High-speed label-free imaging with chemical contrast is effective for non-invasive analysis of the metabolic heterogeneity of single cells. Stimulated Raman scattering (SRS) microscopy enables high-speed label-free image acquisition with molecular vibrational specificity. While single-color SRS microscopy only acquires images at a certain vibrational frequency, multicolor SRS microscopy successively acquires SRS images at different vibrational frequencies, which then can be used to investigate the distributions of different intracellular molecules. However, its imaging speed remains an order of magnitude slower than that of single-color video-rate SRS microscopy. Previous approaches to circumvent this issue used either only two colors with limited chemical specificity or multiplex detection of SRS spectra using a photodiode array at the expense of imaging speed. Here we demonstrate high-speed four-color SRS imaging using a single photodiode by introducing fast wavelength-switched laser pulses. The fast wavelength switching is realized by the use of an optical intensity modulator as a time gate, a diffraction grating, and fiber delay lines. Using the developed system, we demonstrate motion-artifact-free multicolor SRS imaging of polymer beads and living cells. The results firmly support that our method is a powerful tool for the label-free analysis of living cells in microbiology, oncology, plant science, and medicine.
For its label-free imaging capability with chemical specificity, Raman microscopy has found various applications in biology, medicine, pharmacology, and material science. In imaging of moving samples and/or a large number of objects, however, spontaneous Raman imaging falls short due to its slow signal acquisition time. Multicolor stimulated Raman scattering (SRS) microscopy is a promising approach to this end because it can probe the vibrational signatures of molecules at multiple vibrational frequencies with orders of magnitude higher sensitivity than spontaneous Raman scattering. Here we report our two recent achievements that boost the chemical imaging speed of multicolor SRS microscopy. The first one is multicolor SRS imaging that operates at 110 frames per second by frame-by-frame wavenumber tuning. Using this method, we demonstrated video-rate single-cell imaging of intracellular metabolites in live Euglena gracilis, a highly motile microalgal species. We revealed large cell-to-cell variations in the amounts of metabolites, which is effective for efficient biomaterial engineering. The second one is four-color SRS imaging that runs at a pixel dwell time of 0.2 µs per pixel, enabled by fast wavelength switching of laser pulses. We used it to demonstrate SRS-based chemical imaging of polymer beads and living cells. Our SRS system’s fast imaging capability made high-throughput Raman imaging possible as well as blur-free Raman imaging, which are highly useful in diverse applications including cell screening and intraoperative diagnostics.
Photoacoustic microscopy (PAM) has been extensively applied in biomedical study because of its ability to visualize tissue morphology and physiology in vivo in three dimensions (3D). However, conventional PAM suffers from a rapidly decreasing resolution away from the focal plane because of the limited depth of focus of an objective lens, which deteriorates the volumetric imaging quality inevitably. Here, we propose a novel method to synthesize an ultra-long light needle to extend a microscope’s depth of focus beyond its physical limitations with wavefront engineering method. Furthermore, it enables an improved lateral resolution that exceeds the diffraction limit of the objective lens. The virtual light needle can be flexibly synthesized anywhere throughout the imaging volume without mechanical scanning. Benefiting from these advantages, we developed a synthetic light needle photoacoustic microscopy (SLN-PAM) to achieve an extended depth of field (DOF), sub-diffraction and motionless volumetric imaging. The DOF of our SLN-PAM system is up to 1800 µm, more than 30-fold improvement over that gained by conventional PAM. Our system also achieves the lateral resolution of 1.8 µm (characterized at 532 nm and 0.1 NA objective), about 50% higher than the Rayleigh diffraction limit. Its superior imaging performance was demonstrated by 3D imaging of both non-biological and biological samples. This extended DOF, sub-diffraction and motionless 3D PAM will open up new opportunities for potential biomedical applications.
Microalgae have been receiving great attention for their ability to produce biomaterials that are applicable for food supplements, drugs, biodegradable plastics, and biofuels. Among such microalgae, Euglena gracilis has become a popular species by virtue of its capability of accumulating useful metabolites including paramylon and lipids. In order to maximize the production of desired metabolites, it is essential to find ideal culturing conditions and to develop efficient methods for genetic transformation. To achieve this, understanding and controlling cell-to-cell variations in response to external stress is essential, with chemically specific analysis of microalgal cells including E. gracilis. However, conventional analytical tools such as fluorescence microscopy and spontaneous Raman scattering are not suitable for evaluation of diverse populations of motile microalgae, being restricted either by the requirement for fluorescent labels or a limited imaging speed, respectively. Here we demonstrate video-rate label-free metabolite imaging of live E. gracilis using stimulated Raman scattering (SRS) – an optical spectroscopic method for probing the vibrational signatures of molecules with orders of magnitude higher sensitivity than spontaneous Raman scattering. Our SRS’s highspeed image acquisition (27 metabolite images per second) allows for population analysis of live E. gracilis cells cultured under nitrogen-deficiency - a technique for promoting the accumulation of paramylon and lipids within the cell body. Thus, our SRS system’s fast imaging capability enables quantification and analysis of previously unresolvable cell-to-cell variations in the metabolite accumulation of large motile E. gracilis cell populations.
Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.
Wavefront distortion in scattering media can be compensated for using optical wavefront shaping. In this technique, a spatial light modulator (SLM) is used to apply a spatially distributed phase shift to the optical field. A genetic optimization algorithm was used to obtain the SLM pattern which best focuses light within the medium. The target volume is defined by using a focused ultrasound beam to encode light travelling within the acoustic focus. The ultrasonically-encoded light is measured and used as feedback to the algorithm, which then searches for the pattern which maximizes the encoded light intensity. We call this technique ultrasonically-encoded wavefront shaping (SEWS). Using SEWS, we focused light into a scattering medium consisting of ground glass diffuser and a gelatin phantom. The optical intensity at the target was increased by 11 times over the original intensity. These results were validated using fluorescent imaging at the ultrasonic focus.
To achieve localized light delivery beyond turbid layers, TRUE optical focusing has been previously implemented by both analog and digital devices. The digital scheme offers a higher energy gain than the analog version. In many biological applications, the reflection-mode configuration, which uses backscattered light from the sample, is more suitable than the transmission-mode configuration. Although reflection-mode analog TRUE focusing has been demonstrated, its digital implementation has not been explored. Here, we report a reflection-mode digital TRUE focusing to concentrate light through a turbid layer. Further, by simply moving the ultrasound focus, we show the system's dynamic focusing capability.
To focus light beyond one transport mean free path, time-reversed ultrasonically encoded (TRUE) optical focusing has previously been implemented by both analog and digital devices. By allowing wavefront recording with finer resolution and larger aperture, the analog scheme, which uses photorefractive materials as the phase-conjugate mirror, generates a more complete set of time-reversed optical modes than the digital scheme. Here, we report the direct visualization of localized fluorescence excitation inside a turbid medium by photorefractive time reversal. Further, we imaged fluorescent targets embedded in a turbid phantom whose thickness was four transport mean free paths.
Time-reversed ultrasonically encoded (TRUE) optical focusing is an emerging technique that focuses light deep into scattering media by phase-conjugating ultrasonically encoded diffuse light. In previous work, the speed of TRUE focusing was limited to no faster than 1 Hz by the response time of the photorefractive phase conjugate mirror, or the data acquisition and streaming speed of the digital camera; photorefractive-crystal-based TRUE focusing was also limited to the visible spectral range. These time-consuming schemes prevent this technique from being applied in vivo, since living biological tissue has a speckle decorrelation time on the order of a millisecond. In this work, using a Tedoped Sn2P2S6 photorefractive crystal at a near-infrared wavelength of 793 nm, we achieved TRUE focusing inside dynamic scattering media having a speckle decorrelation time as short as 7.7 ms. As the achieved speed approaches the tissue decorrelation rate, this work is an important step forward toward in vivo applications of TRUE focusing in deep tissue imaging, photodynamic therapy, and optical manipulation.
Time-reversed ultrasonically encoded (TRUE) optical focusing focuses light beyond one transport mean free
path by phase-conjugating the ultrasonically tagged light. However, in previous works, only a small portion of the tagged
light was phase-conjugated by using a photorefractive Bi12SiO20 crystal, due to its small active area (1x1 cm2). In this work, we report high-efficiency TRUE focusing using a large-area photorefractive polymer (5x5 cm2), which
demonstrated ~40 times increase in focused energy. Further, we imaged absorbers embedded in a turbid sample of
thickness of ~12 transport mean free paths.
For years, ultrasound-modulated optical tomography (UOT) has been proposed to image optical contrasts deep inside
turbid media (such as biological tissue) at an ultrasonic spatial resolution. The reported imaging depth so far, however,
has been limited, preventing this technique from finding broader applications. In this work, we present our latest
experimental explorations that push UOT to clinically useful imaging depths, achieved through optimizing from different
aspects. One improvement is the use of a large aperture fiber bundle, which more effectively collects the diffused light,
including both ultrasound-modulated and unmodulated portions, from the turbid sample and then sends it to the
photorefractive material. Another endeavor is employment of a large aperture photorefractive polymer film for
demodulating the ultrasound-induced phase modulation. Compared with most UOT detection schemes, the polymer film
based setup provides a much higher etendue as well as photorefractive two-beam-coupling gain. Experimentally, we have
demonstrated enhanced sensitivity and have imaged through tissue-mimicking samples up to 9.4 cm thick at the
ultrasonically-determined spatial resolutions.
Time-reversed ultrasonically encoded (TRUE) optical focusing achieves light focusing into scattering media beyond one transport mean free path, which is desirable in biomedical optics. However, the focused optical energy needs to be increased for broad applications. Here, we report the use of a photorefractive polymer (PRP) as the phase conjugate mirror in TRUE optical focusing. The PRP boosted the focused optical energy by ∼ 40 times in comparison to the previously used photorefractive Bi12SiO20 crystal. As a result, we successfully imaged absorbing objects embedded in the middle plane of a tissue-mimicking phantom having an optical thickness of 120 scattering mean free paths.
KEYWORDS: Point spread functions, Light scattering, Acoustics, Transducers, Monte Carlo methods, Photoacoustic microscopy, Optical resolution, Signal detection, Absorption, Signal generators
The penetration depth of ballistic optical imaging technologies is limited by light scattering. To study the effect of
scattering on optical-resolution photoacoustic microscopy (OR-PAM), we divided the signals in OR-PAM into two
classes: one is from the target volume defined by the optical resolution cell (Class I); the other is from the rest of the
acoustic resolution cell (Class II). We developed a way to simulate the point spread function (PSF) of our OR-PAM
system considering both optical illumination and acoustic detection, then used the PSF to calculate the contributions of
each class of signal to the total signal at different focal depths. Our simulation results showed that: 1) The Class II
signal decays much more slowly than the Class I signal; 2) The full width at half maximum (FWHM) of the PSF for the
focal depth of 0.9 transport mean free path (TMFP) is not broadened much (~10%) compared with that for a clear
medium; 3) Image contrast is degraded with increasing depth when there is a uniform absorption background.
The problem of how to effectively deliver light dynamically to a small volume inside turbid media has been intensively
investigated for imaging and therapeutic purposes. Most recently, a new modality termed Time-Reversed Ultrasonically
Encoded (TRUE) optical focusing was proposed by integrating the concepts of ultrasound modulation of diffused light
with optical phase conjugation. In this work, the diffused photons that travel through the ultrasound focal region are
"tagged" with a frequency shift due to the ultrasound modulation. Part of the tagged light is collected in reflection mode
and transmitted to a photorefractive crystal, forming there a stationary hologram through interference with a coherent
reference optical beam. The hologram is later read by a conjugated optical beam, generating a phase conjugated wavefront
of the tagged light. It is conveyed back to the turbid medium in reflection mode, and eventually converges to the ultrasound
focal zone. Optical focusing effects from this system are demonstrated experimentally in tissue-mimicking phantoms and
ex vivo chicken breast tissue, achieving effective round-trip optical penetration pathlength (extinction coefficient
multiplied by round-trip focusing depth) exceeding 160 and 100, respectively. Examples of imaging optical inclusions with
this system are also reported.
Time-reversed ultrasonically encoded (TRUE) optical focusing was recently proposed to deliver light dynamically to a tight region inside a scattering medium. In this letter, we report the first development of a reflection-mode TRUE optical focusing system. A high numerical aperture light guide is used to transmit the diffusely reflected light from a turbid medium to a phase-conjugate mirror (PCM), which is sensitive only to the ultrasound-tagged light. From the PCM, a phase conjugated wavefront of the tagged light is generated and conveyed by the same light guide back to the turbid medium, subsequently converging to the ultrasonic focal zone. We present experimental results from this system, which has the ability to focus light in a highly scattering medium with a round-trip optical penetration thickness (extinction coefficient multiplied by round-trip depth) as large as 160.
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