Confocal Laser Scanning Microscope
Light microscope is a fundamental tool in science. In biological research, fluorescent labeling of samples, when combined with light microscopy, allows visualization of specific elements of interest; proteins, lipids, nucleic acids, etc. The ability of widefield fluorescent microscope to resolve these elements is limited because of out of focus light that the objective lens unnecessarily captures. In the context of optical systems, confocal means that all visualized objects share the same foci or focus.
Laser Scanning Microscope
Confocal Microscopy Background
In year 1957, Marvin Minsky filed a patent that introduced the first model of an electronic scanning confocal microscope (Fig1). In his microscope model, a point source illumination and accurate moving of the specimen use to achieve point-by-point scanning of the specimen in stepwise manner. Confocality is achieved by an aperture (or pinhole) placed in front of the light detector and permit transmission of light from the focal plane only; thus creating an image that is an optical section of the sample at the focal plane. The microscopic image is generated by projection of the collected "dots" as pixels on a monitor.
- Figure 1. US patent 3013467. Microscopy apertures, By Marvin Minsky.
Principals presented in this model are still implemented in modern confocal microscopes
Confocal Laser Scanning Microscope (CLSM), also known as Point Scanning Confocal Microscope, utilize several elegant techniques to achieve significant enhancement in microscopic image quality and precision. As implicated by the name, CLSM systems use laser-source illumination to excite fluorescent probes in a specific point of the specimen. Motorized mirrors direct the light beam rapidly and precisely over the sample in the necessary stepwise-movement. Set of optical filters and mirrors are used to separate light into different wavelength, and transfer it through an adjustable pinhole to photo-detector that translate the light into electronic signal. Digital signals are processed, presented, and stored as an image by the adjacent software. Over the last 30 years, constant improvement in optics, electronics, mechanics, and signal processing of CLSM platforms increased the scientist ability to collect data from samples in good signal to noise ratio.
The motivated CLSM user should be familiar with the principles of confocality in order obtain meaningful data from samples. Theoretical and practical aspects of the used system should be considered when designing experiments, acquiring data, and analyzing it. Here, at the Cellular Imaging Core Unit at IKI, we provide education and support for interested users.
Many useful links to information sources are well organized in the confocal microscopy page of the University of Arizona.
The 8th generation Confocal instruments offers ultimate sensitivity and resolving power for high demanding samples. Classical CLSM has some inherent limitations. Attempt to increase the resolution by making the pinhole smaller will necessarily lead to significant decrees in detected light - consequently - the signal-to-noise ratio (SNR) drops. To balance between resolution, SNR, and acquisition speed, Carl Zeiss developed a new approach to laser scanning confocal microscopy named Airyscan.
Super Resolution Confocal with Airyscan:
Airyscan is a detector that draws on the fact that a fluorescence microscope will image a point-like source as an extended Airy disk (Airy pattern). Airyscan solves this conundrum between resolution and light efficiency by imaging the Airy disk onto a concentrically arranged hexagonal detector array. Its detection area consists of 32 single elements, each of which acts like a very small pinhole. The confocal pinhole itself remains open and does not block light – thus all photons of the whole Airy disk are collected (Fig 2 left). The signals from all detector elements are then reassigned to their correct position, producing an image with increased SNR and resolution. Because it capitalizes on the scanning and optical sectioning capabilities of a confocal, Airyscan works with standard samples and standard dyes, and even with your thicker samples such as tissue sections.
- Figure 2. Super-Resolution and FAST Airyscan modules
In the additional Fast mode, you profit from yet another advantage of the area detector. The excitation beam is elongated in y and the Airyscan detector, with just one horizontal scanner movement, acquires four lines of image information instead of only one (Fig 2 right). This parallelization delivers a unique combination of high speed, high resolution and high sensitivity. - Figure 3. Drosophila melanogaster neuromuscular junctions stained for Bruchpilot (BRP). Sample courtesy of J. Pielage, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
34 Channels Spectral Imaging:
The Zeiss LSM880 Confocal Microscope is equipped for multi-channel fluorescent imaging. The spectral detector together with the software for spectral unmixing makes it possible to image simultaneously multiple different fluorophores, provided that reference spectra for each of the individual fluorophores are available. The spectral detector also can be used for conventional multi-channel fluorescence imaging. The high sensitivity of the detector allows detection of weak fluorescent signals while employing low levels of laser illumination. In samples with high auto-fluorescent (e.g. chlorophyll in plant cells), the spectral imaging and linear unmixing can be used to record the auto-fluorescent spectral characteristics, and remove it later from the image (Fig 4).
Dr. Uzi Hadad
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