Two-photon microscopy provides sectioned excitation, but in practical settings, it often suffers from contrast limitations. Here, we report the observation of a strong acousto-optic modulation of two-photon excited fluorescence. Harnessing this effect yields enhanced image detail and contrast and improves optical sectioning in deep brain tissue in vivo. Ultrasound-modulation assisted multiphoton imaging (UMAMI) is compatible with standard multiphoton microscopes, without requiring changes to the optical path or image acquisition parameters.
An advantage of two-photon microscopy is its sectioning capabilities, however, in practice it can often suffer from out of focus contamination. Here, we report the new application of strong acousto-optic modulation of two-photon excited fluorescence. We demonstrate how this effect enhances image detail and contrast and improves optical sectioning in vivo. Requiring no changes to the optical path or image acquisition parameters, our method is simple to implement and compatible with standard multiphoton microscopes.
The paper presents a method for investigating temperature fields in space and transparent media using off-axis digital holographic interferometry. This interferometric technique is particularly well-suited for measuring dynamically changing temperature fields due to its capability to automatically evaluate temperature from a single interferogram. The experimental validation of this technique involved measuring temperature variations within a burning candle's flame. To establish a reference point, an initial hologram was captured without a burning candle, followed by subsequent holograms during the burning process. This allowed the observation of changing refractive index gradient states in the surrounding air. The reconstruction of the complex amplitude facilitated the calculation of the distribution of phase changes. By establishing a relationship between phase changes, thermal coefficients of the air's refractive index, and temperature fluctuations, temperature measurements were achieved at these distinct states. The accuracy of the measurement was estimated to be less than 1 °C in our experimental setup, showing the high precision achievable with this technique. In summary, the paper offers analysis of digital holographic interferometry as a tool for temperature measurements in transparent media. Its potential applications extend to combustion studies, space exploration, atmospheric research, and various other scientific disciplines.
Ultrasonic methods hold immense promise for functional measurements and interventions during in vivo studies due to the beneficial scattering properties of acoustic waves. Here we present the design and application of a system that integrates into standard multiphoton microscopes, which co-linearly combines two-photon fluorescence imaging and precise acoustic sonication. By applying this system across a range of samples, we observed a novel gigantic Acousto-Optic (AO) modulation of two-photon excited fluorescence, and also study the mechanisms of functional ultrasound neuromodulation, an emerging non-invasive neurostimulation technique. This work demonstrates simple photonic/acoustic methods to study novel phenomenon in the awake mouse.
Demonstrations of acousto-optic effects in fluorescent samples are widespread but their utility is generally limited due to the small amplitude modulations achievable and concurrently, long acquisition times. Here, we report the observation of a novel, gigantic acousto-optic modulation of two-photon excited fluorescence. The effect’s magnitude when applying low intensity ultrasound allows high SNR modulation of fluorescence in standard multiphoton systems without sophisticated detection schemes and without the need for extensive temporal averaging or signal processing. We observe this effect in the brain of awake behaving mice, demonstrating its utility for future advanced AO applications in vivo.
In single exposure off-axis interferometry, multiple information can be recorded by spatial frequency multiplexing. We
investigate optimum conditions for designing 2D sampling schemes to record larger field of view in off-axis
interferometry multiplexing. The spatial resolution of the recorded image is related to the numerical aperture of the
system and sensor pixel size. The spatial resolution should preserve by avoiding crosstalk in the frequency domain.
Furthermore, the field of view depends on the sensor size and magnification of the imaging system. In order to preserve
resolution and have a larger field of view, the frequency domain should be designed correctly. The experimental results
demonstrate that selecting the wrong geometrical scheme in frequency domain decrease the recorded image area.
Here, we present a triple illumination phase interferometer to have a more flexible unwrapping measurement range for
single shot optical unwrapping. Three beams of this interferometer illuminate a sample at different incident angles, three
phases of the different incident angles are simultaneously recorded using three spatial frequencies. The different
direction phases can be used for dual illumination optical unwrapping; as a result, the phase can be unwrapped by more
than one measurement range. The feasibility of this technique is demonstrated by measuring a stepped object with
heights of 150 and 660 μm. The smaller stepped phase is unwrapped by two measurement range; however, the phase of
larger step is wrapped for only one of the measurement range.
We present a method to reduce the inherent errors caused by band-pass filter in off-axis quantitative phase microscopy and propose the optimum condition that can minimize these errors. We found that phase information of a sample in frequency domain nonlinearly oscillates as a function of the phase-shift correspond to the sample and its medium and the phase information of a sample inside the band-pass filter can be maximized by a proper phase-shift. Through numerical simulations and actual experiments, we demonstrate that the error in phase volume measurement can be effectively reduced by the enhancement of phase signal inside band-pass region using an optimum amount of phase that can be controlled by either changing medium index or wavelength of illumination.
Reduced-phase dual-illumination interferometer (RPDII) is an off axis, single shot and single wavelength phase imaging technique to measure large objects without using unwrapping algorithms. Two beams of this interferometer illuminate a sample at different incident angles, two phases of the different incident angles and their phase difference are recorded. The phase difference between two beams can be controlled by adjusting the incident angles. The angle accuracy that decrease the RPDII accuracy have been studied. We have shown, the groove spacing of the grating and magnification of the lens system before sample, determine the incident angle accuracy. The ability of RPDII to unwrap large phases is shown by reconstructing phase of a step object without using unwrapping algorithms. The reconstructed image shows that the total inaccuracy is much more than the inaccuracy caused by incident angles.
Various quantitative phase microscopy (QPM) techniques for noninvasive and quantitative analysis of samples proposed based on imaging interferometry techniques over the last decade [1-4]. A phase image can be obtained with a single set of interference data in some types of phase microscopes such as diffraction phase microscope [5, 6]. They are suitable for studying rapidly varying phenomena with reduced concern for systematic and sample variations that may occur during the acquisition of the raw data. Dispersion measurements of a sample carry more information than refractive index of measurements at a single wavelength [7]. Knowledge of the optical dispersion for phase objects such as optical fibers, biological cells and micro-particles can provide very useful information about their property. In this work, we report on a common-path and dual wavelength quantitative phase microscope that simultaneously acquires two phase images at different wavelengths. The simultaneous dual-wavelength measurement was performed with a diffraction phase microscope based on a transmission grating and a spatial filter that form a common-path imaging interferometer. With a combined laser source that generates two-color light continuously, a different diffraction order of the grating was utilized for each wavelength component so that the dual-wavelength interference pattern could be distinguished by the distinct fringe frequencies. The refractive index profiles of fiber in both wavelengths were measured adequately by our DW-DPM system.
We present a dual-wavelength diffraction phase microscopy (DW-DPM) that obtains the wavelength-differentiated dual
phase images in a single shot of interference fringe acquisition. For this, the diffraction phase microscopy (DPM) system
was constructed with a transmission grating and a spatial filter that form a common-path interferometer. With a light
source of two spectral components, a different diffraction order of the grating was utilized for each. This resulted in a
combined but distinguishable interference pattern to be acquired by a single image sensor. In this research, our dualwavelength
phase imaging scheme was applied to simultaneously measure dispersion of a sample. Stable and reliable
measurements could be performed in a single shot due to the robust structure of our DW-DPM system.
We propose a single shot and single wavelength phase imaging technique for measuring phase of the transparent objects
without using unwrapping process. A grating between a laser and the object is used to make beams with different angle,
which determines the measurement range of the microscope. The grating pitch and magnification of the lens system
before the sample affect the angle. The angle inside the object is changed according to Snell’s law; therefore, final angle
is related to the refractive index of the object. Magnification of the lens system after sample will control the modulation
frequency of microscope. The interference pattern is constructed at CCD plane and convey information of the sample.
For a phase below the measurement range of the microscope, the reconstructed phase is not wrapped. By increasing the
measurement range accuracy of the system will drop; therefore the magnification of the lenses must choose carefully to
obtain optimal phase. The ability of this technique is demonstrated by reconstructing phases of two transparent step
objects with 150 and 510 μm height. Their refractive indexes for red light are 1.515 and 1.508 , respectively. Therefore,
total optical path length difference is 336 micrometers that is 500 times more than the laser wavelength. The phase is
successfully reconstructed without using unwrapping algorithms.
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