scholarly journals 3D super-resolution deep-tissue imaging in living mice

2019 ◽  
Author(s):  
Mary Grace M. Velasco ◽  
Mengyang Zhang ◽  
Jacopo Antonello ◽  
Peng Yuan ◽  
Edward S. Allgeyer ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Organic Dyes ◽  
3D Visualization ◽  
Water Immersion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Tissue Imaging ◽  
Objective Lens ◽  
Working Distance

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of 2-photon excitation, adaptive optics, far-red emitting organic dyes, and a long-working distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

scholarly journals Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

2019 ◽  
Author(s):  
Aurélien Barbotin ◽  
Silvia Galiani ◽  
Iztok Urbančič ◽  
Christian Eggeling ◽  
Martin Booth
Keyword(s):  
Adaptive Optics ◽  
Molecular Diffusion ◽  
Three Dimensional ◽  
Super Resolution ◽  
Correction Method ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Extreme Sensitivity

Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (3D) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.

scholarly journals PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Super-Resolution Imaging With Lanthanide Luminescent Nanocrystals: Progress and Prospect

2021 ◽  
Vol 9 ◽  
Author(s):  
Hongxin Zhang ◽  
Mengyao Zhao ◽  
István M. Ábrahám ◽  
Fan Zhang
Keyword(s):  
Cell Biology ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Lanthanide Ions ◽  
Fluorescence Probes ◽  
Saturation Intensity ◽  
Resolution Imaging

Stimulated emission depletion (STED) nanoscopy has overcome a serious diffraction barrier on the optical resolution and facilitated new discoveries on detailed nanostructures in cell biology. Traditional fluorescence probes employed in the super-resolution imaging approach include organic dyes and fluorescent proteins. However, some limitations of these probes, such as photobleaching, short emission wavelengths, and high saturation intensity, still hamper the promotion of optical resolution and bio-applications. Recently, lanthanide luminescent probes with unique optical properties of non-photobleaching and sharp emissions have been applied in super-resolution imaging. In this mini-review, we will introduce several different mechanisms for lanthanide ions to achieve super-resolution imaging based on an STED-like setup. Then, several lanthanide ions used in super-resolution imaging will be described in detail and discussed. Last but not least, we will emphasize the future challenges and outlooks in hope of advancing the next-generation lanthanide fluorescent probes for super-resolution optical imaging.

scholarly journals Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

2018 ◽  
Author(s):  
Joshua Yoon ◽  
Colin J. Comerci ◽  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Tim Stearns ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Underlying Structure ◽  
Nanoscale Morphology

ABSTRACTSuper-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2-6 μm long and 200-300 nm diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened (SMO), with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction (PSR) algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, 2-color, 2D STimulated Emission Depletion (STED) microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the SMO-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

scholarly journals Numerically enhanced adaptive optics-based 3D STED microscopy for deep-tissue super-resolved imaging

2019 ◽  
Author(s):  
Piotr Zdankowski ◽  
Maciej Trusiak ◽  
David McGloin ◽  
Jason R. Swedlow
Keyword(s):  
Adaptive Optics ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Thick Layer ◽  
Block Matching ◽  
Stimulated Emission ◽  
Optical Aberrations ◽  
Deep Tissue ◽  
Resolution Imaging ◽  
Induced Pluripotent

AbstractIn stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilising both a high power depletion laser (increasing risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here we demonstrate a custom-built 3D STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating, tissue. We introduce and investigate image denoising by block-matching and collaborative filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well-established Fourier ring correlation metric. Experimental validation of the proposed method is achieved through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under a 80µm thick layer of tissue with lateral and axial resolution of 256nm and 300nm, respectively.

Development of a digital SEM

1991 ◽  
Vol 49 ◽  
pp. 358-359
Author(s):  
A. Yamada ◽  
A. Shibano ◽  
K. Harasawa ◽  
T. Kobayashi ◽  
H. Fukuda ◽  
...  
Keyword(s):  
Large Diameter ◽  
Objective Lens ◽  
Large Specimen ◽  
Backscattered Electrons ◽  
Digital Scanning ◽  
Junction Type ◽  
High Resolution Images ◽  
Working Distance ◽  
Type Detector ◽  
Short Working

A newly developed digital scanning electron microscope, the JSM-6300, has the following features: Equipped with a narrower conical objective lens (OL), it allows high resolution images to be obtained easily at a short working distance (WD) and a large specimen tilt angle. In addition, it is provided with automatic functions and digital image processing functions for ease of operation.Conical C-F lens: The newly developed conical C-F objective lens, having low aberration characteristics over a wide WD range, allows a large-diameter (3-inch) specimen to be tilted up to 60° at short WD, and provides images with low magnifications starting at 10*. On the bottom of the lens, a p n junction type detector is provided to detect backscattered electrons (BE) from the specimen. As the narrower conical 0L increases the secondary electron (SE) detector's field intensity on the specimen surface, high SE image quality is obtained.

Defocus ramp correction in spot-scan imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 130-131
Author(s):  
Kenneth H. Downing
Keyword(s):  
Computer Control ◽  
Three Dimensional ◽  
Sufficient Accuracy ◽  
Objective Lens ◽  
Electron Crystallography ◽  
Contrast Transfer Function ◽  
Tilt Axis ◽  
Specimen Height ◽  
The Difference ◽  
Envelope Functions

Three-dimensional structures of a number of samples have been determined by electron crystallography. The procedures used in this work include recording images of fairly large areas of a specimen at high tilt angles. There is then a large defocus ramp across the image, and parts of the image are far out of focus. In the regions where the defocus is large, the contrast transfer function (CTF) varies rapidly across the image, especially at high resolution. Not only is the CTF then difficult to determine with sufficient accuracy to correct properly, but the image contrast is reduced by envelope functions which tend toward a low value at high defocus.We have combined computer control of the electron microscope with spot-scan imaging in order to eliminate most of the defocus ramp and its effects in the images of tilted specimens. In recording the spot-scan image, the beam is scanned along rows that are parallel to the tilt axis, so that along each row of spots the focus is constant. Between scan rows, the objective lens current is changed to correct for the difference in specimen height from one scan to the next.

Low-light-level three-dimensional video microscope with long working distance objective lenses

1994 ◽  
Vol 52 ◽  
pp. 226-227
Author(s):  
W. Lin ◽  
J. Gregorio ◽  
T.J. Holmes ◽  
D. H. Szarowski ◽  
J.N. Turner
Keyword(s):  
Three Dimensional ◽  
Light Level ◽  
Stereo Pair ◽  
Light Illumination ◽  
Initial Image ◽  
Low Light ◽  
Image Recording ◽  
Depth Discrimination ◽  
Video Microscope ◽  
Working Distance

A low-light level video microscope with long working distance objective lenses has been built as part of our integrated three-dimensional (3-D) light microscopy workstation (Fig. 1). It allows the observation of living specimens under sufficiently low light illumination that no significant photobleaching or alternation of specimen physiology is produced. The improved image quality, depth discrimination and 3-D reconstruction provides a versatile intermediate resolution system that replaces the commonly used dissection microscope for initial image recording and positioning of microelectrodes for neurobiology. A 3-D image is displayed on-line to guide the execution of complex experiments. An image composed of 40 optical sections requires 7 minutes to process and display a stereo pair.The low-light level video microscope utilizes long working distance objective lenses from Mitutoyo (10X, 0.28NA, 37 mm working distance; 20X, 0.42NA, 20 mm working distance; 50X, 0.42NA, 20 mm working distance). They provide enough working distance to allow the placement of microelectrodes in the specimen.

High-speed brightfield 3-D imaging and 3-D image analysis of thick and overlapped cell clusters in cytological preparations

1994 ◽  
Vol 52 ◽  
pp. 218-219
Author(s):  
Robert W. Mackin
Keyword(s):  
Image Analysis ◽  
High Speed ◽  
Three Dimensional ◽  
Image Data ◽  
Ccd Camera ◽  
Objective Lens ◽  
Array Processor ◽  
Vaginal Smears ◽  
Low Contrast ◽  
Computer Controlled

This paper presents two advances towards the automated three-dimensional (3-D) analysis of thick and heavily-overlapped regions in cytological preparations such as cervical/vaginal smears. First, a high speed 3-D brightfield microscope has been developed, allowing the acquisition of image data at speeds approaching 30 optical slices per second. Second, algorithms have been developed to detect and segment nuclei in spite of the extremely high image variability and low contrast typical of such regions. The analysis of such regions is inherently a 3-D problem that cannot be solved reliably with conventional 2-D imaging and image analysis methods.High-Speed 3-D imaging of the specimen is accomplished by moving the specimen axially relative to the objective lens of a standard microscope (Zeiss) at a speed of 30 steps per second, where the stepsize is adjustable from 0.2 - 5μm. The specimen is mounted on a computer-controlled, piezoelectric microstage (Burleigh PZS-100, 68/μm displacement). At each step, an optical slice is acquired using a CCD camera (SONY XC-11/71 IP, Dalsa CA-D1-0256, and CA-D2-0512 have been used) connected to a 4-node array processor system based on the Intel i860 chip.

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