We have developed a new shot noise limited light source for Coherent Raman microscopy and other nonlinear microscopy modalities such as SHG and multiphoton fluorescence. Compared to the market leading SRS light source it achieves 100x faster tuning for random wavelength access including power control and dispersion compensation. Further switching from 80 to 40MHz repetition rate with subharmonic modulation frequency of 20MHz reduces the pixel dwell time in theory by a factor of 8 for the same signal to noise ratio. We will present a comparison to the prevailing SRS light source along with fingerprint images of relevant biological samples.
We present a ready to use label free coherent Raman microscope (CARS/SRS) to perform instantaneous virtual histology and chemical imaging for scientific applications. The system is based on a picosecond laser source that can address two vibrational wavenumbers simultaneously across the entire vibrational spectrum (400cm-1 – 4000cm-1). The laser source is coupled to a ready to use beam scanning microscope enabling SRS, CARS and multiphoton microscopy. Targeting the CH2 and CH3 chemical bonds, we demonstrate instantaneous stimulated Raman histology in the brain and the GI. This new, ready to use, SRS/CARS and multiphoton microscope enables SRS detection with exquisite sensitivity (shot noise limited) and ratio-metric measurement by targeting simultaneously two vibrational frequencies that can be tuned anywhere across the vibrational spectrum.
We present a shot noise limited, three-color SRS implementation to address two molecular vibrations simultaneously. The system allows fast, high quality stimulated Raman histology as well as background-free SRS imaging. It is ready to be tested in hospitals for its viability and image quality in comparison to classical rapid histology.
It is based on a mode-locked fs-laser from which 2 narrow-band Stokes laser beams are extracted and subsequently modulated at 13 and 20MHz. The center part of the fs-laser is frequency doubled to pump a picosecond optical parametric oscillator, which can be tuned from 500 to 5000 cm−1.
Coherent Raman and infrared microscopy have a huge potential in life science applications since both technologies probe the specific vibrational properties of samples and thus can identify and differentiate molecules. Easy to use, rugged and computer controlled light sources are essential for the translation into life science and medicine. We will present our latest developments on light sources dedicated to these applications.
We present a MgO:PPLN- and AgGaSe2-based, picosecond OPG/OPA scheme, enabling the generation of mid-to-long-infrared continuously tunable, microjoule energy pulses with a narrow spectral bandwidth of sub-8 cm-1 over the full tuning range. The approach combines a 1-µm-pumped, double-pass OPG setup with a 2-µm-pumped parametric booster amplifier stage. As pump laser is applied a CPA-free Ho:YLF laser delivering 32 µJ at 2051 nm wavelength and 20 kHz repetition rate. The OPG/OPA system specifications of µJ-level pulse energies in combination with narrowband and widely tunable, mid-to-long-IR spectra enable numerous possibilities for mid-IR spectroscopy and wavelength-specific mid-IR material processing.
We employed an HgGa2S4 nonlinear crystal to achieve the highest average power from a high-repetition rate mid-infrared femtosecond optical parametric oscillator (OPO), synchronously pumped near 1 μm. The continuous tuning of the nonresonated idler extended from below 4.4 to beyond 12 μm. The OPO was pumped by a 6.5-W, 1.035-μm, 81-MHz, 220-fs Yb-fiber laser. The idler output reached an average power of over 355 mW at 5.2 μm (at a signal power of > 2 W at 1.25 μm). The peak quantum efficiency of the OPO approached 40%. The signal output was diffraction limited with M2 < 1.1. No crystal damage was observed at average pump intensities as high as 60 kW/cm2.
We employed mercury thiogallate (HgGa2S4 or HGS) to achieve the highest average output power from a 1 µm synchronously pumped optical parametric oscillator (SPOPO) generating femtosecond idler pulses continuously tunable in the mid-IR. HGS is one of the most promising wide band-gap non-oxide nonlinear crystals without two-photon absorption (TPA) losses at such pump wavelengths. The SPOPO was pumped by an 80 MHz mode-locked Yb-fiber laser with 6.5 W of average power at 1.035 µm and a pulse duration of 220 fs. Continuous tuning of the non-resonated idler was achieved from 4.4 to beyond 12 µm with an average output power of over 350 mW at 5.2 µm (>2 W at 1.25 µm for the signal) and tens of mW at the edges of the idler tuning range (hundreds of mW for the signal). The quantum efficiency of the device exceeded 20 % up to 8 µm (> 30% in the maximum). The output of the OPO was diffraction limited with a beam quality factor (M²) below 1.1. No crystal damage was observed at average power densities as high as 60 kW / cm² although using lower power densities was necessary to achieve the best possible beam quality. The only other crystals that were successfully employed in lower power SPOPOs in the past, CdSiP2 (CSP) and OPGaP, lack either the tuning range due to limited mid-IR transparency or the capability for continuous tuning.
We present an efficient coherent source widely tunable in the mid-infrared spectral range consisting of a commercial picosecond Yb-fiber laser operating at 80 MHz repetition rate, a synchronously-pumped OPO (SPOPO) and differencefrequency generation (DFG) in AgGaSe2. With an average input pump power of 7.8 W at 1032 nm and at 80 MHz, the SPOPO outputs are tunable from 1380 to 1980 nm (Signal) and from 2.1 to ~4 μm (Idler) with pulse durations between 2.1 and 2.6 ps over the entire tuning range. After temporally overlapping Signal and Idler through a delay line, the two beams are spatially recombined with a dichroic mirror (reflecting for the Signal in s-polarization and transmitting for the Idler in p-polarization), and focused by a 150 mm CaF2 lens to a common focus. For DFG we employ an AR-coated 10- mm thick AgGaSe2 nonlinear crystal cut for type-I interaction at θ =52°. The generated mid-infrared picosecond pulses are continuously tunable between 5 and 18 μm with average power up to 130 mW at 6 μm and more than 1 mW at 18 μm. Their spectra and autocorrelation traces are measured up to 15 μm and 11 μm, respectively, and indicate that the input spectral bandwidth and pulse duration are maintained to a great extent in the nonlinear frequency conversion processes. The pulse duration slightly decreases from 2.1 to 1.9 ps at 6.7 μm while the spectral bandwidth supports ~1.5 ps (~10 cm-1)durations across the entire mid-infrared tuning range. For the first time narrow-band mid-infrared pulses with energy exceeding 1 nJ are generated at such high repetition rates.
In narrow-bandwidth coherent Raman scattering (CRS) microscopy, efficient signal generation is accomplished with two-color laser sources providing synchronized picosecond pulses whose frequency difference and spectral widths match the molecular Raman frequency and bandwidth, respectively. With vibrational bandwidths of typically 10 cm-1, the optimum laser pulse durations thus correspond to about 2 ps. Here, we present a new light source consisting of an amplified Yb-fiber oscillator providing 2-ps pulses at 1031 nm and a synchronously green-pumped optical parametric oscillator (OPO). The OPO slightly shortens the pulses to < 2 ps while maintaining a bandwidth of 10 cm-1. Output power levels of 1 W in both the 1031-nm and the OPO-branch with continuously tunable frequency differences between the two beams covering a broad range from 700 to 4500 cm-1 are achieved. In addition to CARS microscopy, this light source allows for SRS imaging via an integrated electro-optical modulation of the 1031-nm beam at 20 MHz with a depth of >95%, locked to the laser repetition rate of 80 MHz. The OPO noise at 20 MHz was found to be only 60% above the combined detector and laser noise of a conventional Nd:YVO pump source. This represents a significant reduction in laser noise when compared to other fiber-based laser sources previously proposed for SRS microscopy. When SRS imaging with this new light is compared with a Nd:YVO pumped OPO (delivering 7 ps and 5 ps pulses, respectively), a 5- to 6-fold increase in SRS signal strength and signal-to-noise ratio has been achieved. Video-rate SRS and the capability of multi-spectral SRS imaging are demonstrated.
We present an efficient coherent source widely tunable in the mid-infrared (mid-IR) spectral range consisting of a novel femtosecond Yb-fiber laser operating at ~50 MHz repetition rate, a synchronously-pumped OPO (SPOPO) and difference-frequency generation (DFG) in AgGaSe2. With an average input power of 5 W for ~260 fs pump pulses at 1034 nm, the SPOPO outputs are tunable from ~1710 to 1950 nm (signal) and from 2200 to 2600 nm (idler) with pulse durations between 200 and 250 fs over the entire tuning range. After temporally overlapping signal and idler through a delay line, the two beams are spatially recombined with a dichroic mirror and focused to a beam diameter of ~75 μm. For DFG we employ an uncoated 2-mm-thick AgGaSe2 nonlinear crystal cut for type-I interaction at θ=57°. The generated femtosecond mid-IR pulses are continuously tunable between 5 and 17 μm with average power of up to 69 mW at 6 μm and more than 1 mW at 17 μm. Their spectra and autocorrelation traces are measured up to 12 μm and 8 μm, respectively, and indicate that the input spectral bandwidth and pulse duration are maintained to a great extent in the nonlinear frequency conversion processes. The DFG pulse width measured at 7.2 μm amounts to ~300 fs (FWHM). The measured spectral bandwidth supports ~150 fs Gaussian pulse durations across the entire DFG tuning range. For the first time mid-IR pulses with energy exceeding 1 nJ are generated at such high repetition rates.
We present a new Optical Parametric Oscillator (OPO) based on collinear, quasi phase-matched interaction in a
periodically poled crystal (PP-crystal) with an integrated extra-cavity prism compressor delivering Signal pulses with
durations as low as 30 fs around 1150 nm center wavelength. The design matching between the Ti:Sapphire pump laser
(Coherent Micra-10 or MiraV10), the PP-crystal and the intra-cavity dispersion compensation of the OPO enables stable
Signal emission covering a spectral region from 1050 nm to 1250 nm with pulse energies exceeding 4.5nJ. Tunability of
the Signal pulses between 1130 nm and 1200 nm is given at reduced bandwidth.
Recent development of optically-pumped semiconductor laser (OPSL) technology provide a Ti:sapphire pump source
reducing cost and complexity while maintaining a high standard of performance and reliability. In this paper we report
on the performance of a compact (930 x 330 x 170 mm³), cavity-dumped ultrafast Ti:Sapphire laser oscillator pumped by
an OPSL and using negative dispersion mirrors. The system generates broadband pulses of more than 45nJ pulse energy
from single shot to 2 MHz repetition rate. At higher repetition rates the pulse energy decreases as it is typical for cavity
dumped laser systems, but with 10 nJ at 27 MHz (division ratio of 1:2) the pulse energy is at the level of the Mantis in
standard configuration (8 nJ). FWHM spectrum of the Mantis is specified >70nm and bandwidth over 100nm could be
achieved by fine adjustment of the dispersion with intracavity wedges. Compressed pulse duration down to 12.5fs was
measured. The system is based on the standard Coherent Mantis laser with a cavity dumper extension, making the
footprint only 350 mm longer. It can be user configured either as standard oscillator or as a cavity dumped oscillator.
We will present a new flexible laser source for multimodal Multiphoton excitation microscopy including CARS. It
consists of a tuneable femtosecond-Ti:Sapphire laser and an optical parametric oscillator (OPO). The new OPO-design
allows for high flexibility in pump- and output wavelengths giving rise to for instance image EGFP with the Ti:Sapphire
and tdRFP with the OPO simultaneously. This is presented on living mouse brain tissue.
The minimum energy difference between Ti:Sapphire and
OPO-wavelengths achievable is 2500cm-1. Thus CARS
imaging of lipids is possible. Due to synchronous pumping of the OPO the pump- and OPO pulses are intrinsically
locked in time to each other thus they can be brought to perfect overlap of pump and stokes pulses. Uncaging
multiphoton microscopy is also possible with this system due to the low minimum OPO pump wavelength of 730nm.
We demonstrate heterodyne detection of CARS signals using a cascaded phase-preserving chain to generate the CARS input wavelengths and a coherent local oscillator. The heterodyne amplification by the local oscillator reveals a window for shot noise limited detection before the signal-to-noise is limited by amplitude fluctuations. We demonstrate an improvement in sensitivity by more than 3 orders of magnitude for detection using a photodiode. This will enable CARS microscopy to reveal concentrations below the current mMolar range.
A new, broadly tuneable synchronously pumped picosecond optical parametric oscillator (OPO) for Coherent anti-Stokes
Raman Scattering (CARS) microscopy is presented. It is based on a non-critically phase-matched LBO crystal, pumped
by the second harmonic (532 nm) of a mode-locked Nd:Vanadate laser.
The tuning range covers 680 nm to 990 nm (Signal beam) and 1150 nm to 2450 nm (Idler beam), thus completely
substituting picosecond - Ti:Sapphire lasers. By using the Signal and Idler as pump and Stokes beams for CARS
microscopy, this translates into a vibrational frequency range of ~1350 - >10.000 cm-1.
Both beams are extracted from the same cavity mirror and therefore propagate collinearly. Due to the mechanism of
their generation, Signal and Idler are optically synchronized, and thus, perfectly overlap in space and in time with no
jitter.
The 5 ps pulses generated are close to transform limited and of excellent beam quality (M2 < 1,1) and show a high
pointing stability. The output power for Signal and Idler is about 2 W @ 4 W pump power leading to 50% overall
conversion efficiency.
The perfect spatial and temporal overlap, stable operation, and broad tuneability makes the described OPO an ideal and
nearly hands-free laser source for CARS microscopy. The longer operational wavelength range results in higher
penetration depths and lower sample photodamage than previously reported systems. Thus, our CARS source is
optimized to image highly heterogeneous tissue samples, as will be shown in several applications.
The latest methods for further sensitivity improvements will be presented.
Here we present a multiphoton excitation microscopy setup extending the excitation wavelengths far beyond one micron.
A synchronously pumped fs-OPO (OPO PP-Automatic, APE) pumped by a fs-Ti:Sapphire oscillator is used as the light
source. The biological relevant wavelength range from <1050 to >1350 nm can be covered with a fixed pump frequency
and a single optics set through hands free, automated tuning. Together with the Ti:Sapphire pump laser (Coherent
Chameleon) excitation wavelengths from 680 to 1600nm are achieved.
Two separate scanners (LaVision BioTec) are optimized for Ti:Sapphire and OPO wavelength ranges respectively
including dispersion compensation for maintaining the short pulses at the sample site. An overall transmission of 30-38%
up to 1400 nm was verified.
Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of
the penetration depths, strongly reduced photo damage, a 30fold increased excitation efficiency of red fluorescent dyes,
including RFP and Cy5.5. Excitation at 1100 nm further leads to a 4fold decrease in autofluorescence, resulting in a
significantly improved signal-to-noise ratio. The resolution is slightly reduced in comparison to Ti:Sapphire excitation,
which corresponds well to the longer excitation wavelength used. Phototoxicity and photobleaching is reduced by 80-
95%.
An OPO pump wavelength around 800nm opens up the possibility to use the Ti:Sapphire laser to pump the OPO and to
excite the sample simultaneously giving the opportunity to excite dyes such as GFP with the pump laser and red shifted
fluorophores (for instance RFP) with the OPO at the same time.
Here we present the first results of a new multi photon excitation microscopy setup which extends the excitation
wavelengths far beyond one micron. A synchronously pumped femtosecond-OPO (OPO PP-Automatic, APE) pumped
by a femtosecond-Ti:Sapphire oscillator is used as the light source. Up to 500nm tuning can be achieved in the NIR (for
instance 1100-1600nm) with fixed pump frequency and a single optics set. Automated tuning makes it an ideal tool for
MPE-, SHG- and THG microscopy, which is demonstrated in combination with an optimized scanner / microscope /
detection system. Together with the Ti:Sapphire pump laser (Coherent Chameleon) excitation wavelengths from 700 to
1600nm are achieved.
A single-beam scanner (LaVision BioTec) was optimized for Ti:Sapphire and OPO wavelength ranges including
dispersion compensation for maintaining the short pulses at the sample site as well as an overall transmission of 30-38%
for the OPO range (measured up to 1400nm).
Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of
the penetration depths, strongly reduced photo damage, and by 30fold increased excitation efficiency and 10fold reduced
photobleaching of red fluorescent dyes, including RFP and Cy5.5. 1100nm-excited SHG efficiency of collagen was 10
to 30fold stronger, compared to 880 nm, yet autofluorescence was decreased by up to 4 times resulting in a significantly
improved signal-to-noise ratio for the detection of fluorescent dyes. The resolution is slightly reduced in comparison to
Ti:Sapphire excitation, which corresponds well to the longer excitation wavelength used.
A new, synchronously pumped picosecond OPO for CARS microscopy is presented. It is based on non-critically
phasematched interaction in LBO pumped by a frequency-doubled modelocked Nd:Vanadat laser at 532 nm.
Within the parametric process a tuneable pair of two different wavelengths in the NIR range is generated (Signal <680
...990 nm, Idler 1150...>2450 nm). In this system they are extracted from the cavity at the same mirror and therefore
propagating collinear at the same beam path. Due to the mechanism of their generation there is no jitter between Signal
and Idler. Though the wavelengths are different the GVD is negligible for this picosecond pulse duration. As a result the
two pulse trains are spatially and temporally perfectly matched.
The pulses generated are close to transform limit with about 5-6 ps pulse duration, excellent beam quality (M2 < 1,1) and
high pointing stability. The output power for Signal and Idler is about 1 W each @ 4 W pump power. The tuning
mechanism is split into two parts - temperature tuning for rough variations and fast angular BRF tuning for the fine
adjustment of the output wavelength.
The perfect spatial and temporal overlap make the described OPO an ideal and nearly hands-free laser source for CARS
microscopy with a tuneable energy difference 1,400 ... >10,000 cm-1. The absolute wavelength range is resulting in high
penetration depth and low photo damage of the analyzed samples.
Finally some CARS-images are presented and the latest results and methods for further sensitivity enhancements are
shown.
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