses to the EOM can be engineered to have appropriate voltage levels such that the whole sweep after the MOPA has nearly constant amplitude. It should be noted for the above that any amplitude mismatch becomes quadratic in SFIG signal height. In order to adjust the powers of the buffered sweep versions, the individual buffer stages allow precise power adjustment through attenuation of single branches. Thereby, all sweeps can be brought to the same amplitude.

[0084] A corrected gain profile or a corrected pulse height over the whole sweep can also be achieved by superimposing a swept bias voltage to the modulation device. For example, when an optical amplifier is employed, this will lead to a time-dependent gain or time-dependent pulse gain in the amplifier and thereby the pulse height can be controlled individually.

[0085] The gain profile can also be controlled by employing SRS gain using various pump wavelengths. The gain profiles of each SRS pump mix can be used to modify the gain curve.

[0086] FIG. 13 illustrates an example embodiment 330 of imaging different areas of interest in a sample, such as imaging a larger section at lower speed. For imaging, it may be advantageous to image a larger section of a sample at a lower speed, for example imaging a 256x256 image at only few frames per second, which means low duty cycles comparable to Mode- locked laser system. For example, a pulse length of 150fs at 80MFIz repetition rate corresponds to a duty cycle of 1.2 x 10^-5, which would require a repetition rate of 80 kFIz for a 150 ps laser. Thus, a 256x256 pixel image can be recorded at 1.4 Flz. Then, faster imaging of a subset of the image, such as an area of interest (AOI), can be achieved by only employing pulses to this particular part of the image. This is achieved through a flexible pulse pattern applied to the MOPA laser. The result is, by way of example and not limitation, that only some parts of the wavelength sweep are addressed by active pulsing, and also the secondary scanning element is driven at higher speeds, while the duty cycle, and thus the high signal strength, is preserved.

[0087] This concept is illustrated in FIG. 13, with a 256x256 image being acquired at 1.4 Hz, while an example 16x16 pixel AOI is then imaged at 361 Hz, or an 8x8 AOI imaged at 1.4 kHz. This solution can make fast imaging possible in a practicable manner, by intelligently and flexibly reducing the image size to an AOI in order to increase speeds. Thus, neither average power nor pixel dwell time need to be changed, which are the typical bottlenecks when trying to increase imaging speed.

[0088] FIG. 14 illustrates an example embodiment 350 of the wavelength swept laser utilizing Raman scattering. Another embodiment of the present disclosure performs the modulation and amplification of the wavelength swept laser through stimulated Raman scattering (SRS). The optical fiber output 356 of a Fourier domain mode locking (FDML) laser 352, and the optical fiber output 358 of a laser oscillator 354 (Pico or Femto second) used as a Raman pump are coupled to a Raman shifter 360. The laser oscillator is configured with short pulse durations and a high repetition rate which is utilized to amplify the wavelength sweep to short pulses. In the example shown the oscillator operates at a wavelength around 1010 to 1040 nm, thus yielding a Raman gain in the glass-fiber for the wavelength swept laser at a wavelength range of approximately 1030 to 1090nm. This will also only amplify short pulses of duration of the Raman pump pulses with gain in the order of approximately 50 to 60 dB, if co-propagation in glass-fibers is applied. The short pulse output 361 is directed to a splitter, preferably a wavelength division multiplexer (WDM) or another wavelength- selective filter 362 with both Raman amplified FDML pulses 364 and residual pump pulses 366 being output in different directions.

[0089] FIG. 15A through FIG. 15D illustrate an embodiment showing pulse distributions 370, 380, 390, 400 over multiple laser sweeps. A lower repetition rate can be achieved by distributing the pulses over several sweeps of the laser. In imaging, this can be advantageous to provide lower repetition rate illumination while maintaining image resolution and/or image size. In an imaging application, the desired number of pixels along a line can be distributed over several sweeps, for example a single sweep 370, two sweeps for one line 380, four sweeps 390, eight sweeps 400 or even more sweeps. The patterns in the circles indicate different colors of the pixels meaning that different parts of the output spectrum can be distributed over several consecutive sweeps to stitch together a whole line illumination pattern. The image size can be kept constant and only the total imaging rate decreased by distributing the spectral pulses over multiple sweeps, which can help to lower the repetition rate on the sample.

[0090] FIG. 16 illustrates a flow imaging embodiment 410 showing a beam 412 from a swept source laser being diffracted by a diffraction element 414 with its diffracted rays 416 directed to a splitter 417 directing the visible light to a lensing element 418 whose output is directed through a sample retention device, exemplified by transparent substrate 420 with flow path 422 for directing a flow of particles (e.g., cells) 424 to a reading area 426. Reflections and/or fluorescence returning from these particles is received back through lens 418 as a light pattern 428 which passes through splitter 417 as light 430 which is registered by the detector 432 that outputs electrical signals which are amplified 434 and converted to digital signal(s) by an analog-to-digital converter 436, which is optionally synchronized by a phase-locked loop (PLL) synch signal 438, which outputs a digital stream 440 configured for being digitally processed. The PLL is derived from the frequency swept laser source such as to form a phase locked detection where the emanating signals generated within the flow chamber have a direct phase relationship to the excitation light. This is particularly useful when recording very fast occurring events, such as fluorescent lifetime imaging (FLIM), where the direct phase-locked detection enables well- defined mapping of the excitation pattern to the detection time trace.

[0091] By way of example and not limitation, one application of this technology is in the field of flow imaging. For instance when screening a large number moving particles (e.g., cells) in a short time. The imaging speed which can be obtained using this technology can provide very significant benefits to both regular imaging as well as to fluorescence and fluorescence lifetime imaging. In a preferred embodiment, this imaging capability can lead to high speed cell sorting systems which provide a wealth of information. In addition, the disclosed technology can be used for in-vivo or in-situ flow cytometry where the imaging can be performed using an optical endoscope. The technology is particularly suited because of the fiber-based laser design.

[0092] The embodiment described in the presented technology can be

readily implemented within various optical scanning devices (apparatus), systems and methods. It should also be appreciated that optical scanning devices are often implemented to include one or more computer processor devices (e.g., CPU, microprocessor, microcontroller, computer enabled ASIC, etc.) and associated memory storing instructions (e.g., RAM, DRAM, NVRAM, FLASH, computer readable media, etc.) whereby programming (instructions) stored in the memory are executed on the processor to perform the steps of the various process methods described herein.

[0093] The computer and memory devices were not depicted in the

diagrams for the sake of simplicity of illustration, as one of ordinary skill in the art recognizes the use of computer devices for carrying out steps involved with optical scanning. The presented technology is non-limiting with regard to memory and computer-readable media, insofar as these are non-transitory, and thus not constituting a transitory electronic signal.

[0094] Embodiments of the present technology may be described herein with reference to flowchart illustrations of methods and systems according to embodiments of the technology, and/or procedures, algorithms, steps, operations, formulae, or other computational depictions, which may also be implemented as computer program products. In this regard, each block or step of a flowchart, and combinations of blocks (and/or steps) in a flowchart, as well as any procedure, algorithm, step, operation, formula, or computational depiction can be implemented by various means, such as hardware, firmware, and/or software including one or more computer program instructions embodied in computer-readable program code. As will be appreciated, any such computer program instructions may be executed by one or more computer processors, including without limitation a general purpose computer or special purpose computer, or other programmable processing apparatus to produce a machine, such that the computer program instructions which execute on the computer processor(s) or other programmable processing apparatus create means for

implementing the function(s) specified.

[0095] Accordingly, blocks of the flowcharts, and procedures, algorithms, steps, operations, formulae, or computational depictions described herein support combinations of means for performing the specified function(s), combinations of steps for performing the specified function(s), and computer program instructions, such as embodied in computer-readable program code logic means, for performing the specified function(s). It will also be understood that each block of the flowchart illustrations, as well as any procedures, algorithms, steps, operations, formulae, or computational depictions and combinations thereof described herein, can be implemented by special purpose hardware-based computer systems which perform the specified function(s) or step(s), or combinations of special purpose hardware and computer-readable program code.

[0096] Furthermore, these computer program instructions, such as

embodied in computer-readable program code, may also be stored in one or more computer-readable memory or memory devices that can direct a computer processor or other programmable processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or memory devices produce an article of manufacture including instruction means which implement the function specified in the block(s) of the flowchart(s). The computer program instructions may also be executed by a computer processor or other programmable processing apparatus to cause a series of operational steps to be performed on the computer processor or other programmable processing apparatus to produce a computer-implemented process such that the instructions which execute on the computer processor or other programmable processing apparatus provide steps for implementing the functions specified in the block(s) of the flowchart(s), procedure (s) algorithm(s), step(s), operation(s), formula(e), or computational

depiction(s).

[0097] It will further be appreciated that the terms "programming" or

"program executable" as used herein refer to one or more instructions that can be executed by one or more computer processors to perform one or more functions as described herein. The instructions can be embodied in software, in firmware, or in a combination of software and firmware. The instructions can be stored local to the device in non-transitory media, or can be stored remotely such as on a server, or all or a portion of the instructions can be stored locally and remotely. Instructions stored remotely can be downloaded (pushed) to the device by user initiation, or automatically based on one or more factors.

[0098] It will further be appreciated that as used herein, that the terms

processor, hardware processor, computer processor, central processing unit (CPU), and computer are used synonymously to denote a device capable of executing the instructions and communicating with input/output interfaces and/or peripheral devices, and that the terms processor, hardware processor, computer processor, CPU, and computer are intended to encompass single or multiple devices, single core and multicore devices, and variations thereof.

[0099] From the description herein, it will be appreciated that the present disclosure encompasses multiple embodiments which include, but are not limited to, the following:

[00100] 1. A frequency-shifted pulsed swept laser apparatus, comprising: (a) a frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency wq ; (b) a pulse modulator coupled to the output of the frequency swept laser and configured to modulate the broadband periodical sweep from the frequency-swept laser into a pulsed output; and (c) a frequency shifter that shifts the pulsed output into pulsed outputs centered around a different frequency.

[00101] 2. A frequency-shifted pulsed swept laser apparatus, comprising: (a)

(a) a frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency ooO ; (b) a pulse modulator coupled to the output of the frequency swept laser and

configured to modulate pulses from the frequency-swept laser; and (c) a frequency-shifting mechanism that shifts the pulsed output to different frequencies.

[00102] 3. A frequency-doubled pulsed swept laser apparatus, comprising:

(a) a near-infrared frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency wq ; (b) a pulse modulator coupled to the output of said frequency swept laser and configured to modulate each periodical sweep from said frequency swept laser to a number of n pulses at an output of said pulse modulator; (c) an amplifier coupled to said output of said pulse modulator and configured to amplify the n pulses to high levels of instantaneous power at an output of said amplifier; and (d) a second harmonic generator coupled to said output of said amplifier; (e) wherein said second harmonic generator is configured to frequency-double the n pulses at the output from said amplifier and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

[00103] 4. A frequency-doubled pulsed swept laser apparatus, comprising:

(a) a near-infrared frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency wq ; (b) a pulse modulator coupled to the output of the frequency swept laser and configured to modulate each sweep from the frequency swept laser to a number of n pulses; (c) an amplifier coupled to the output of the pulse modulator and configured to amplify the pulses to high instantaneous power; and (d) a second harmonic generator coupled to the output of the amplifier; (e) said second harmonic generator configured to frequency- double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

[00104] 5. A frequency-shifted pulsed swept laser apparatus, comprising: (a) a frequency swept laser configured to generate an output as a broadband periodical sweep in a wavelength centered around a frequency ooO ; (b) a pulse modulator coupled to the output of said frequency swept laser and configured for modulating pulses from said frequency-swept laser; and (c) a frequency-shifter configured to shift the modulated pulses to different frequencies.

[00105] 6. A frequency-shifted pulsed swept laser apparatus, comprising: (a) a frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency wq ; (b) a pulse modulator coupled to the output of the frequency swept laser and configured to modulate pulses from the frequency-swept laser; and (c) a frequency-shifter configured to shift the modulated pulses to different frequencies.

[00106] 7. The apparatus of any preceding embodiment, wherein the

frequency swept laser has an instantaneous linewidth of less than about one hundredth of the instantaneous frequency.

[00107] 8. The apparatus of any preceding embodiment, wherein the

frequency swept laser has an instantaneous linewidth of less than about one thousandth of the instantaneous frequency.

[00108] 9. The apparatus of any preceding embodiment, wherein the

frequency swept laser has an instantaneous linewidth of less than about 100s part per million (ppm).

[00109] 10. The apparatus of any preceding embodiment, wherein the pulse modulator is configured for pulse durations of less than about 10ns.

[00110] 11. The apparatus of any preceding embodiment, wherein the pulse modulator is configured for pulse durations of less than about 1 ns.

[00111] 12. The apparatus of any preceding embodiment, wherein the pulse modulator is configured for pulse durations of less than about 100ps.

[00112] 13. The apparatus of any preceding embodiment, wherein said

frequency shifter is configured to shift frequencies by a method selected from the group of frequency shifting mechanisms consisting of second harmonic generation, sum frequency generation, difference frequency generation, parametric oscillation, Raman scattering, Brillouin scattering, second-harmonic generation.

[00113] 14. The apparatus of any preceding embodiment, wherein said frequency shifter comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

[00114] 15. The apparatus of any preceding embodiment, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

[00115] 16. The apparatus of any preceding embodiment, further comprising an optical amplifier coupled to the output of the pulse modulator and configured to amplify the pulsed output to increase instantaneous power levels, wherein instantaneous power can exceed average power.

[00116] 17. The apparatus of any preceding embodiment, wherein said frequency shifter comprises an optical parametric oscillator (OPO).

[00117] 18. The apparatus of any preceding embodiment, wherein said frequency shifter is configured for Raman scattering and/or Brioullin scattering.

[00118] 19. The apparatus of any preceding embodiment, wherein said apparatus is configured as a component of a fluorescence imaging system by supplying the excitation light to generate fluorescence light.

[00119] 20. The apparatus of any preceding embodiment, wherein said apparatus is configured as a component of a fluorescence lifetime imaging system by supplying the excitation light to generate fluorescence light and subsequently determine the fluorescence decay time and thus the fluorescence lifetime.

[00120] 21. The apparatus of any preceding embodiment, further comprising a high bandwidth detection system configured for recording analog exponential decay of a fluorescence signal in order to extract a

fluorescence lifetime signal. [00121] 22. The apparatus of any preceding embodiment, further comprising a synchronizer configured for recording generated signals synchronously to their generation.

[00122] 23. The apparatus of any preceding embodiment, wherein the

synchronizer comprises a phase locked loop (PLL).

[00123] 24. The apparatus of any preceding embodiment, wherein said apparatus is a component of a rapid flow cytometry system.

[00124] 25. The apparatus of any preceding embodiment, wherein the

apparatus is a component of a rapid flow cytometry imaging system configured to capture images.

[00125] 26. The apparatus of any preceding embodiment, wherein imaging contrast is obtained for the captured images as selected from the from a group of contrast sources consisting of absorption, Raman scattering, Brioullin scattering, phase change, fluorescence and fluorescence lifetime.

[00126] 27. The apparatus of any preceding embodiment, wherein said pulse modulator is configured to generate a modulation pattern to modify and/or flatten intensity of the broadband periodical sweep.

[00127] 28. The apparatus of any preceding embodiment, wherein said pulse modulator is configured to generate time-varying pulse modulation to control amplitude, width, or shape of the pulses.

[00128] 29. The apparatus of any preceding embodiment, further comprising an amplifier having a gain spectrum, wherein the pulse modulator is configured to perform time-varying pulse modulation by shaping gain spectrum of the amplifier.

[00129] 30. The apparatus of any preceding embodiment, further comprising optical fibers over which at least a portion of visible light utilized in said apparatus are carried.

[00130] 31. The apparatus of any preceding embodiment, wherein said optical fibers comprise single-mode fibers.

[00131] 32. The apparatus of any preceding embodiment, further comprising a buffering mechanism within said frequency swept laser.

[00132] 33. The apparatus of any preceding embodiment, wherein said buffering mechanism is configured to provide a substantially linear sweep in wavelength.

[00133] 34. The apparatus of any preceding embodiment, wherein said

frequency swept laser is configured with a sweep range that is adjustable to fall within an absorption bandwidth of a chosen fluorophore.

[00134] 35. The apparatus of any preceding embodiment, wherein said

frequency swept laser is configured with a pulse repetition rate of laser pulses which approximately corresponds to fluorescence lifetime of a sample being imaged.

[00135] 36. The apparatus of any preceding embodiment, wherein said

pulse repetition rate of the laser pulses is configured to provide sufficient time in between consecutive pulses for complete signal decay of the fluorescence lifetime.

[00136] 37. The apparatus of any preceding embodiment, wherein pulses from said frequency swept laser have a repetition rate that is adjusted to avoid bleaching/triplett state formation.

[00137] 38. The apparatus of any preceding embodiment, wherein said

frequency swept laser is configured with a programmable pulse modulation pattern along the sweep whereby a warped pulse pattern can be created.

[00138] 39. The apparatus of any preceding embodiment, wherein said

programmable pulse modulation pattern is configured for allotting denser or sparser sampling to regions of interest.

[00139] 40. The apparatus of any preceding embodiment, wherein the

frequency swept laser has a pulse pattern that extends over several sweeps.

[00140] 41. The apparatus of any preceding embodiment, wherein said

frequency swept laser is configured with a pulse pattern that is

reprogrammable during a time in which measurements are being made to intelligently choose the pulses and/or pulse pattern and/or pulse length and/or pulse repetition rate in real-time according to a sample dependent factor.

[00141] 42. The apparatus of any preceding embodiment, wherein said pulse modulator comprises an electro-optical modulator (EOM).

[00142] 43. The apparatus of any preceding embodiment, wherein said electro-optical modulator (EOM) has a high extinction ratio.

[00143] 44. The apparatus of any preceding embodiment, wherein a time- dependent swept control signal is applied to said pulse modulator to synchronously adjust pulse to background contrast in response to instantaneous frequency of the frequency swept laser.

[00144] 45. The apparatus of any preceding embodiment, wherein the time- dependent control signal modifies the transient input light into an optical amplifier to achieve a time-dependent output level of the light.

[00145] 46. The apparatus of any preceding embodiment, wherein said apparatus is configured to generate an output wavelength on the order of 530 nm.

[00146] 47. The apparatus of any preceding embodiment, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

[00147] 48. The apparatus of any preceding embodiment, wherein the

apparatus is a component of a fluorescence lifetime imaging system (FLIM).

[00148] 49. The apparatus of any preceding embodiment, wherein said frequency-shifter shifts frequency by a method selected from a group of frequency shifting elements consisting of second harmonic generation, sum frequency generation, and difference frequency generation.

[00149] 50. The apparatus of any preceding embodiment, wherein said frequency-shifter comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

[00150] 51. The apparatus of any preceding embodiment, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

[00151] 52. The apparatus of any preceding embodiment, wherein said frequency-shifted pulsed swept laser apparatus is a component of a fluorescence lifetime imaging system (FLIM).

[00152] 53. The apparatus of any preceding embodiment, wherein the

frequency swept laser has an instantaneous linewidth of less than about one hundredth of the instantaneous frequency, preferably less than about one thousandth of the instantaneous frequency and most preferably less than about 100 part per million (ppm).

[00153] 54. The apparatus of any preceding embodiment, wherein the pulse modulator employs pulse durations of less than about 10ns, preferably less than about 1 ns, most preferably less than about 100ps.

[00154] 55. The apparatus of any preceding embodiment, wherein said frequency-shifting mechanism shifts frequency by a method selected from group consisting of second harmonic generation, sum frequency

generation, and difference frequency generation.

[00155] 56. The apparatus of any preceding embodiment, wherein said frequency-shifting mechanism comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

[00156] 57. The apparatus of any preceding embodiment, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

[00157] 58. The apparatus of any preceding embodiment, further comprising an optical amplifier coupled to the output of the pulsed light source, configured to amplify the pulses to high instantaneous power.

[00158] 59. The apparatus of any preceding embodiment, wherein the

frequency-shifting mechanism comprises an optical parametric oscillator (OPO).

[00159] 60. The apparatus of any preceding embodiment, wherein the

frequency-shifting mechanism makes use of Raman scattering and/or Brioullin scattering.

[00160] 61. The apparatus of any preceding embodiment, wherein the

apparatus is a component of a fluorescence imaging system.

[00161] 62. The apparatus of any preceding embodiment, wherein the apparatus laser is a component of a fluorescence lifetime imaging system.

[00162] 63. The apparatus of any preceding embodiment, further comprising a high bandwidth detection system that is used to record analogue exponential decay of a fluorescence signal in order to extract a

fluorescence lifetime signal.

[00163] 64. The apparatus of any preceding embodiment, further comprising a synchronization mechanism to record generated signals synchronously to their generation.

[00164] 65. The apparatus of any preceding embodiment, wherein the

synchronization mechanism comprises a phase locked loop (PLL).

[00165] 66. The apparatus of any preceding embodiment, wherein the

apparatus is a component of a rapid flow cytometry system.

[00166] 67. The apparatus of any preceding embodiment, wherein the

apparatus is a component of a rapid flow cytometry imaging system.

[00167] 68. The apparatus of any preceding embodiment, wherein imaging contrast is sourced from the group of sources consisting of absorption, scattering, phase change, fluorescence and fluorescence lifetime.

[00168] 69. The apparatus of any preceding embodiment, wherein the pulse modulator generates a modulation pattern to modify and/or flatten intensity over the frequency sweep.

[00169] 70. The apparatus of any preceding embodiment, wherein the pulse modulator employs time-varying pulse modulation by means of amplitude, width or shape of the pulses.

[00170] 71. The apparatus of any preceding embodiment, further comprising an amplifier having a gain spectrum, wherein the pulse modulator employs time-varying pulse modulator by shaping the gain spectrum of the amplifier.

[00171] 72. The apparatus of any preceding embodiment, wherein the

frequency swept laser comprises at least partially optical fibers.

[00172] 73. The apparatus of any preceding embodiment, wherein the

optical fibers are comprised of single-mode fibers.

[00173] 74. The apparatus of any preceding embodiment, wherein the

frequency swept laser includes a buffering mechanism. [00174] 75. The apparatus of any preceding embodiment, wherein the buffering mechanism provides an almost linear sweep in wavelength.

[00175] 76. The apparatus of any preceding embodiment, wherein the

frequency swept laser has a sweep range that is adjustable to fall within the absorption bandwidth of a chosen fluorophore.

[00176] 77. The apparatus of any preceding embodiment, wherein the

pulses from the frequency swept laser have a repetition rate of the laser pulses chosen according to the fluorescence lifetime of the sample.

[00177] 78. The apparatus of any preceding embodiment, wherein time in between consecutive pulses is sufficient for complete signal decay.

[00178] 79. The apparatus of any preceding embodiment, wherein said

signals comprise phosphorescent signals.

[00179] 80. The apparatus of any preceding embodiment, wherein pulses from the frequency swept laser have a repetition rate that is adjusted to avoid bleaching/triplett state formation.

[00180] 81. The apparatus of any preceding embodiment, wherein the

frequency swept laser has a pulse modulation pattern is freely

programmable along the frequency sweep such as to create a warped pulse pattern.

[00181] 82. The apparatus of any preceding embodiment, wherein said

programmability allows allotting denser/sparser sampling to regions of interest.

[00182] 83. The apparatus of any preceding embodiment, wherein the

frequency swept laser has a pulse pattern that extends over several frequency sweeps.

[00183] 84. The apparatus of any preceding embodiment, wherein the pulse pattern can be employed for interleaved sampling.

[00184] 85. The apparatus of any preceding embodiment, wherein the

frequency swept laser has a pulse pattern that is reprogrammable on-the-fly such as to intelligently choose the pulses and/or pulse pattern and/or pulse length and/or pulse repetition rate in real-time according to a sample dependent factor. [00185] 86. The apparatus of any preceding embodiment, wherein the pulse modulator comprises an electro-optical modulator (EOM).

[00186] 87. The apparatus of any preceding embodiment, wherein the EOM has a high extinction ratio.

[00187] 88. The apparatus of any preceding embodiment, wherein a time- dependent swept bias voltage is applied the pulse modulator to

synchronously adjust the bias voltage in tune with the instantaneous frequency of the frequency swept laser.

[00188] 89. The apparatus of any preceding embodiment, wherein the time- dependent bias voltage achieve a time-dependent pulse gain in an optical amplifier.

[00189] 90. The apparatus of any preceding embodiment, wherein said frequency-shifter shifts frequency by a method selected from group consisting of second harmonic generation, sum frequency generation, and difference frequency generation.

[00190] 91. The apparatus of any preceding embodiment, wherein said frequency-shifter comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2wo in the visible wavelength range.

[00191] 92. The apparatus of any preceding embodiment, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

[00192] 93. The apparatus of any preceding embodiment, wherein the laser is a component of a fluorescence lifetime imaging system (FLIM).

[00193] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more."

[00194] As used herein, the term "set" refers to a collection of one or more objects. Thus, for example, a set of objects can include a single object or multiple objects.

[00195] As used herein, the terms "substantially" and "about" are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ± 10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. For example, "substantially" aligned can refer to a range of angular variation of less than or equal to ±10°, such as less than or equal to ±5°, less than or equal to ±4°, less than or equal to ±3°, less than or equal to ±2°, less than or equal to ±1 °, less than or equal to ±0.5°, less than or equal to ±0.1 °, or less than or equal to ±0.05°.

[00196] Additionally, amounts, ratios, and other numerical values may

sometimes be presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[00197] Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure but as merely providing illustrations of some of the presently preferred embodiments. Therefore, it will be appreciated that the scope of the disclosure fully encompasses other embodiments which may become obvious to those skilled in the art.

[00198] All structural and functional equivalents to the elements of the disclosed embodiments that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Furthermore, no element,

component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed as a "means plus function" element unless the element is expressly recited using the phrase "means for". No claim element herein is to be construed as a "step plus function" element unless the element is expressly recited using the phrase "step for".

Claims

CLAIMS What is claimed is:

1. A frequency-shifted pulsed swept laser apparatus, comprising:

(a) a frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency wq ;

(b) a pulse modulator coupled to the output of the frequency swept laser and configured to modulate the broadband periodical sweep from the frequency- swept laser into a pulsed output; and

(c) a frequency shifter that shifts the pulsed output into pulsed outputs centered around a different frequency.

2. The apparatus of claim 1 , wherein the frequency swept laser has an instantaneous linewidth of less than about one hundredth of the instantaneous frequency.

3. The apparatus of claim 1 , wherein the frequency swept laser has an instantaneous linewidth of less than about one thousandth of the instantaneous frequency.

4. The apparatus of claim 1 , wherein the frequency swept laser has an instantaneous linewidth of less than about 100s part per million (ppm).

5. The apparatus of claim 1 , wherein the pulse modulator is configured for pulse durations of less than about 10ns.

6. The apparatus of claim 1 , wherein the pulse modulator is configured for pulse durations of less than about 1 ns.

7. The apparatus of claim 1 , wherein the pulse modulator is configured for pulse durations of less than about 100ps.

8. The apparatus of claim 1 , wherein said frequency shifter is configured to shift frequencies by a method selected from the group of frequency shifting mechanisms consisting of second harmonic generation, sum frequency generation, difference frequency generation, parametric oscillation, Raman scattering, Brillouin scattering, second-harmonic generation.

9. The apparatus of claim 1 , wherein said frequency shifter comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

10. The apparatus of claim 9, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

11. The apparatus of claim 1 , further comprising an optical amplifier coupled to the output of the pulse modulator and configured to amplify the pulsed output to increase instantaneous power levels, wherein instantaneous power can exceed average power.

12. The apparatus of claim 1 , wherein said frequency shifter comprises an optical parametric oscillator (OPO).

13. The apparatus of claim 1 , wherein said frequency shifter is configured for Raman scattering and/or Brioullin scattering.

14. The apparatus of claim 1 , wherein said apparatus is configured as a component of a fluorescence imaging system by supplying the excitation light to generate fluorescence light.

15. The apparatus of claim 1 , wherein said apparatus is configured as a component of a fluorescence lifetime imaging system by supplying the excitation light to generate fluorescence light and subsequently determine the fluorescence decay time and thus the fluorescence lifetime.

16. The apparatus of claim 15, further comprising a high bandwidth detection system configured for recording analog exponential decay of a fluorescence signal in order to extract a fluorescence lifetime signal.

17. The apparatus of claim 1 , further comprising a synchronizer configured for recording generated signals synchronously to their generation.

18. The apparatus of claim 17, wherein the synchronizer comprises a phase locked loop (PLL).

19. The apparatus of claim 1 , wherein said apparatus is a component of a rapid flow cytometry system.

20. The apparatus of claim 1 , wherein the apparatus is a component of a rapid flow cytometry imaging system configured to capture images.

21. The apparatus of claim 20, wherein imaging contrast is obtained for the captured images as selected from the from a group of contrast sources consisting of absorption, Raman scattering, Brioullin scattering, phase change, fluorescence and fluorescence lifetime.

22. The apparatus of claim 1 , wherein said pulse modulator is configured to generate a modulation pattern to modify and/or flatten intensity of the

broadband periodical sweep.

23. The apparatus of claim 22, wherein said pulse modulator is configured to generate time-varying pulse modulation to control amplitude, width, or shape of the pulses.

24. The apparatus of claim 22, further comprising an amplifier having a gain spectrum, wherein the pulse modulator is configured to perform time-varying pulse modulation by shaping gain spectrum of the amplifier.

25. The apparatus of claim 1 , further comprising optical fibers over which at least a portion of visible light utilized in said apparatus are carried.

26. The apparatus of claim 25, wherein said optical fibers comprise single-mode fibers.

27. The apparatus of claim 1 , further comprising a buffering mechanism within said frequency swept laser.

28. The apparatus of claim 27, wherein said buffering mechanism is configured to provide a substantially linear sweep in wavelength.

29. The apparatus of claim 1 , wherein said frequency swept laser is configured with a sweep range that is adjustable to fall within an absorption bandwidth of a chosen fluorophore.

30. The apparatus of claim 1 , wherein said frequency swept laser is configured with a pulse repetition rate of laser pulses which approximately corresponds to fluorescence lifetime of a sample being imaged.

31. The apparatus of claim 30, wherein said pulse repetition rate of the laser pulses is configured to provide sufficient time in between consecutive pulses for complete signal decay of the fluorescence lifetime.

32. The apparatus of claim 1 , wherein pulses from said frequency swept laser have a repetition rate that is adjusted to avoid bleaching/triplett state formation.

33. The apparatus of claim 1 , wherein said frequency swept laser is configured with a programmable pulse modulation pattern along the sweep whereby a warped pulse pattern can be created.

34. The apparatus of claim 33, wherein said programmable pulse modulation pattern is configured for allotting denser or sparser sampling to regions of interest.

35. The apparatus of claim 1 , wherein the frequency swept laser has a pulse pattern that extends over several sweeps.

36. The apparatus of claim 1 , wherein said frequency swept laser is configured with a pulse pattern that is reprogrammable during a time in which measurements are being made to intelligently choose the pulses and/or pulse pattern and/or pulse length and/or pulse repetition rate in real-time according to a sample dependent factor.

37. The apparatus of claim 1 , wherein said pulse modulator comprises an electro-optical modulator (EOM).

38. The apparatus of claim 37, wherein said electro-optical modulator (EOM) has a high extinction ratio.

39. The apparatus of claim 1 , wherein a time-dependent swept control signal is applied to said pulse modulator to synchronously adjust pulse to background contrast in response to instantaneous frequency of the frequency swept laser.

40. The apparatus of claim 39, wherein the time-dependent control signal modifies the transient input light into an optical amplifier to achieve a time- dependent output level of the light.

41. A frequency-doubled pulsed swept laser apparatus, comprising:

(a) a near-infrared frequency swept laser configured to output a broadband periodical sweep in a wavelength centered around a frequency ooO ;

(b) a pulse modulator coupled to the output of said frequency swept laser and configured to modulate each periodical sweep from said frequency swept laser to a number of n pulses at an output of said pulse modulator;

(c) an amplifier coupled to said output of said pulse modulator and configured to amplify the n pulses to high levels of instantaneous power at an output of said amplifier; and

(d) a second harmonic generator coupled to said output of said amplifier;

(e) wherein said second harmonic generator is configured to frequency- double the n pulses at the output from said amplifier and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

42. The apparatus of claim 41 , wherein said apparatus is configured to generate an output wavelength on the order of 530 nm.

43. The apparatus of claim 41 , wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

44. The apparatus of claim 43, wherein the apparatus is a component of a fluorescence lifetime imaging system (FLIM).

45. A frequency-shifted pulsed swept laser apparatus, comprising:

(a) a frequency swept laser configured to generate an output as a broadband periodical sweep in a wavelength centered around a frequency wq ;

(b) a pulse modulator coupled to the output of said frequency swept laser and configured for modulating pulses from said frequency-swept laser; and

(c) a frequency-shifter configured to shift the modulated pulses to different frequencies.

46. The apparatus of claim 45, wherein said frequency-shifter shifts frequency by a method selected from a group of frequency shifting elements consisting of second harmonic generation, sum frequency generation, and difference frequency generation.

47. The apparatus of claim 45, wherein said frequency-shifter comprises a second harmonic generator configured to frequency-double the pulses and produce a pulsed swept output centered around a frequency 2 wq in the visible wavelength range.

48. The apparatus of claim 47, wherein said second harmonic generator comprises a non phase-matched Beta Barium Borate (BBO) crystal.

49. The apparatus of claim 45, wherein said frequency-shifted pulsed swept laser apparatus is a component of a fluorescence lifetime imaging system (FLIM).