Are you confused by the terms light sheet and confocal? Are they completely unrelated or does one fall under the definition of the other?
In this article I will define the terms and explain exactly how they are related, along with additional information such as comparisons between the two.
In general terms, in light sheet microscopy samples are illuminated by a thin sheet of light emitted from the side. In confocal microscopy, point illumination as well as a pinhole to block out-of-focus light is used. Both methods use fluorescence and are capable of producing stacks and 3D renderings, with light sheet being more capable of scanning larger tissue volumes at higher speed while confocal being better at depicting subcellular detail at higher magnification.
To understand how this works, you first need to know how a standard SPIM works. As its name implies, in SPIM, the specimen is illuminated with a thin, wide sheet of light to observe the specimen’s fluorescence over time.
In confocal SPIM, there is an additional feature: a pinhole positioned between the detector and the objective lens that allows only fluorescence from one focal plane to be detected at a time.
The pinhole blocks out-of-focus light from above and below the focal plane, which improves image resolution compared to non-confocal SPIMs.
Light Sheet Microscopy Basics
Light-sheet microscopy is a method for non-invasive, high-resolution imaging of biological samples. In this technique, cells are illuminated by a thin sheet of light propagated orthogonally to the light path for image acquisition.
This results in high penetration with minimal photobleaching and phototoxicity and thus allows long-term imaging of live samples.
Light-sheet microscopy benefits from the use of several specialized optical components. These include a condenser to generate the light sheet, an objective to collect emitted fluorescence or reflected light, and a tube lens in between to relay the image from one to the other.
The system can be arranged in two configurations: single plane illumination (SPIM) microscopy, where only one side of the sample is illuminated at a time, and dual-plane illumination (DPIM) microscopy, where both sides of the sample are illuminated simultaneously.
In addition, SPIM can be divided into two types: off-axis SPIM and reflective SPIM. Off-axis SPIM illuminates the sample from 90° with respect to the detection axis.
Reflective SPIM illuminates the sample from below through a specially designed glass-bottom dish or a coverslip coated with a scattering material.
Uses of Light sheet
Cell biology
If you want to visualize organelles in 3D, you can do that with a light-sheet microscope.
This is the most common application that people use it for.
To observe cellular dynamics
The microscope is designed to observe cellular dynamics over a long period in living specimens.
It employs a laser light sheet, a technique that uses a sheet of laser light illuminating the sample from an angle, as opposed to conventional microscopy, where the specimen is bombarded with illumination from all directions.
This means that the cells are exposed to less light than conventional microscopy to be observed for more extended periods without damage.
The critical advance lies in adaptive optics, a technique first developed to improve astronomical images taken by ground-based telescopes.
By measuring distortions caused by turbulence in the atmosphere, adaptive optics can alter the wavefront of incoming light using a deformable mirror.
The result is an image that is much sharper and more detailed than possible.
To visualize gene expression
Light-sheet fluorescence microscopy (LSFM) is a fluorescence imaging technique. It uses a thin sheet of light to illuminate a single plane of the specimen, while fluorescence from other aircraft is suppressed.
LSFM can provide optical sectioning similar to confocal microscopy or multiphoton microscopy by scanning the light sheet with less photodamage and photobleaching.
Moreover, LSFM enables fast 3D imaging of entire cells and organisms without damaging them through photobleaching and phototoxicity. It also allows for visualizing gene expression in whole embryos, with minimal disturbing light penetration into the specimen and surrounding area.
To perform super-resolution imaging
Light-sheet microscopy has been used to perform super-resolution imaging. This is achieved by using a pulsed laser to illuminate a thin section of the sample and taking images at the axial focal plane.
The sample is moved slightly in the z-axis, and the process is repeated. The resulting images are then combined using computational methods, such as iterative deconvolution or super-resolution optical fluctuation imaging (SOFI) analysis, to obtain an image with resolution better than that achievable with conventional microscopes.
Light sheet vs Confocal- Differences and Similarities
Light-sheet and confocal microscopes have several differences and similarities. Here are some of the differences.
Differences
Image Quality
With confocal microscopy the specimen must be illuminated through a pinhole and viewed under a highly narrow angle of view. This results in a high-quality image because it blocks scattered light from outside the focal plane, which can degrade image resolution.
In contrast, a light sheet is a fragile sheet of light that illuminates the entire sample from the side, creating a more uniform image. However, because the light is much brighter than in a confocal microscope, it is harder to control the scattered light outside the focal plane (especially for large specimens).
Light Efficiency
The efficiency of a laser can be increased by spreading it over a large surface area. A light sheet microscope takes advantage of this by using lasers to create fragile sheets of light that can cover large areas.
These sheets are created using cylindrical lenses and cylindrical mirrors and go through the specimen one by one. In contrast, only a tiny fraction of the laser energy makes it through the pinhole aperture to illuminate the sample in confocal microscopy.
With confocal microscopes, there are issues of light penetration through thick tissues. Even if the tissue is fully permeated by a fluorescent antibody marker, it may not be visible for this reason.
Speed
In traditional confocal microscopy, the laser beam is scanned across one sample axis, which is also moved along an orthogonal axis.
In light-sheet fluorescence microscopy (LSFM), the specimen is illuminated by a thin sheet of light that sweeps across it in one direction. At the same time, a detector scans in the perpendicular direction.
This arrangement makes it possible to acquire images at high speed, with very low photo-bleaching and phototoxicity.
LSFM is well suited to live cell imaging and other applications where dynamic processes need to be observed and recorded over time without damaging the sample.
The speed of lightsheet microscopes is what makes them perfect for studying large tissues or even entire organs such as rodent brains that have been cleared with methods like CLARITY. This is best for studying things such as neuron projections from one part of the organ to another. While light sheet microscopes are achieving better and better resolution, confocal microscopes still dominate in terms of observing cell detail under very high magnification (up to 150x objective).
Similarities
Both types of microscopes can be used to create image stacks and ultimately 3D volume or surface renderings of the objects studied.
Both light-sheet microscopy and confocal microscopes are types of fluorescence microscopes. Both techniques involve laser scanning of a specimen to produce an image.
The main difference is that confocal microscopes use a series of pinholes to exclude light outside the focal plane. In contrast, light-sheet microscopy combines illumination and detection along the same axis using a thin light sheet.
Light-sheet microscopy uses a thin sheet of laser light to illuminate the specimen below or above rather than illuminating it with a point source (as in a conventional microscope).
The thin layer of light reduces photobleaching and phototoxicity, which are significant problems when imaging live specimens over time. The advantage over confocal microscopy is speed; because fluorescence emitted from all points on the specimen are collected at once.
Both light sheet and confocal microscopes can be used to image cleared specimens.
How old is light sheet microscopy?
The Ultramicroscope was the first light-sheet microscope, and was first created in 1903 by Richard Adolf Zsigmondy and Henry Siedentopf. It consisted of a rectangular slit combined with white light. It produced images of microscopic particles and enabled scientists to closely observe the interaction of individual molecules. However, it was not a fluorescent light sheet microscope. Those did not appear until the 1990s.
Price of light sheet vs confocal
When it comes to light-sheet microscopy, a typical system costs around 200,000- $400,000, depending on what features are included. The lattice light-sheet approach for in-cell imaging costs about $700,000.
Confocal microscopes are still more popular and more available than light sheet and can be purchased used for a much lower price, but a modern confocal microscope from a well known brand such as Olympus or Nikon, with an average of 4 lasers, will cost around $300,000.
Pricing of either can vary greatly based on the type of imaging you want to do, the type of specimen holders, objective types which can have different characteristics even with the same magnification, and attachments. You can even buy a light sheet attachment to a confocal microscope, but those can be pricey at $200,000.
Here is a useful video on light sheet microscopy basics:
I hope you found this article useful. Click the following link to learn the maximum magnification of a confocal microscope.
