Jianyong Tang

Multilayer three-dimensional super resolution imaging of thick biological samples

Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 μm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ≈10 μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.

Two-photon single-cell optogenetic control of neuronal activity by sculpted light

Recent advances in optogenetic techniques have generated new tools for controlling neuronal activity, with a wide range of neuroscience applications. The most commonly used approach has been the optical activation of the light-gated ion channel channelrhodopsin-2 (ChR2). However, targeted single-cell-level optogenetic activation with temporal precessions comparable to the spike timing remained challenging. Here we report fast (≤1 ms), selective, and targeted control of neuronal activity with single-cell resolution in hippocampal slices. Using temporally focused laser pulses (TEFO) for which the axial beam profile can be controlled independently of its lateral distribution, large numbers of channels on individual neurons can be excited simultaneously, leading to strong (up to 15 mV) and fast (≤1 ms) depolarizations. Furthermore, we demonstrated selective activation of cellular compartments, such as dendrites and large presynaptic terminals, at depths up to 150 μm. The demonstrated spatiotemporal resolution and the selectivity provided by TEFO allow manipulation of neuronal activity, with a large number of applications in studies of neuronal microcircuit function in vitro and in vivo.

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.

Multilayer and Three-dimensional Super-Resolution Imaging of Thick Biological Samples

We have demonstrated super-resolution imaging of protein distributions in cells at depth at multiple layers with a lateral localization precision better than 50nm. The approach is based on combining photoactivated localization microscopy with temporal focusing.

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.Light-sheet fluorescence microscopy enables high resolution imaging of biological samples. Here the authors use reflective coverslips to obtain multiple sample views simultaneously, improving the speed of acquisition and resolution compared to dual-view selective plane illumination microscopy.

Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

Three-dimensional nanoscopy of biological samples

We have demonstrated super-resolution imaging of protein distributions in cells at depth at multiple layers with a lateral localization precision better than 50nm. The approach is based on combining photoactivated localization microscopy with temporal focusing.

Multilayer Three-dimensional Super-resolution Imaging of Thick Biological Samples

Recent advances in optical microscopy have created the capability of creating images in biological samples beyond the diffraction limit at nanometre resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples and a sample geometry using total internal reflection that limits the imaging depth to a fraction of an optical wavelength. However, in order to study whole cells or organelles which are typically up to ∼15μm deep into the cell, the extension of these methods to a 3D super-resolution technique is required.Here we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super-resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ∼10μm in both mitochondrially-labeled fixed cells, and in the membranes of living S2 Drosophila cells.View Large Image | View Hi-Res Image | Download PowerPoint Slide