Enhanced axial resolution of the cytoskeleton in fluorescence microscopy by standing-wave excitation

1993 ◽  
Vol 51 ◽  
pp. 88-89
Author(s):  
Frederick Lanni ◽  
Brent Bailey ◽  
Daniel L. Farkas ◽  
D. Lansing Taylor
Keyword(s):  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Focal Plane ◽  
Information Transfer ◽  
High Speed ◽  
Depth Resolution ◽  
Depth Of Field ◽  
Imaging Method ◽  
Axial Resolution ◽  
Direct Imaging

When the depth-of-field of a microscope is less than the axial dimension of the specimen, 3d information can be derived from a set of images recorded as the specimen is stepped through the object focal plane of the microscope. This procedure, known as optical sectioning microscopy (OSM), is the same in direct imaging and confocal scanning. For both of these cases in fluorescence microscopy, axial (depth) resolution is more limited than transverse resolution, for fundamental reasons. Our research aim has been to enhance axial resolution in fluorescence OSM (FOSM) while retaining the high-speed information transfer characteristics of direct imaging that are necessary for 3d studies of living cells in culture.Standing-wave fluorescence microscopy (SWFM) is a direct imaging method in which the object is illuminated by a three-dimensional field of planar interference fringes (standing waves) oriented parallel to the focal plane of the microscope. This field is produced in the specimen by crossing two coherent, collimated, s-polarized beams of equal amplitude directed through the specimen at complementary angles (θ, π -θ) relative to the axis of the microscope.

Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation

Nature ◽  
1993 ◽  
Vol 366 (6450) ◽  
pp. 44-48 ◽  
Author(s):  
Brent Bailey ◽  
Daniel L. Farkas ◽  
D. Lansing Taylor ◽  
Frederick Lanni
Keyword(s):  
Fluorescence Microscopy ◽  
Standing Wave ◽  
Wave Excitation ◽  
Axial Resolution

Optical subsectioning and multiple focal-plane imaging in the standing-wave fluorescence microscope

1994 ◽  
Vol 52 ◽  
pp. 158-159
Author(s):  
Brent Bailey ◽  
Vijay Krishnamurthi ◽  
Frederick Lanni
Keyword(s):  
Refractive Index ◽  
Wave Field ◽  
Standing Wave ◽  
Focal Plane ◽  
Imaging Method ◽  
Intensity Pattern ◽  
Polarized Beams ◽  
System A ◽  
Standing Wave Field ◽  
Dual Beam

Standing-wave fluorescence microscopy (SWFM) is a direct-imaging method through which very high axial resolution can be obtained in certain types of biological specimens. In the microscope, interference of light is used to produce a standing-wave field through the specimen, so that it is illuminated in a pattern of planar zones, rather than uniformly. Fluorescence is excited with spatial selectivity in proportion to the intensity pattern. In the instruments in use at present, two nearlycollimated s-polarized beams from a laser are directed into the specimen from opposite sides at mirrorimage angles with respect to the microscope axis (Figure). The alternating nodal and antinodal planes of the field are in this case parallel to the object focal plane, with a node spacing equal to λo/(2n cos θ), where θ is the beam angle, and n is the refractive index of the specimen. The minimum node spacing, λo/2n, is obtained when the beams are counterpropagating on-axis, and is typically 0.18 μm. In the simplest SWFM system, a mirror is placed behind the specimen such that an on-axis beam interferes with its reflection. In our dual-beam instrument, the laser output is split, and the beams directed into the specimen through a pair of opposed high-NA objectives. In both cases, the standing-wave field planes can be shifted axially through the specimen, by control of the phase of one of the beams. This can be done with high precision by use of a piezoelectric mirror drive. For SWFM to work well, it is necessary that the specimen be a weak phase object, i.e., that the refractive index heterogeneity be small.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

scholarly journals Recurrent neural network-based volumetric fluorescence microscopy

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Luzhe Huang ◽  
Hanlong Chen ◽  
Yilin Luo ◽  
Yair Rivenson ◽  
Aydogan Ozcan
Keyword(s):  
Neural Network ◽  
Image Reconstruction ◽  
Fluorescence Microscopy ◽  
Recurrent Network ◽  
Sample Volume ◽  
Depth Of Field ◽  
Objective Lens ◽  
Wide Field ◽  
Volumetric Imaging ◽  
3D Image Reconstruction

AbstractVolumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over an extended depth-of-field. Using experiments on C. elegans and nanobead samples, Recurrent-MZ is demonstrated to significantly increase the depth-of-field of a 63×/1.4NA objective lens, also providing a 30-fold reduction in the number of axial scans required to image the same sample volume. We further illustrated the generalization of this recurrent network for 3D imaging by showing its resilience to varying imaging conditions, including e.g., different sequences of input images, covering various axial permutations and unknown axial positioning errors. We also demonstrated wide-field to confocal cross-modality image transformations using Recurrent-MZ framework and performed 3D image reconstruction of a sample using a few wide-field 2D fluorescence images as input, matching confocal microscopy images of the same sample volume. Recurrent-MZ demonstrates the first application of recurrent neural networks in microscopic image reconstruction and provides a flexible and rapid volumetric imaging framework, overcoming the limitations of current 3D scanning microscopy tools.

scholarly journals Coherence tomography with broad bandwidth extreme ultraviolet and soft X-ray radiation

2021 ◽  
Vol 127 (4) ◽  
Author(s):  
S. Skruszewicz ◽  
S. Fuchs ◽  
J. J. Abel ◽  
J. Nathanael ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Near Infrared ◽  
Extreme Ultraviolet ◽  
Infrared Light ◽  
Imaging Method ◽  
Optical Coherence ◽  
Axial Resolution ◽  
Cross Sectional ◽  
X Ray ◽  
Broad Bandwidth

AbstractWe present an overview of recent results on optical coherence tomography with the use of extreme ultraviolet and soft X-ray radiation (XCT). XCT is a cross-sectional imaging method that has emerged as a derivative of optical coherence tomography (OCT). In contrast to OCT, which typically uses near-infrared light, XCT utilizes broad bandwidth extreme ultraviolet (XUV) and soft X-ray (SXR) radiation (Fuchs et al in Sci Rep 6:20658, 2016). As in OCT, XCT’s axial resolution only scales with the coherence length of the light source. Thus, an axial resolution down to the nanometer range can be achieved. This is an improvement of up to three orders of magnitude in comparison to OCT. XCT measures the reflected spectrum in a common-path interferometric setup to retrieve the axial structure of nanometer-sized samples. The technique has been demonstrated with broad bandwidth XUV/SXR radiation from synchrotron facilities and recently with compact laboratory-based laser-driven sources. Axial resolutions down to 2.2 nm have been achieved experimentally. XCT has potential applications in three-dimensional imaging of silicon-based semiconductors, lithography masks, and layered structures like XUV mirrors and solar cells.

scholarly journals High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity

2021 ◽  
Vol 118 (8) ◽  
pp. 081104
Author(s):  
Andrew J. Bower ◽  
Carlos Renteria ◽  
Joanne Li ◽  
Marina Marjanovic ◽  
Ronit Barkalifa ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Neuronal Activity ◽  
High Speed ◽  
Label Free ◽  
Two Photon ◽  
Metabolic Transients ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence
Sign in / Sign up

Export Citation Format

Share Document