Light sheetmicroscopy

Lattice light sheet microscopy is a novel combination of techniques from Light sheet fluorescence microscopy, Bessel beam microscopy, and Super-resolution microscopy (specifically structured illumination microscopy, SIM[4]).

The collected fluorescence signals are then processed and used to construct an image of the sample. The scanning mechanism allows for the acquisition of multiple optical sections at different depths within the sample, which can be used to generate a three-dimensional image.

Light sheetfluorescence microscopy

To generate a three-dimensional image, the laser beam is scanned across the sample in a raster pattern. The reflected light is collected at each point and used to build up a two-dimensional image. By scanning the laser beam through multiple planes, a stack of images is acquired, which can be reconstructed into a three-dimensional image of the sample.

A laser scanning confocal microscope (LSCM) is an advanced imaging technique that allows for high-resolution, three-dimensional imaging of biological samples. It works by using a laser beam to scan the sample and collect fluorescence signals, which are then used to construct an image.

In recent years, there have been advancements in LSCM technology. For example, the use of multiple lasers with different wavelengths allows for the simultaneous imaging of multiple fluorophores, enabling the study of multiple cellular components or processes in a single experiment. Additionally, the development of faster scanning systems and sensitive detectors has improved the speed and sensitivity of LSCM, making it more suitable for live-cell imaging.

Light sheetmicroscopy using an Airy beam

As the laser beam scans across the sample, it excites fluorescent molecules within the sample. These molecules emit light at a longer wavelength, which is then collected by a detector. However, to ensure that only light from the focal plane is detected, a pinhole aperture is placed in front of the detector. This pinhole blocks out-of-focus light, resulting in improved image contrast and resolution.

A laser scanning confocal microscope (LSCM) is an advanced imaging technique that provides high-resolution, three-dimensional images of biological samples. It works by using a laser beam to scan the sample and a confocal pinhole to eliminate out-of-focus light, resulting in improved image quality and optical sectioning.

Overall, LSCM is a powerful imaging technique that provides high-resolution, three-dimensional images of biological samples. Its ability to eliminate out-of-focus light and its compatibility with various fluorophores make it an essential tool in biological research and medical diagnostics.

Lattice light sheet microscopy can be viewed as an improvement of Bessel beam light sheet microscopes[8] in terms of axial resolution (also termed resolution in z). In Bessel beam light sheet microscopes, a non-diffracting Bessel beam is first created then dithered in the x direction to produce a sheet. However, the lobes of a Bessel functions carry as much energy as the central spot, resulting in illumination out of the depth of field of the observation objective.

The detector collects the emitted light and converts it into an electrical signal, which is then processed and used to generate an image. The scanning mirrors, confocal pinhole, and detector are all synchronized to ensure that only light from the focal plane is detected, resulting in a sharp, high-resolution image.

By scanning the laser beam across the sample in a raster pattern, a series of optical sections are obtained at different depths. These sections are then combined to create a three-dimensional image of the sample. The confocal microscope provides high-resolution images with improved contrast and reduced background noise compared to conventional microscopes. It is widely used in various fields of research, including biology, medicine, and materials science, for studying the structure and function of biological samples and other materials at the cellular and subcellular levels.

Finally, to obtain a uniform intensity at the sample rather than a lattice, the sheet is dithered using a galvanometer oscillating in the x direction.

Practically, the lattice of interfering Bessel beams is engineered by a spatial light modulator (SLM), a liquid-crystal device whose individual pixels can be switched on and off to display a binary pattern. Due to the matrix nature of the SLM, the generated pattern contains many unwanted frequencies. Thus, these are filtered out by the means of an annulus placed in a plane conjugated with the back focal plane of the objective (Fourier domain).

Lattice Light-Sheet Microscopy combines high resolution and clarity at high image acquisition speed, without damaging samples through photobleaching.[1] Photobleaching is a major and highly common problem in fluorescence microscopy wherein fluorescent tags will lose their ability to emit photons upon repeated excitation. Unlike common fluorescence microscopes, samples in a Lattice Light-Sheet Microscope experience photobleaching at a rate drastically reduced when compared to conventional techniques (In conventional techniques, this results in an image signal that gets weaker over the course of multiple excitations). This allows for longer exposures without loss of signal, which in turn allows for video to be captured at over longer periods of time. The Lattice method also has the ability to resolve 200 to 1000 planes per second, an extremely fast imaging rate that allows continuous video capture. This capture rate is one order of magnitude faster than Bessel beam excitation, and two orders of magnitude faster than Spinning Disk Confocal Microscopy.[1] These two advantages combine to allow researchers to take very detailed movies over long periods of time.

The principle of laser scanning confocal microscopy involves the use of a laser beam to illuminate a sample and a pinhole aperture to eliminate out-of-focus light. This technique allows for the acquisition of high-resolution, three-dimensional images of biological samples.

Latticelight sheetmicroscopy

Next, a set of scanning mirrors is used to move the laser beam across the sample in a raster pattern. These mirrors can rapidly scan the laser beam in both the x and y directions, allowing for precise control over the scanning process.

The scanning mechanism in LSCM is a key component that enables the microscope to capture images with high spatial resolution. The laser beam is focused onto a small spot on the sample, and the spot is then scanned across the sample in a raster pattern. This scanning is typically achieved using a pair of galvanometer mirrors that rapidly move the laser beam in the x and y directions.

Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes.[1][2]

Confocal microscopy

The basic principle of LSCM involves focusing a laser beam onto a specific point on the sample. The laser light excites fluorophores in the sample, causing them to emit fluorescent light. However, instead of collecting all the emitted light, LSCM uses a pinhole aperture to block out-of-focus light. This pinhole is placed in front of a detector, which only detects the light that is in focus. By scanning the laser beam across the sample and detecting the emitted light at each point, a high-resolution image is formed.

Lattice light sheet microscopy aims at reducing the intensity of the outer lobes of the Bessel functions by destructive interference. To do so, a two-dimensional lattice of regularly spaced Bessel beams is created. Then, destructive interference can be triggered by carefully tuning the spacing between the beams (that is, the period of the lattice).

In a laser scanning confocal microscope, a laser beam is focused onto a specific point on the sample. The laser light is then reflected off the sample and collected by a detector. The detector measures the intensity of the reflected light, which is used to generate an image of the sample.

The key component of a confocal microscope is the pinhole aperture. This aperture is placed in front of the detector and blocks out-of-focus light from reaching the detector. By eliminating this out-of-focus light, the confocal microscope can produce images with improved contrast and resolution compared to conventional microscopes.

Latticelight sheetmicroscopy: imaging molecules to embryos at high spatiotemporal resolution

The technique, along with chemical and genetic manipulation techniques, was used to capture a live image of a virus (a virus that was engineered to spike COVID-19 proteins) infecting a cell, by injecting its genetic material into the cell's endosome for the first time, at Harvard Medical School, in cooperation with other institutions.[9][10]

Recent advancements in LSCM technology have focused on improving imaging speed and sensitivity. For example, the use of resonant scanning mirrors allows for faster scanning rates, enabling real-time imaging of dynamic processes. Additionally, the development of highly sensitive detectors and advanced image processing algorithms has further enhanced the capabilities of LSCMs.

Overall, laser scanning confocal microscopy provides a powerful tool for studying biological samples with high resolution and three-dimensional imaging capabilities. Its ability to eliminate out-of-focus light and its compatibility with various imaging techniques make it an essential tool in modern biological research.

The aggregation of T cell and target cells was observed, along with the subsequent formation of the immunological synapse. The advancements of the lattice sheet method revealed three-dimensional movement patterns of actin as well as lamellipodial protrusion in these interactions. The increase in imaging speed also allowed the observation of fast moving neutrophils through the extracellular matrix in another study[citation needed].

In lattice light sheet microscopy, very similarly to light sheet microscopy, the illumination of the sample occurs perpendicular to the image detection. Initially the light sheet is formed by stretching the linearly polarized circular input beam with a pair of cylindrical lenses along the x axis and then compressing it with an additional pair of lenses along the z axis.[5] This modification creates a thin sheet of light that is then projected onto a binary ferroelectric spatial light modulator (SLM). The SLM is a device that spatially varies the waveform of a beam of light. The light that is reflected back from the SLM is used to eliminate unwanted diffraction. Diffraction is eliminated by the transform lens that creates a Fraunhofer diffraction pattern from the reflected light at an opaque mask containing a transparent annulus.[5] Optical lattices are two or three dimensional interference patterns, which here are produced by the transparent annular ring. The mask is conjugate to x and z galvanometers. This quality of the microscope is important for the dithered mode of operation, where the light sheet must be oscillated within the x axis.

A laser scanning confocal microscope (LSCM) is an advanced imaging technique that allows for high-resolution, three-dimensional imaging of biological samples. It works by using a laser beam to scan the sample point by point and then detecting the emitted light from each point.

light sheetmicroscopy中文

As the laser beam interacts with the sample, it excites fluorescent molecules present in the sample. These molecules emit light at a longer wavelength, which is then collected by a detector. The emitted light passes through a confocal pinhole, which is placed in front of the detector. The pinhole acts as a spatial filter, allowing only the light emitted from the focal plane to pass through while blocking out-of-focus light.

The pinhole aperture in LSCM is a crucial component as it eliminates the out-of-focus light, resulting in improved image contrast and resolution. This technique, known as optical sectioning, allows for the visualization of thin optical sections of the sample, which can be reconstructed into a three-dimensional image.

It was developed in the early 2010s by a team led by Eric Betzig.[1] According to an interview conducted by The Washington Post, Betzig believes that this development will have a greater impact than the work that earned him the 2014 Nobel Prize in Chemistry for "the development of super-resolution fluorescence microscopy".[3]

Lattice light sheet microscopy is useful for in-vivo cellular localization and super resolution. Lattice light sheets' confined excitation band keeps nearly all illuminated cells in focus. The reduction of large, out of focus spots allow precise tracking of individual cells at a high molecular density, a capability unattainable through previous microscopy methods.[1] Consequently, lattice light sheet is being used for a number of dynamic cellular interactions. The decrease in phototoxicity has created opportunities to study the subcellular processes of embryos without damaging their living tissues. Studies have examined and quantified the extent of the highly variable growth patterns of microtubules throughout mitosis. Dictyostelium discoideum (slime mold) cells were imaged during their rapid chemotactic movement toward one another and the initial contact.

A guide to light-sheet fluorescence microscopy for multiscale imaging

In conclusion, a laser scanning confocal microscope works by using a laser beam to scan a sample and a confocal pinhole to eliminate out-of-focus light. This technique provides high-resolution, three-dimensional images of biological samples and has seen significant advancements in recent years.

Overall, the scanning mechanism in laser scanning confocal microscopy plays a crucial role in capturing high-resolution images of biological samples. The continuous advancements in technology continue to enhance the capabilities of LSCM, making it an invaluable tool in biological research.

The latest advancements in laser scanning confocal microscopy include the use of multiple lasers with different wavelengths to excite different fluorophores simultaneously. This allows for the visualization of multiple cellular components or molecules within a sample. Additionally, confocal microscopes can now be equipped with advanced imaging techniques such as fluorescence lifetime imaging or fluorescence correlation spectroscopy, which provide further insights into the dynamics and interactions of biological molecules.

A laser scanning confocal microscope works by using a laser beam to illuminate a sample and then collecting the emitted light through a pinhole aperture. The laser beam is focused onto a specific point on the sample, and the emitted light is detected by a photomultiplier tube or a detector array. The pinhole aperture allows only the light from the focal plane to pass through, while blocking out-of-focus light.

The basic principle of an LSCM involves several optical components. Firstly, a laser is used as the light source, typically a high-intensity, monochromatic laser such as a helium-neon or argon-ion laser. The laser emits a focused beam of light that is directed onto the sample.

The lattice light-sheet microscope has two modes of operation: In the dithered mode, the light sheet is rapidly scanned along the x axis and only one image is recorded per Z plane, at normal diffraction limited resolutions.[1] The second mode of operation is the structured illumination microscopy mode (SIM). SIM is a technique where a grid pattern of excitation light is superimposed on the sample and rotated in steps between the capture of each image.[4][6][7] These images are then processed via an algorithm to produce a reconstructed image past the limit of diffraction that is built into our optical instruments.

In recent years, there have been advancements in LSCM technology. For example, the use of resonant scanners has allowed for faster scanning speeds, enabling real-time imaging of dynamic processes. Additionally, the development of adaptive optics has improved the resolution and image quality by correcting for aberrations in the optical system.

The technique is being actively developed at the Janelia Research Campus of the Howard Hughes Medical Institute.[11] Eric Betzig has stated that his goal is to combine his work on microscopy to develop a "high-speed, high-resolution, low-impact tool that can look deep inside biological systems."[3] Penetration deeper than 20–100 μm may be achieved by combining lattice light-sheet microscopy with adaptive optics.[1]

Lattice light sheet microscopy is limited to transparent and thin samples to achieve good image quality. The quality of image acquired degrades with imaging depth. This phenomenon occurs due to sample-induced aberrations, and it has been proposed that imaging samples to beyond 20 to 100 μm will require adaptive optics.[1]