Single shot SPIFI operating in the Fourier Domain is demonstrated for the first time to our knowledge. We present initial results capturing Fourier Domain single shot images with both one and two-dimensional detectors and demonstrate that the same enhanced frequency support that is characteristic of classical SPIFI translates directly into single shot SPIFI architectures as well. Linear Fourier Domain single shot SPIFI is systematically analyzed, for both types of detectors. Finally, we show that despite the complex pulse structure imposed on the illumination beam by SPIFI, nonlinear single shot SPIFI can be realized, and third harmonic generation imaging is demonstrated. Prepared by LLNL under Contract DE-AC52-07NA27344. LLNL-ABS-860152.
Photodiodes are commonly used in Laser Powder Bed Fusion (LPBF) for process monitoring. However, they spatially average out features within the melt pool. We introduce Spatial Frequency Modulation Imaging (SPIFI), a technique enabling 1D spatially resolved imaging with photodiodes. SPIFI can produce high-resolution images using blackbody radiation or coherent illumination. In LPBF monitoring, we use three photodiodes at different wavelengths (780, 920, and 1070 nm) to obtain 1D SPIFI images of the melt pool temperature and reflected fusing beam. Our experimental results aim to benchmark LPBF physics models for accurate melt process simulations and serve as an in-situ process diagnostic.
Spatial frequency modulated imaging (SPIFI) employs a structured line-shaped illumination, able to resolve beyond conventional resolution limits for coherent light with high speed. It produces image harmonics, with each order carrying a higher resolution. To date this method used rotating reticles to produce the necessary structured illumination, limiting image acquisition to about 100 μs. Here, we introduce a single-shot approach. We show that a super-resolved 1D image can be acquired with a single femtosecond pulse, with potential acquisition rates in the tens of kHz.
Lately, the use of ultrafast in-volume laser-based processing of transparent materials has gained ground as a 3D-printing method of functional materials, photonics devices and high-density storage media. In this talk, we discuss the use of wide-field third-harmonic imaging that offers a non-destructive means for investigating and characterizing laser-written in-volume complex structures. Specifically, the method is used for identifying laser-induced modifications and establishing their taxonomy over a large area of a material. Unlike confocal arrangements, its ability to capture both the direct and scattered signal enables the collection of comprehensive information related to the local laser-induced modifications. Its inline nature allows for in situ monitoring of the material's response to various laser exposure conditions. As future prospect, it offers a pathway towards the implementation of closed-loop control algorithms, guaranteeing the accuracy and consistency of the desired modifications.
SignificanceMultiphoton microscopy is a powerful imaging tool for biomedical applications. A variety of techniques and respective benefits exist for multiphoton microscopy, but an enhanced resolution is especially desired. Additionally multiphoton microscopy requires ultrafast pulses for excitation, so optimization of the pulse duration at the sample is critical for strong signals.AimWe aim to perform enhanced resolution imaging that is robust to scattering using a structured illumination technique while also providing a rapid and easily repeatable means to optimize group delay dispersion (GDD) compensation through to the sample.ApproachSpatial frequency modulation imaging (SPIFI) is used in two domains: the spatial domain (SD) and the wavelength domain (WD). The WD-SPIFI system is an in-line tool enabling GDD optimization that considers all material through to the sample. The SD-SPIFI system follows and enables enhanced resolution imaging.ResultsThe WD-SPIFI dispersion optimization performance is confirmed with independent pulse characterization, enabling rapid optimization of pulses for imaging with the SD-SPIFI system. The SD-SPIFI system demonstrates enhanced resolution imaging without the use of photon counting enabled by signal to noise improvements due to the WD-SPIFI system.ConclusionsImplementing SPIFI in-line in two domains enables full-path dispersion compensation optimization through to the sample for enhanced resolution multiphoton microscopy.
Using the structured illumination, single pixel detection imaging technique SPatIal Frequency modulation Imaging (SPIFI), we demonstrate a cascaded Wavelength Domain and Spatial Domain (WD-SD-SPIFI) system enabling real-time, in-line, second order dispersion compensation optimization for multiphoton imaging. Enhanced resolution is demonstrated by imaging a sub-diffractive 140 nm fluorescent nanodiamond with Two Photon Excitation Fluorescence (2PEF) to measure the Point Spread Function (PSF). With a 1034 nm pulsed laser through a Numerical Aperture (NA) of 0.5, a PSF Full Width at Half Max (FWHM) of 780 nm was measured with minimal post processing analysis that only requires Fast Fourier Transforms (FFTs).
In recent years, new super resolution imaging methods based on the anti-bunching properties of photons emitted by single quantum emitters have emerged. Thus far, these methods have been extremely limited in speed as they rely on very low repetition lasers to match the speed of cameras or use high-speed photon counting at individual points scanned across the surface of the object. Here, we study the use of spatio-temporally modulated illumination light to acquire photon counts from an extended region of the object. Thus, we combine high speed photon detection with extended illumination to enhance the imaging speed of anti-bunching super resolution microscopy.
New spatial domain (SD) frequency modulation imaging (SPIFI) architectures are developed that enable the use of the full numerical aperture of long working distance excitation optics without sacrificing the field-of-view. When multiplexed with wavelength domain (WD) SPIFI, multiple advantages follow. One, the WD-SPIFI signals can be used to optimize the multiphoton SD-SPIFI signals. Two, these new SD-SPIFI architectures enable video rate SPIFI. Finally, applications of these new architectures to advanced manufacturing will be presented.
Imaging of the interior of object with light has long been a challenge for optical imaging. Optical diffraction tomography (ODT) is able to obtain three-dimensional (3D) object information through object rotation. We will discuss harmonic optical tomography (HOT) that exploits a defocused illumination beam for nonlinear optical tomography. We will also discuss our demonstration of coherent ODT with incoherent light emission in a new optical tomography technique called fluorescent diffraction tomography (FDT) and the use of spatial frequency imaging for high speed nonlinear optical microscopy.
Raman microscopy is a powerful mode of label free nonlinear optical microscopy that is hampered by weak cross-sections, leading to slow imaging. We will discuss two advances in coherent Raman microscopy: 1) high speed coherent anti-Stokes Raman scattering microscopy imaging based on spatial frequency modulation imagining (SPIFI) where a structured line focus is used to image is used to image specimens with a single pixel detector. 2) Doppler Raman microscopy that exploits the extremely low timing jitter of modelocked lasers for ultrasensitive Raman spectroscopy and microscopy.
Multidimensional, multicontrast, spatial frequency modulation imaging with built-in femtosecond pulse characterization and dispersion compensation is presented. We present wavelength domain imaging which produces one-dimensional imaging with single element detection, and a multiplexed spatial and wavelength domain system that produces two-dimensional images and is compatible with single element detection imaging.
In recent years, we demonstrated a new approach to super-resolution microscopy based on driving a nonlinear interaction with a Spatial Frequency Modulated Imaging (SPIFI). SPIFI is a line imaging technique that linearly sweeps all the frequencies supported by the band-pass of the objective lens. Here we introduce a new method of unrestricted super-resolution imaging based on driving saturated absorption in the specimen excitation. The saturated absorption drives harmonic distortions of the spatial frequencies used to illuminate the sample. These harmonics manifest themselves as temporal harmonic frequencies allowing for easy detection and separation of the super-resolution information in the far field.
Recently we have demonstrated that spatial frequency modulation imaging can use extended excitation sources in linear and nonlinear image modalities, is compatible with single element detection, and results in enhanced lateral resolution across the excitation beam. In this paper, we will present new methods where the SPIFI platform goes from one-dimensional to two-dimensional imaging while still exhibiting the enhanced resolution across the added dimension. Significantly, we present the physical mechanism responsible for the resolution enhancement for all imaging modalities, we provide computational models that support the physical model for the increased resolution, and finally, present experimental verification of the resolution enhancement.
We present results comparing simultaneous spatial and temporal focusing beams with traditional Gaussian beams during femtosecond laser processing. We establish the importance of accounting for aberrations in refractive focusing elements and present a feedback mechanism for material processing.
The majority of optical super-resolution imaging methods have been developed for thin or transparent biological samples, where the effects of scattering are minimized. Moreover, most of these techniques are based on the manipulation of fluorescent probes, or other molecular real-energy states. Multiphoton spatial frequency modulated imaging (MP-SPIFI) provides a pathway for super-resolving fine structures through multiple scattering lengths by making use of a modulated line focus and nonlinear excitation, and is applicable to both fluorescence and harmonic generation imaging. The technique works by projecting a set of 1D spatial frequencies onto the object, and utilizing the multiphoton interaction to drive harmonics of the spatial frequencies. The result is that an n-photon interaction yields frequency support of nearly 2n beyond the lens NA in the lateral x-dimension, along the line focus. However, the axial resolution of the object is limited in the conventional way by how tightly the line is focused. Here, we improve axial resolution by employing a limited-angle diffraction tomography, where the illumination is rotated in the x-z plane relative to the sample. The set of angular measurements are coherently combined in spatial-frequency space. Using a priori information about the location of each measurement in this kx-kz space greatly enhances the signal-to-noise ratio of the reconstructed object. We expect the method to be a useful way to improve resolution in deep-tissue imaging, or with any sample that exhibits strong scattering.
Through spatial frequency modulated imaging (SPIFI), multimodal, multiphoton microscopy (MPM) benefits from an extended excitation source without compromising the key performance characteristics afforded by point scanning MPM platforms. For example, the introduction of an in-house custom machined mask, which imparts a spatially distinct, temporal amplitude modulation to the extended excitation source, allows one and two-dimensional images to be captured with single element detection. This enables extended source imaging methods to retain a key feature of the point scanning systems; namely, the ability to image within scattering media, at depth.
Further, the range of contrast mechanisms for the extended source techniques presented here are not limited and readily extend to both linear and nonlinear imaging modalities. The SPIFI method developed here enables facile detection of such images with the added benefit of enhanced resolution. Notably, the resolution improvement holds across contrast mechanisms, and is independent of whether the contrast is generated through linear or nonlinear processes. Significantly, phase also comes into play as we present new SPIFI geometries that illustrate the role of phase in strategically controlling the source geometry and/or generating image contrast.
Superresoltuion (SR) microscopy is a valuable tool for biological studies. While the ability to resolve features to 20 nm and below is now routine in transparent specimens, such as cell cultures and cleared specimens, many areas of biological study have not been probed with SR imaging. Further, SR microscopy has thus far been limited primarily to contrast mechanisms that rely on real energy states of a target molecule, with fluorescence being the dominant modality. We recently demonstrated that spatial-frequency modulated imaging (SPIFI) enables superresolved imaging for both multiphoton fluorescence and nonlinear coherent scattering with single-pixel detection. The technique operates by projecting a set of spatial frequencies in one dimension along a spatiotemporally modulated line-focus that illuminates the specimen. Harmonics of the spatial frequencies projected onto the specimen encode spatial information beyond the diffraction limit of the illumination light. This additional information scales with the order of the nonlinearity, but is limited to the single dimension in which the grating sequence is projected. Consequently, 2D images collected with SR SPIFI are diffraction limited in the dimension perpendicular to the line focus. In this work, we extend our technique to a two-dimensional resolution enhancement with an inverse-domain lateral computed tomography. By enabling 2D SR SPIFI while maintaining single-pixel detection, we anticipate more widespread use of this method for imaging in turbid media.
Spatial Frequency Modulated Imaging (SPIFI) with single element detection has previously been demonstrated with a time varying amplitude spatial frequency. This has been shown in a variety of modalities (linear, TPEF, SHG) and also with variations on the base design to provide additional dimensions of information. SPIFI is also capable of providing enhanced resolution images. However, the signal-to-noise is a limiting factor in the quality of the resolution enhancement. We present a microscope design which uses a nematic spatial light modulator to provide a time varying amplitude from an amplitude or phase grating. Twophoton excitation fluorescence images of 10-µm fluorescent polystyrene beads are presented using a phase grating. Additionally, the microscope can provide spatial gratings in polarization which provide an alternative means of imaging in third harmonic generation (THG). THG images are provided using an amplitude and polarization-grating modulation pattern.
MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes — namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.
Simultaneous spatially and temporally focussing (SSTF) of ultrashort pulses allows for an unprecedented control of the intensity distribution of light. It has therefore a great potential for widespread applications ranging from nonlinear microscopy, ophthalmology to micro-machining. SSTF also allows to overcome many bottlenecks of ultrashort pulse micro-machining, especially non-linear effects like filamentation and self-focussing. Here, we describe and demonstrate in detail how SSTF offers an additional degree of freedom for shaping the focal volume. In order to obtain a SSTF beam, the output of an ultrafast laser is usually split by a grating into an array of copies of the original beam, which we refer to as beamlets. The ratio of the beamlet array width to the width of the invidual beamlet is the beam aspect ratio. The focal volume of the SSTF beam can now be tailored transversally by shaping the cross-section of the beamlets and axially by choosing the right beam aspect ratio. We will discuss the requirements of the setup for a successful implementation of this approach: Firstly, the group velocity dispersion and the third order dispersion have to be compensated in order to obtain a high axial confinement. Secondly, the beamlet size and their orientation should not vary too much spectrally. Thirdly, beamlet and SSTF focus should match. We will hence demonstrate how SSTF allows to inscribe tailored three-dimensional structures with fine control over their aspect ratio. We also show how the SSTF focus can be adapted for various glasses and crystals.
We present a laser scanning microscope capable of producing multiple focal volumes. These volumes can be displaced
vertically, to acquire simultaneous images from multiple planes, or superimposed at the same depth but with different
polarization states. We call this last implementation, differential multiphoton laser scanning microscopy (dMPLSM). To
our knowledge, this constitutes the first report of a multifocal microscope with this capacity. The microscope is able to
take images in different modalities, two-photon excited fluorescence, second, and third harmonic generation. In this
work, we demonstrate several capabilities of our microscope: simultaneous acquisition of two and six images from two
focal planes separated by several microns, and a pair of simultaneous images taken at the same focal plane but with two
different polarizations. Some potential applications include following microorganism motion, studies of phase matching
in microscopic environments, studies of blood flow, etc. The microscope is based on a pulsed ultrafast laser. The pulses
are split, manipulated and recombined in an interlaced pattern in order to generate a sequence of pulses with different
divergences, and possibly different polarizations. This pulse train is sent to the objective and focused at different depths.
The signal is recorded using a photoncounting photomultiplier tube. Images from different foci are separated using time
demultiplexing based on a low cost field programmable gate array. The use of a single element detector, instead of a
multi-element (CCD camera), allows for imaging of scattering media. The use of photon counting leads to lower signal
to noise ratio in the images.
Micro-molding can be used for the cost-effective fabrication of elements such as active or passive components in MEMS
devices, hydrophobic surfaces, cell-growth scaffolds or optical components such micro-lens arrays and gratings. This
method is also particularly interesting for examining high-aspect ratio laser-machined structures fabricated in glass
material. Thanks to this technique, surfaces not accessible with common imaging techniques can be observed on their
molded negative structure with very high fidelity. As an illustration, we issue the use of the PDMS molding technique to
analyze the quality of high aspect ratio holes and channels structures. Furthermore, we show preliminary results on the
molding of a novel type of complex structures formed in glass using temporal and spatial beam shaping.
It has recently been demonstrated that diode laser bars can be used to not only optically trap red blood cells in flowing
microfluidic systems but also, stretch, bend, and rotate them. To predict the complex cell behavior at different locations
along a linear trap, 3D optical force characterization is required. The driving force for cells or colloidal particles within
an optical trap is the thermal Brownian force where particle fluctuations can be considered a stochastic process. For
optical force quantification, we combine diode laser bar optical trapping with Gabor digital holography imaging to
perform subpixel resolution measurements of micron-sized particles positions along the laser bar. Here, diffraction
patterns produced by trapped particles illuminated by a He-Ne laser are recorded with a CMOS sensor at 1000 fps where
particle beam position reconstruction is performed using the angular spectrum method and centroid position detection.
3D optical forces are then calculated by three calibration methods: the equipartition theorem, Boltzmann probability
distribution, and power spectral density analysis for each particle in the trap. This simple approach for 3D tracking and
optical control can be implemented on any transmission microscope by adding a laser beam as the illumination source
instead of a white light source.
We report red blood cell (RBC) stretching using a Zeiss Axioplan microscope, modified for phase contrast and optical
trapping using a 808 nm diode laser bar, as a tool to characterize RBC dynamics along a linear optical trap. Phase
contrast offers a convenient method of converting small variations of refractive index into corresponding amplitude
changes, differentially enhancing the contrast near cell edges. We have investigated the behavior of RBCs within both
static and dynamic microfluidic environments with a linear optical stretcher. Studies within static systems allow
characterization of cell interactions with the line optical force field without the complicating forces associated with
hydrodynamics. In flowing, dynamic systems, cells stretch along the optical trap down microfluidic channels and are
eventually released to recover their original shape. We record the dynamic cell response with a CMOS camera at 250 fps
and extract cell contours with sub-pixel accuracy using derivative operators. To quantify cell deformability, we measure
the major and minor axes of individual cells both within and outside of the trap, which also allows measurement of cell
relaxation. In these studies, we observe that cell rotation, stretching, and bending along the linear optical trap, are tightly
coupled to the modulation of optical power and cell speed inside our microfluidic systems.
The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry.
We show that local fields associated both with overall structural features and with unintended defects can be important in
the second-order nonlinear response of metal nanostructures. We first consider noncentrosymmetric T-shaped gold
nanodimers with nanogaps of varying size. The reflection symmetry of the T-shape is broken by a small slant in the
mutual orientations of the horizontal and vertical bars, which makes the sample chiral and gives rise to a different
nonlinear response for left- and right-hand circularly-polarized fundamental light. Measurements of achiral and chiral
second-harmonic signals as well as the circular-difference response exhibit a nontrivial dependence on the gap size. All
results are explained by considering the distribution of the resonant fundamental field in the structure and its interaction
with the surface nonlinearity of the metal. We also prepared arrays of ideally centrosymmetric circular nanodots.
Second- and third-harmonic generation microscopies at normal incidence were used to address polarization-dependent
responses of individual dots. Both signals exhibit large differences between individual dots. This is expected for second-harmonic
generation, which must arise from symmetry-breaking defects. However, similar results for third-harmonic
generation suggest that both nonlinear responses are dominated by strongly localized fields at defects.
We demonstrate a two-photon absorption scanning microscope capable of imaging two focal planes simultaneously. The
23MHz fundamental laser is split in two, one part delayed in time while the other is focused with a deformable mirror to
change its divergence. Both parts are then recombined to form a 46MHz pulse train consisting of two interlaced trains
with different divergences that after the objective are focused at different sample depths. At the detection path, photon
counting techniques allow photons coming from each depth to be separated based on their relative timing with respect to
the 46MHz train. The separation is accomplished using a field-programmable gate array that has been programmed to
switch back and forth between two counters at a rate provided by a master clock generated by the 46MHz pulse train.
The computer that controls the scanners reads and resets the counters before moving to a new pixel. The scheme is
demonstrated for two depths but can be extended to a larger number, the ultimate limit being the fluorescence lifetime.
This technique could also be implemented for second or third harmonic generation microscopy, in this case the ultimate
achievable number of focal planes would be determined by the electronics speed.
The recently developed technique of ultrafast third harmonic generation (THG) micro-spectroscopy is discussed. The approach is easily adapted to a standard laser scanning microscope and allows for two and three photon resonances to be identified in non-fluorescent unlabeled samples. This work provides nonlinear microscopists with a tool for further understanding the contrast and damage mechanisms they will encounter under nonlinear excitation. Here, we use THG micro-spectroscopy to investigate the nonlinear optical properties of hemoglobin over the spectral range of 770 -1000 nm with 100-fs duration, ~1-nJ energy laser pulses. We demonstrate the ability of this approach to distinguish different ligand binding states in physiological solutions of human hemoglobin.
Our newly developed multimodal microscope enables simultaneous collection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) signals. The signals can be generated within different or the same intercellular structures. In comparing two signals, traditional methods of image crosscorrelation analysis using Pearson's coefficient provide a general parameter as to whether the images are similar, however it does not give detailed information about correlation of different structures inside the images. We present here a new technique that employs a pixel by pixel analysis over an entire area or volume that is used to correlate the structures appearing in the images. The result of the analysis reveals structures within the sample that are generated by both nonlinear signals as well as highlighting the structures that are generated by only one of the nonlinear signals. The algorithm provides a means to colocalize different structures revealed by the different nonlinear contrast mechanisms. Structural correlation maps are useful in identifying the origin of structures in one nonlinear contrast mechanism when the origin of structures in another is known. Image analysis has also been exploited for sequences of images taken in time. The intensity fluctuations in time for each pixel reveal regions of intense physiological activity in biological samples. Correlation of time dependent fluctuations from different pixels in the image time series allows construction of the structural map that undergoes similar time behavior or appears out of phase. These structural correlation analysis techniques are demonstrated based on polystyrene beads and cardiomyocytes.
Dynamic imaging of cardiomyocytes was performed with a simultaneous detection second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) microscope. The fast scanning system of ~12 frames/second synchronized with multichannel detection provided the possibility of imaging three dimensional static and two dimensional dynamic structures of cardiomyocytes. The SHG images highlighted the myofibrils of the cardiomyocytes while THG images revealed the locations of mitochondria. Dynamic data showed that during imaging, chaotic nanocontractions took place inside the cardiomyocytes. The time series of THG images reveled large intensity fluctuations "flickering" in the regions of mitochondria. The flickering in THG correlated with the flickering in MPF. Addition of the uncoupler FCCP inhibited flickering in THG and MPF, and also inhibited nanocontractions. The simultaneous imaging with SHG, THG and MPF proved to be a very powerful microscopy tool for investigation of interactions of different organelles inside a cell.
Simultaneous detection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence with ultrafast laser pulses from a Nd:Glass laser was used to image isolated adult rat cardiomyocytes. The simultaneous detection enabled visualization of different organelles of cardiomyocytes, based on the different contrast mechanisms. It was found that SHG signal depicted characteristic patterns of sarcomeres in a myofilament lattice. The regular pattern of the THG signal, which was anticorrelated with the SHG signal, suggested that the third harmonic is generated within mitochondria. By labeling the cardiomyocytes with the mitochondrial dye tetramethylrhodamine methyl ester (TMRM), comparisons could be made between the TMRM fluorescence, THG, and SHG images. The TMRM fluorescence had significant correlation with THG signal confirming that part of the THG signal originates from mitochondria.
We show that a simple plane wave analysis can be used even under tight focusing conditions to predict the dependence of third-harmonic generation on the polarization state of the incident beam. Exploiting this fact, we then show that circularly polarized beams may be used to spatially characterize the beam focus and temporally characterize ultrashort pulses in high numerical aperture systems by experimentally demonstrating, for the first time, novel collinear, background-free, third-harmonic intensity autocorrelations in time and space in a high numerical aperture microscope.
Our ability to study the complex interactions between macromolecules within living cells has been greatly enhanced by the development of biophysical techniques such as fluorescence correlation spectroscopy (FCS) and multiphoton microscopy. One area of great interest to cell biologists is the molecular mechanism that governs cellular adhesion. Direct physical and chemical measurements on intact living cells will be important for obtaining a better understanding of how cells control their adhesive properties at the molecular level in order to control tissue development, maintain tissue integrity, and regulate cellular migration. Cells dynamically regulate the formation and disassembly of macromolecules in focal adhesions within the basal membrane so it would be advantageous to be able to measure such phenomena in situ. By combining two-photon microscopy imaging of living cells expressing fusion proteins of adhesion molecules and mutants of the green fluorescent protein, and image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) analysis, we have been able to perform direct studies of the molecular transport and clustering. We report on the characterization of flow, diffusion, aggregation, and co-localization of adhesion macromolecules/fluorescent protein constructs in living cells by two-photon ICS and ICCS experiments at 37 degree(s)C.
We report on the application of femtosecond x-ray scattering to experimental studies of the photo-induced, structural phaser transition in VO2. The transition between the two crystalline phases of the material occurs, for sufficiently intense excitation, within 500 fs.
Advances in laser-scanning microscopy and the advent of confocal microscopy permitted the development of image correlation spectroscopy (ICS). ICS is an imaging analog of fluorescence correlation spectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. The ICS method entails spatial autocorrelation analysis of fluorescence fluctuations within an image sampled from an area of the sample as well as temporal autocorrelation analysis of fluorescence fluctuations through a time series of images. Together, the spatial/temporal autocorrelation analysis enables measurement of fluorophore concentration, aggregation state and transport properties. ICS was first implemented on a confocal laser-scanning microscope (CLSM) using single photon excitation. More recently we have extended the method for two-photon ICS as well as image cross-correlation spectroscopy (ICCS). ICCS allows measurement of co-localization of non-identical molecules labeled with fluorophores of different emission wavelengths. We present a variety of applications of the ICS and ICCS methods in cellular systems. We will discuss the measurement of the transport and clustering properties of membrane receptors by single photon ICS and two-photon ICCS. As well, we will describe how spatial ICS may be used to quantify the distribution of fluorescently labeled dendritic spines in neurons.
We use third harmonic generation (THG) microscopy to image waveguides and single-shot structural modifications produced in bulk glass using femtosecond laser pulses. THG microscopy reveals the internal structure of waveguides written with a femtosecond laser oscillator, and gives a three-dimensional view of the complicated morphology of the structural changes produced with single, above-threshold femtosecond pulses. We find that THG microscopy is as sensitive to refractive index change as differential interference contrast microscopy, while also offering the three-dimensional sectioning capabilities of a nonlinear microscopy technique. It is now possible to micromachine three-dimensional optical devices and to image these structures in three dimensions, all with a single femtosecond laser oscillator.
Recent advances in femtosecond laser plasma x-rays sources have resulted in several experiments to explore the dynamics of physical and chemical processes on the femtosecond time scale. We present our most recent progresses on the development of an intense broadband x-ray source in the multi-keV range, for application to time-resolved EXAFFS experiments. Experiments have been realized with two different CPA laser systems having different pulse durations and characteristics. X-ray emissions in the 5KeV range generated form solid targets with the INRS Nd:Glass laser and the UCSD Ti:Sapphire laser have been characterized through high resolution and time resolved x-ray spectroscopy. The application of this source to time resolved EXAFS measurements with a sub-picosecond time resolution will also be discussed.
Temporally decorrelated multifocal arrays eliminate the spatial interference of adjacent foci that occurs in multifocal arrays and allow multifocal imaging with the diffraction-limited resolution of a single focus even with closely spaced foci. To date, we have produced 1-D temporally decorrelated multifocal arrays using low throughput etalons, which limited the efficiency of the arrays. In this work, we demonstrate a 2-D high-efficiency cascaded-beamsplitter array for producing the beamlets. Using the cascaded beamsplitters, we split the 800-nm light from an ultrashort-pulsed Ti:Al2O3 laser into a 2-D array of beamlets in which the pulses arrive at a plane perpendicular to the propagation direction at different times. We then overlap the collimated beams with slightly different angles at the entrance aperture to a 1.25 NA oil-immersion objective and produce 2-dimensional array of foci that are temporally decorrelated. This allows multiphoton imaging with diffraction-limited focusing, even for pulses as short as 20-fs. This new method of imaging will make it possible to completely overlap the foci and eliminate the need for scanning. This makes highly efficient use of the power available from typical ultrafast lasers, increasing the frame rate in multiphoton microscopy and the throughput in micromachining applications.
Image correlation spectroscopy (ICS) was developed as an imaging analog of fluorescence correlation spectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. Ics was first implemented on a confocal laser scanning microscope (CLSM) and entails spatial autocorrelation analysis of fluorescence fluctuations within an image sampled from an area of the cell. Spatial and temporal autocorrelation analysis of image time series enables measurement of both the molecular dynamics and aggregation state of the imaged molecules. The parallel nature inherent in the collection of multiple fluctuations in an imaging scheme improves the signal to noise ratio of the correlation analysis, which enhances dynamic measurements for slowly moving species in membrane systems. We outline our development of two-photon ICS and describe recent applications of the method for measurements of flow, diffusion and aggregation behavior of green fluorescent protein/integrin receptor constructs in living cells. We also describe the use of two-photon excitation to perform two-color image cross-correlation spectroscopy to measure the dynamics and colocalization of non-identical species labeled with different fluorophores.
In this paper we study the spatial distribution and wavefront characteristics of third harmonic generation in relation to some material and interface conditions over the focal region of the fundamental beam. We investigate, mostly from an experimental point of view, the implications the physics of the THG generation process has in situations where THG may be employed for 3D imaging. Due to the non- linear character of the THG generation process it is inherently suitable for this application. For the first time images of the distribution of the THG radiation, as the interface is moved through focus, are shown. Experiments on closely spaced interfaces or bilayers confirm unambiguously the correctness of the vector model for THG generation (Ward et al. 1969) in uniform media. In view of these and other data the image formation, especially for biological objects, with THG radiation will be discussed.
The interaction of macromolecules in space and time are known to be important for the regulation of many biochemical reactions. Image correlation spectroscopy (ICS) was recently introduced as an imaging analog of fluorescence correlation spectroscopy optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. We present two novel developments of dynamic ICS that will greatly enhance our abilities to measure molecular interactions as a function of time and space in living cells. We illustrate the use of a rapid scan two-photon microscope system to collect image series at high time resolution (30 frames/s) for dynamic ICS analysis. Secondly, we demonstrate the implementation of two-color image cross-correlation spectroscopy (ICCS) with a CLSM using multiple wavelength excitation, and with two-photon excitation of samples containing two different fluorescent species. Cross-correlation analysis allows the degree of co- localization of two different fluorophores to be measured directly. By performing two-color ICCS, we can monitor the interactions of non-identical labeled macromolecules as a function of time and space. We describe the experimental setup for both methods and illustrate the application for measurements of the diffusion coefficients of singly and doubly labeled fluorescent microspheres in aqueous solutions.
As ultrafast multiphoton microscopes become more useful for biological imaging, a major challenge for researchers is to determine the exposure conditions that provide the best combination of image resolution, contrast and specimen viability. To do this requires an accurate understanding of the spatial and temporal evolution of ultrashort pulses at the focus produced by a microscope objective. The objective itself, however, can significantly alter the pulses. Some effects, such as the broadening of pulses due to group delay dispersion in materials along the path, are understood and partial compensation for them can be made. Other effects, such as radial variations in the propagation time and variations in the pulse width, are less well understood. In this work, we investigate the radially dependent propagation and focusing of ultrashort pulses through a Zeiss CP- Achromat 100X, 1.25 NA, infinity-corrected, oil immersion microscope objective. We also extend to this high numerical aperture case the technique of collinear type II second harmonic generation frequency-resolved optical grating which has previously been used to measure the temporal intensity and phase of ultrashort pulses at the focus of air objectives with lower numerical aperture.
High intensity chirped pulses can be used for probing microscopic chemical environments through the use of a particular choice of dye, for instance SNAFL2. The basis for this technique is that the excited state populations can be manipulated through control over the temporal order of the excitation frequencies in the excitation pulse -- i.e. chirp - - with the outcoming fluorescence as the reporting parameter. A chirp dependent fluorescence response can also be observed in larger molecular systems with more degrees of freedom like for instance green fluorescent proteins. In preparation for application of the technique to microscopy we use a facility permitting observation of this phenomenon in various dyes with high sensitivity. High power, 30 fs pulses from an OPA, tunable from 400 nm to 1.5 micron are used. These pulses with a repetition rate of 1 kHz are sufficiently intense that a relatively large sample region can be excited to saturation from which then a sub-region with uniform excitation conditions can be selected for signal collection.
Recently a novel imaging technique based on third-harmonic generation (THG) was introduced. This technique relies on a third-order non-linear interaction to generate a coherent signal response on the third-harmonic frequency with respect to the fundamental input radiation. Here we report on the input NA dependence of the THG signal and examine the resulting imaging characteristics of this novel technique in terms of resolution and contrast generation. We'll demonstrate the potential of the technique through a number of imaging examples, with special emphasis on in vivo applications. The latter illustrates the non-invasive character of the technique.
This contribution discusses some biological applications of ultrashort laser pulses. Some examples are given of recently developed techniques that exploit the special features offered by ultrashort laser pulses: real time two-photon microscopy with multipoint excitation, fluorescence lifetime measurement by double pulse saturation excitation and pH-sensing by multiphoton quantum control.
Over the last few years a number of microscopical techniques have been developed that take advantage of ultrashort optical pulses. All these techniques rely on temporal pulse integrity at the focal point of a high-numerical aperture (NA) focusing system. We have investigated the dispersion induced broadening for pulses on the optical axis, using the two-photon absorption autocorrelation (TPAA) technique. We demonstrate that the induced broadening can be pre- compensated for by a properly designed dispersion pre- compensation unit for pulses as short as 15 femtosecond. Another source of pulse broadening in high-NA focusing systems is due to radial variations in the dispersion over the pupil of the objective. This may cause differences in the group delay between on-axis and outer ray wave packets, as well as differences in the broadening of the wave packets themselves. In this paper we present experimental results on the measurement of these radial variations in the dispersion characteristics over the aperture of high-NA microscope objectives, using a slightly modified TPAA technique.
Because of low operating speed and excessive collateral damage, lasers have not succeeded in replacing conventional tools in many surgical and dental applications. Recent developments now allow the new generation of amplified ultrashort pulse lasers to operate at high repetition rates and high single pulse energies. A Titanium:sapphire Chirped Pulse Regenerative Amplifier system operating at 1 KHz and 50 fs pulse duration, was used to demonstrate ultrashort pulse ablation of hard and soft tissue. Maximum ablation rates for enamel and dentin were approximately 0.650 micrometers /pulse and 1.2 micrometers /pulse respectively. Temperature measurements at both front and rear surface of a 1 mm dentin and enamel slices showed minimal increases. Scanning electron micrographs clearly show that little thermal damage is generate by the laser system. If an effective delivery system is developed, ultrashort pulse system may offer a viable alternative as a safe, low noise dental tool.
Pulse broadening of ultrashort optical pulses, as short as 15 femtoseconds, due to the propagation through high- numerical-aperture microscope objectives can be pre- compensated to ensure temporal pulse integrity at the focal point. The predictions from dispersive ray-tracing calculations show excellent agreement with the experimental results from two-photon absorption autocorrelation for the Zeiss CP-Achromat 100X/1,25 oil microscope objective. From this, general predictions can be inferred for dispersion in most types of microscope objectives. Key element to the work is a carefully designed dispersion pre- compensation configuration, which minimizes pulse broadening due to residual third order dispersion. The capability to focus these ultrashort pulses with control of the pulse definition at the focal point is important for two-photon absorption and time-resolved microscopy.
We summarize recent progress aimed at observing biochemical and biological dynamics using confocal microscopy with 3D spatial resolution down to a few hundred nm and temporal resolution to 15 fs. We also review recent control of population dynamics using tailored ultrafast pulses, i.e. quantum control. Progress is described for i) feedback control, ii) multiphoton control, and iii) molecular (pi) pulse. Finally, using ultrafast light pulses, we combine confocal and quantum control techniques to produce a new way to measure the microscopy chemical environment, int his case pH, potentially with a spatial resolution of a few hundred nanometers.
Optical pump, x-ray diffraction probe measurements have been used to study the lattice dynamics of single crystals with picosecond-milliangstrom resolution by employing a table- top, laser-driven x-ray source. The x-ray source, consisting of an approximately 30 fs, 75 mJ/pulse, 20 Hz repetition rate, terawatt laser system and a moving Cu wire target assembly, generates approximately 5 X 1010 photons (4π steradians s)-1 of Cu Kα radiation. Lattice spacing changes of as small as 1 X 10-3 Å in a few picoseconds have been detected, utilizing Bragg diffraction from GaAs single crystals. Enhancement of the diffraction intensity associated with degradation of the crystals during and after the laser irradiation has been observed, likely due to a transition from dynamic to kinematic diffraction.
It is shown that nanosecond to picosecond fluorescence relaxation phenomena can be accessed for imaging after double pulse saturation excitation. This new technique has been introduced before as fluorescence lifetime imaging (DPFLIm) (Mueller et al, 1995). An OPA laser system generating ultra short, widely tunable, high power optical pulses provides the means for the selective excitation of specific fluorophores at sufficient excitation levels to obtain the necessary (partial) saturation of the optical transition. A key element in the developed method is that the correct determination of fluorescence relaxation times does allow for non-uniform saturation conditions over the observation area. This is true for the validation demonstration experiments reported here as well as for imaging applications at a later stage. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique. As only integrated signal detection is required no fast electronics are needed, making the technique suitable for fluorescence lifetime imaging in confocal microscopy, especially when used in combination with bilateral scanning and cooled CCD detection.
Regenerative pulse shaping is used to overcome gain narrowing during ultrashort pulse amplification. We have demonstrated multiple spectral filters for broadening the amplified spectrum. We have produced amplified pulses with an energy of approximately 5 mJ and bandwidths of approximately 100 nm, or nearly 3 times wider than the gain narrowing limit of Ti:sapphire.
We show that a pair of grisms (transmission gratings written onto prisms) exhibits second- and third-order dispersion opposite to that of an optical fiber. Using a fiber stretcher and a grism- pair compressor, we demonstrate chirped-pulse amplification and transform-limited compression of 135-fs pulses. This scheme can be used to produce 50-fs, microjoule pulses in a compact and simple system.
We propose a method for producing sub-femtosecond VUV pulses by compressing the high order harmonic radiation emitted from a nonlinear medium driven by an intense, ultrafast laser pulse. We present single atom calculations of the harmonic amplitudes and phases as well as simulations of a VUV compressor. Single harmonics from a 27 fs driving pulse can be compressed to less than 2 fs duration, and several adjacent harmonics with similar phase structure can be combined within the same compressor to produce an attosecond pulse.
Techniques for the production of multiterawatt, sub-20-fs, optical pulses via chirped pulse amplification are discussed. Regenerative pulse shaping is used to control gain narrowing during amplification and an optimized, quintic-phase-limited, dispersion compensation scheme is used to control higher order phase distortions over a bandwidth of approximately 100 nm. Transform-limited, 18-fs pulses of 4.4-TW peak power have been produced in a Ti:sapphire- based, chirped pulse amplification system at a repetition rate of 50 Hz. Extensions to shorter durations and peak powers approaching 100 TW are also described.
Single-shot laser induced breakdown, in wide band gap materials such as SiO2 and MgF2, has been studied over almost 5 orders of magnitude in duration from 150 fs to 7 ns. A Ti:sapphire chirped pulse amplification system was used in this experiement, so the pulse duration could be continuously adjusted without changing any other parameters. The damage threshold was detected by looking at the plasma formation and the change of material transmission coefficient. The avalanche mechanism was found to dominate over the entire pulse-width range even for 150 fs pulses where we would expect multi-photon processes to take over. A strong departure from the conventional fluence threshold scaling law is observed for pulses shorter than 10 ps, where beyond this point the fluence threshold increases. Also, it is observed for the first time that for short pulses the damage threshold becomes very accurate and less statistical than that for longer pulses.
We describe a simple method for compensating large amounts of second- and third-order material dispersion, and we present two compact and robust stretcher-compressor systems for microjoule-and millijoule-level chirped pulse-amplification.
Microjoule pulse energies are achieved from a single stage upconversion fiber amplifier for the first time in this demonstration of chirped pulse amplification using a multimode Tm:ZBLAN fiber. A Ti:sapphire laser system provides the seed pulse for the upconversion fiber amplifier which produces subpicosecond pulse trains with energies as great as 16 (mu) J at repetition rate of 4.4 kHz. The compressed pulse peak power is more than 1 MW, and the pulse is characterized both temporally and spatially.
Efficient second harmonic conversion (70 - 80%) using Type II and Type I crystals is demonstrated with 400-fs, 1.053-micrometers laser pulses at intensities up to several hundreds of GW/cm2. The experimental results generally agree with the predictions of the code MIXER. For the Type II predelay scheme, evidence is obtained of pulse shortening down to approximately 100 fs.
Compact all-solid state laser sources are developed for femtosecond pulse generation tunable around 193 nm utilizing high peak power Ti:sapphire oscillator/amplifier systems and phase matched sequential sum frequency conversion in three (beta) -barium-borate (BBO) crystals arranged in different schemes. Using thin crystals and a delay line for optimization of the temporal overlap of the interacting pulses in the last conversion stage 190 fs optical pulses with pulse energies of more than 2 (mu) J at 193 nm at 20 Hz repetition rate and 170 fs pulses with pulse energies of up to 4 (mu) J at 200 nm (0.8 (mu) J at 193 nm) for 1 kHz repetition rate are produced with excellent spectral, temporal, and spatial stability.
We discuss the use and importance of phase-sensitive pulse diagnostics for the optimization of ultrafast lasers and for implementing quantum control. Knowledge of the exact phase structure is needed in order to determine the phase and amplitude transformations that are required to produce a desired tailored pulse. A special case of pulse shaping is the phase and amplitude compensation of amplified pulses to yield transform-limited pulses. Full intensity and phase measurements of a kHz amplified femtosecond laser system are presented. These measurements provide considerable information about the phase structure which is acquired during amplification and the changes needed to minimize the pulse width. In addition, a versatile light pulse synthesizer which is currently under development is described for producing amplified femtosecond pulses tailored in phase and amplitude.
The combination of broadly tunable solid-state lasers and the technique of chirped pulse amplification has made it possible to produce energetic, femtosecond pulses capable of focused intensities > 1018 W/cm2. In addition, these lasers, which have multi-terawatt peak power capability, can be designed in very compact and robust configurations. Further, the average power capability of solid-state chirped pulse amplification sources can now exceed 1 W, as compared to the 10 mW average power limitation of previous femtosecond lasers.
We have developed a novel series of femtosecond amplifiers based on a combination of materials including Ti:Al2O3, Cr:LiSrAlF6, alexandrite, and Nd:glass with the intent of increasing the average power capability of high intensity laser sources.
We describe our work on the amplification of short pulses in tunable solid state materials; specifically alexandrite and Ti:sapphire.
Our goal is to amplify femtosecond range pulses to the joule level in a table top size laser. We will describe our results which show
that such a laser is now feasible.
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