KEYWORDS: Second-harmonic generation, Holography, Holograms, Microscopy, Signal to noise ratio, 3D image processing, Scattering, Spatial resolution, 3D acquisition, Camera shutters
We apply holography to second harmonic generation (SHG) microscopy to obtain three-dimensional images of
biological specimens without the use of external labeling. Nonlinear scanning microscopy techniques like SHG,
has proven to be a powerful technique for imaging biological samples such as collagen, myosin, and muscle fibrils,
but it lacks in acquisition speed for video rate 3D imaging. In nonlinear holography however, full 3D images can be
numerically reconstructed from one hologram, the recorded intensity interference pattern between the endogenous
SHG and coherent reference fields. The femtosecond Yb:KGW laser oscillator used in these experiments appears
in biologically compatible window at a center wavelength near 1 μm. The limited absorption helps prevent
damage to our samples, even for prolonged exposures. Holograms were recorded with exposure times as short
as 1 ms using a fast mechanical shutter. Extrapolating the trend measured above this limit, we anticipate a 10
dB SNR at 40 μs integration time. Such high 3D image frame rates would yield a great deal of information of
chemical waves, neurological networks, and other biological systems.
We present an imaging technique that acquires one-dimensional cross-section through a sample by imposing a
chirped spatial frequency amplitude modulation on the probing beam. The spatial distribution of the sample is
directly mapped to modulation frequency components of the spatially-integrated temporal signal from a singleelement
detector. The electronic time-domain signals are the auto-correlation of the spatial frequency distribution
of an image. The method is demonstrated by imaging both absorptive and florescent objects.
In optical microscopy, the polarization state of the focal field strongly influences formed images due to its interactions
with the sample and the effective focal spot size. We demonstrate experimentally that control over the spatial profile of the
focal field polarization state improves spatial resolution in
laser-scanning third harmonic generation (THG) microscopy.
The focal field is manipulated by imaging a spatial light modulator to the focal plane of a moderate-numerical aperture
microscope. The resolution enhancement arises from exploiting the suppression, in isotropic media, of THG for circularlypolarized
field polarization. By synthesizing a focal field whose polarization state changes from linear at the beam center
to circular beyond radius rs, we quench THG beyond rs. A transverse spatial resolution of up to 2 times is demonstrated.
Targeted manipulation necessitates measurement techniques that allow us to determine of the focal field polarization state.
We develop two such techniques to characterize the field. We use a nano-particle with known third-order susceptibility
to localize THG scattering to a small focal volume. Scanning this nano-probe through the focal volume of the microscope
allows for complete reconstruction of the vector point spread function. Under moderate focusing conditions, where
the recorded THG signal is dominated by the incident paraxial polarization component, the spatial polarization state is
determined non-iteratively via three linear-polarization projection THG measurements. Under tight focusing conditions,
polarization scrambling occurs such that the input and focal fields are dissimilar, and we introduce an algorithm for focal
field retrieval through the collection of far-field THG images.
The performance of collinear and noncollinear pulsed barium borate optical parametric oscillators is analyzed under third harmonic Nd:YAG pumping. In the collinear case the effects of output coupling, cavity length and pump spotsize on threshold and slope efficiency have been measured and compared to a simple model. Tuning ranges, bandwidths and efficiencies are compared for collinear operation and for both directions of noncollinear operation. For a low pump divergence collinear operation is more efficient, but noncollinear operation is favored for a pump divergence in excess of 4 milliradians. Single frequency OPO operation with less than 250 MHz bandwidth is obtained using a simple new injection seeding technique for both signal and idler. Precise crystal temperature control is used to simultaneously manipulate the gain envelope and mode frequencies, ensuring stable seeding on the strongest mode.
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