Here we achieved record-high >500 volumes/second two-photon imaging by improving lateral and axial scanning speed via 32-channel multifocal excitation/detection, and a tunable acoustic gradient-index lens, respectively. We developed a deconvolution process to reduce scattering-induced crosstalk in multifocal detection scheme, thus enabling whole brain imaging of Drosophila with millisecond and micrometer spatiotemporal resolution. Potential applications toward brain science include studying millisecond dynamics in a neuronal network, and resolving 3D microfluidics in blood vessels.
Studying neuronal connections and activities in vivo is critical for understanding the brain. Optical microscopy, with the capability of specific fluorescent labeling and sub-cellular spatial resolution, has become an indispensable tool in neuroscience. However, the major limitation of optical imaging is penetration depth and imaging speed to capture neural signal dynamics in deep brain regions. Recently, by applying adaptive optics, high-energy laser, or long wavelength lasers for nonlinear imaging, penetration depth around 1mm has been achieved in living mouse brains. Nevertheless, this depth barely pierces through the mouse cortex and is far from reaching the bottom of the centimeter-thick mouse brain. For studying deeper regions of the brain, brain slice is one possible approach, yet it is invasive and cut away many neuron connections. In this study, a home-built two-photon microscope is integrated with both a gradient refractive index (GRIN) lens and a tunable acoustic gradient (TAG) lens. The GRIN lens serves as a micro-endoscope which extends the imaging depth to a centimeter while minimizing the invasiveness, and the TAG lens provides ~100kHz axial scanning which enables high-speed volumetric imaging of neuronal response. This novel high-speed volumetric endoscopy system offers an unprecedented opportunity towards studying three-dimensional neuronal dynamics in deep brains regions of a living mouse.
Cell samples flowing along a microfluidic tube are scanned with an optical coherence tomography (OCT) system and their correlation times in M-mode scans are calibrated. In particular, the variations of correlation time with waiting time after 5 and 10 % ethanol are applied to the cell samples are compared for understanding the evolution of cell morphology in the cell death pathways of apoptosis and necrosis, respectively. Also, Au nanorings (NRIs) are taken up by cells for increasing the scattering strength in OCT scanning and hence increasing the signal-to-noise ratio. It is found that when cells are incubated with 5 % ethanol, the correlation time keeps decreasing with waiting time and then increases at 7 hours. On the other hand, when cells are incubated with 10 % ethanol, the correlation time keeps decreasing with waiting time all the way up to 7 hours. This difference may imply that the correlation time from OCT scanning may be determined not only by the size of cell fragment, but also by the smoothness of the cell fragment in a scale of several hundred nm during the apoptosis and necrosis processes. In particular, the results imply that the surface smoothness of the apoptotic bodies formed at the final stage of an apoptosis process is higher than that of the cell fragments formed at the final stage of a necrosis process. This OCT scanning technique has the potential application to the determination of cell death condition with the function similar to cell flow cytometry.
Photosensitizer can be taken up directly by cells or through the linkage with gold nanoparticles (NPs). When gold NPs linked with a photosensitizer are taken up by cells, both photodynamic (PDT) and photothermal effects for damaging cells can be generated under the illumination of a light source of an appropriate wavelength. The gold NPs and photosensitizer can escape from cells through an exocytosis process such that the efficiency of cell damage may decay with time. In this study, we investigate the dependencies of cell damage efficiency, gold nanoring (NRI) and photosensitizer amounts stayed in cells on the delay time. In particular, we differentiate the two possible escape pathways of the photosensitizer, AlPcS, i.e., AlPcS escape alone after its linkage with Au NRI is broken and AlPcS escape together with the linked Au NRI. Also, we intend to understand the dependence of the aforementioned AlPcS escape pathway on the location of Au NRIs inside a cell after they are taken up. It is found that generally, the PDT-induced cell damage efficiency decays more slowly when AlPcS is taken up together with linked Au NRI, compared with the case that AlPcS is taken up alone by cells. This trend indicates that the photosensitizer is more stable when it is linked with an Au NP. This result is confirmed by the observations of Au NRI and photosensitizer escapes through mass spectrometry, flow cytometry, and confocal fluorescent microscopy.
A cell spheroid can be as large as 1 mm in diameter, containing more than 300,000 cells. It can be used for simulating a cancer tumor in cell treatment study. In this study, the cell uptake behaviors in cell spheroids are investigated. In particular, the penetration depths of Au nanoring, Au nanorod, and photosensitizer (AlPcS) in cell spheroids are evaluated based on a newly proposed technique of using confocal fluorescent microscopy. By using a geometric computation process, we can estimate the width of a fluorescent belt region, which corresponds to the uptake depth. Based on such evaluations, we can understand whether the quiescent viable cells in the middle layer of a cell spheroid. Also, by using a cell culture insert, we can estimate the uptake Au nanoparticle number per cell through mass spectrometry measurement. It is found that after 48-hour incubation time, the Au nanoparticles and photosensitizer all have the penetration depths in the range of 110-140 m. Such uptake penetration depths control the cell damage depths under laser illuminations. For observing the uptake depths, we use different fluorescent dyes to incubate the cell spheroids. It is found that the penetration depth of a dye also relies on its molecular weight. The smaller uptake penetration depths of Au nanoparticles into a cell spheroid can be attributed to the settlement of Au nanoparticles in the incubation solutions such that the upper portion of a cell spheroid has no access to the Au nanoparticles.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.