Swept Confocally Aligned Planar Excitation (SCAPE) Microscopy Provides High Speed 3D Images

A new microscope that can image cells in living things at high speed and in 3D has been developed at Columbia University Medical Center (CUMC). Swept Confocally Aligned Planar Excitation Microscopy or SCAPE is 10 to 100 times faster in 3D imaging than other conventional microscopy systems.

Elizabeth Hillman who is an associate professor of biomedical engineering at Columbia Engineering and of radiology at CUMC developed the microscope. She believes that the advancement of biomedical and neuroscience research has its foundation on real-time 3D imaging at the cellular resolution. She says that, ""With SCAPE, we can now image complex, living things, such as neurons firing in the rodent brain, crawling fruit fly larvae, and single cells in the zebrafish heart while the heart is actually beating spontaneously--this has not been possible until now.""

Unlike other microscopes that uses two objective lenses to help produce the 3d image, SCAPE uses a single-objective lens. A light sheet is swept through the sample producing the 3D image without even moving the lens or the sample.

The image on the left shows that the sample is lit up by a thin sheet of blue light at an angle which is swept back and forth within the sample. This area is then imaged onto a high speed sCMOS (Scientific Complementary Metal–Oxide–Semiconductor) camera through the objective lens. The illuminated plane is always co-aligned with the plane of the camera through de-scanning and image rotation optics. The end result is data equivalent to conventional light-sheet microscopy, but requiring a single, stationary objective lens, no sample translation, and consequently very high speed 3-D imaging. (See image on the left).

Although SCAPE currently does not have the penetration depth of a two photon microscope, the next-generation versions of SCAPE are in development that will deliver even better speed, resolution, sensitivity, and penetration depth.

Aside from neuroscience and biomedical research, where capturing live neurons can now be possible, SCAPE also has future potential in molecular biology and other clinical applications.

MIT News: Researchers Improve Electron Microscopy Using Engineered Protein Labels

A fluorescent microscope is an optical microscope that is used to observe specimens that undergo a fluorescence or phosphorescence stage. The specimen has small molecules called fluorophores attached to it. These fluorophores emit light when irradiated with a specific wavelength of light. This causes the specimen to be illuminated from within the specimen itself, generating a much more detailed image.

The molecule used in making the specimen fluorescent is the green fluorescent protein (GFP). It gives off a bright green fluorescence when exposed to light in the blue to ultraviolet range. GFP is generally safe to use when illuminating live cells.

This imaging technique has revolutionized molecular biology. But since fluorescent microscopes are optical in nature, this technique cannot be used with electron microscopes. Optical microscopes uses light beams to image a specimen, electron microscopes use beams of electrons to produce a magnified image.

High Speed Digital Microscope Developed For High Resolution Imaging

Scientists at the University of Leicester have developed a rapid scanning microscope that images without the loss of quality. The new form of digital confocal microscope that can create an image 100 times faster than a regular microscope.

The digital microscope can be attached on to a regular microscope and projects light through a system of mirrors on to the microscopic sample. Mimicking a television, the device projects patterns of illumination onto the specimen, and only light that is precisely in the plane of focus returns along the same path and is reflected by the mirror onto a camera to form an image.

The ability to be able to program the mirror device allows the illumination pattern to be adjusted easily for different types of specimens and conditions giving ease of use and flexibility. Unwanted light that comes from regions of the specimen which are out of focus are rejected, improving the image quality.

The resulting images can be scanned on a computer at around 100 frames per second, showing biological processes such as cell activity at much higher speeds than regular microscopes - which tend to be capped at around 1 frame per second.

The Leicester team's microscope has no moving parts, making it robust, and the use of a programmable, digital micromirror allows the user to alter the size and spacing of mirrors in order to choose the quality of the image and adapt to different imaging conditions. Consequently, it has much greater flexibility than other microscopes capable of similar speeds.

The findings are due to be published on the online journal PLOS ONE on August 24.

The researchers believe this technology will be a big help to those working in many scientific fields, including biomedical research and neuroscience.

Virtual Nanoscopy Enables Large Scale Composite Images

The electron microscope is a type of microscope that uses a beam of electrons to illuminate and create an image of a target specimen. The electron microscope has a greater resolving power than a light powered optical microscope allowing it to view smaller objects in greater detail.

Electron microscopes are able to do this because electrons have wavelengths about 100,000 times shorter than photons (light). They can achieve better than 50 picometer resolution and magnifications of up to about 10,000,000x whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nanometer resolution and useful magnifications below 2000x.

It uses electrostatic and electromagnetic "lenses" to control the electron beam and focus it to form an image. These lenses operate somewhat similar to the glass lenses of an optical microscope that form a magnified image by focusing light on or through the specimen. Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is often used for quality control and failure analysis.

Image: Advances in “virtual nanoscopy” enable the generation of large-scale composite images of biological tissues, as described in The Journal of Cell Biology (JCB) and made accessible through an upgrade to JCB’s JCB DataViewer web application. Users can “zoom in” from a high-resolution, composite image of a zebrafish embryo (top) to detailed images of tiny subcellular structures (bottom). Credit: © Williams et al, 2012

Virtual nanoscopy: Like 'Google Earth' for cell biologists

Just as users of Google Earth can zoom in from space to a view of their own backyard, researchers can now navigate biological tissues from a whole embryo down to its subcellular structures thanks to recent advances in electron microscopy and image processing, as described in The Journal of Cell Biology (JCB). An upgrade to the JCB DataViewer, JCB's browser-based image presentation tool, now also makes these data publicly accessible for exploration and discovery.

The JCB DataViewer is an image hosting and presentation platform for original image data associated with articles published in JCB. Developed in a collaboration between Glencoe Software, Inc. and the Rockefeller University Press, the JCB DataViewer was the first browser-based viewer for multidimensional microscopy image data. It is based on open source software built by the Open Microscopy Environment (OME).

Since the early days of cell biology, electron microscopy has revealed cellular structures in exquisite detail. The technique has always been limited, however, by the fact that it can only capture a tiny portion of the cell in a single image at high resolution, making it difficult for researchers to relate the structures they see to the cell as a whole, let alone to the tissue or organ in which the cell is located. Viewing samples at lower resolution, on the other hand, can reveal the larger picture of a cell or tissue, but researchers then lose the benefit of seeing fine details.