General Scanning - In the News
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 A better understanding of cell function could provide vital assistance to researchers working on therapies for a wide range of diseases. Confocal laser scanning microscopes (CLSMs) provides a powerful method of observing cellular activity. Resolving dynamic changes in living cells or tissues requires observation at video rates of 30 frames per second, but commercially available CLSMs frequently have slower acquisition rates of only 1 to 5 frames per second. Fast CLSMs have been developed, but their cost is very high. Dr. Ian Parker, University of California, Irvine, and Dr. Michael Sanderson at the University of Massachusetts Medical School, Worcester, Massachusetts, have helped overcome this problem by developing a design for a much less expensive custom CLSM. Their design generates images at a rate of 30 frames per second. The key innovation in the design is the replacement of the slow horizontal scanning system with a resonant scanner or galvanometer that oscillates at 7910 Hz from General Scanning Inc., of Billerica, Massachusetts. Dr. Michael Sanderson explained “this not only provides the necessary speed to scan individual horizontal lines but also has the advantage that the fly-back scan can be used for data collection of the next horizontal line. By reducing the number mirror rotations by half the image acquisition rate is doubled.” Observing Cell Function  The development of a wide range of fluorescent reporter dyes has greatly enhanced the potential ability to observe cell function through a CLSM. Cell activities involving changes in ion concentrations, such as Ca2+ or pH, or the expression of proteins can be readily detected. The challenge is that this application requires high resolution, both in terms of time and space. Fluorescent molecules that are outside the plane of focus reduce the contrast and resolution of the image. CLSMs address this problem by using a small aperture that rejects out of focus light so that the final image represents a thin slice through the specimen with greatly improved axial resolution. CLSMs, however, require that the image be illuminated with a scanning point of light, so the temporal resolution is governed by the time required to span the specimen.  A fast acquisition time is essential when studying rapid biological processes. According to Luc Leybaert, Professor of Physiology, Ghent University, Belgium, “The conclusion is that resonant scanners are still the most versatile technology for fast laser scanning microscopy, because they allow both confocal and two-photon imaging and have many additional advantages over other laser scanning technologies.”The conventional approach of generating the 2D scan across the image is to utilize the reflection of two moving mirrors, aligned at 90 degrees, with the motion of each mirror being driven by a linear saw-tooth control signal. The side-to-side rotation of one mirror creates the horizontal scan line while the up-down rotation of the second mirror creates the vertical deflection. Inertia limits the speed at which mirrors can accurately follow rapidly changing saw-tooth control signals. The researchers overcame this limitation by replacing one of the moving mirrors with a CRS resonant scanner from General Scanning. The scanner oscillates with sinusoidal motion. The mirror gradually accelerates and decelerates as it progresses through the oscillatory cycle, avoiding the need to rapidly reset the scan to the beginning of each line. Dr. Michael Sanderson emphasized this point by noting that “this autonomous oscillation of the horizontal mirror greatly simplifies the design of the control electronics because it eliminates the need for the generation of fast and linear saw-tooth command signals.” The result of this sinusoidal motion is that the image is progressively stretched towards the edges. Fortunately this distortion is predictable and easy to correct by using a simple look-up table to relocate the pixel locations of incoming data to remove image distortion in real time and record the images to hard disk. The image correction process developed by Dr. Sanderson consists of four steps: 1) determine a phase constant for the mirror and frame grabber 2) determine the center pixel of the final image 3) determine the correction factor for each pixel or phase position relative to the center pixel and 4) relocate the pixel position as indicated by the correction factor Dr Sanderson described the utility of this approach to correcting the image distortion as “a neat solution” because “it is independent of hardware and the algorithm works at any angular rotation of the CRS mirror to provide a distortion free-zoom capability for the microscope” Although the CRS scanner provides the key hardware necessary to make fast imaging possible, Dr Sanderson emphasized that “that the real-time acquisition, correction and storage of 4 channels of video data at 30 frames per second is only made possible by employing the advanced recording software provided by Video Savant from IO Industries.” Recent Research CLSM imaging at high temporal and spatial resolution opens up promising new avenues of research. Resonant scanning based CLSM has the potential to determine the complex signaling acting at the center of the brain. It can be used to resolve the dynamics of fast vascular responses to neuronal or glial activation in the brain, leading the way towards better understanding of the neurovascular unit with its neurons, astrocytes, and vessel cells all linked in an intricate communications network. Ian Parker, of the University of California Irvine has extensively used his microscopes to understand, at the subcellular level, elementary calcium-signaling events such as calcium sparks, blips and puffs. More recently at the whole tissue level, Parker and co-workers have used 2-photon CLSM to track the behavior of immune cells in lymph nodes in order to understand how our immune system recognizes and responds to foreign elements. Dr. Sanderson and co-workers have used resonant scanning based CLSM to study in situ the Ca2+ signaling that occurs with airway epithelial and smooth muscle cells (SMCs) In a lung slice, the airway SMCs retain the ability to perform repetitive contractions over several days. The researchers have shown that a variety of drugs such as acetylcholine (ACH) induce a rapid increase in Ca2+ followed by Ca2+ oscillation. Dr. Sanderson reasons “by understanding these fundamental control signals in normal airway smooth muscle cells, we hope to get an insight into the dysfunction associated with smooth muscles in asthma” These oscillations persist while the drug is present and the the cessation of the oscillation is accompanied by the relaxation of the muscle. This research is apparently the first time that Ca2+ signaling in airway SMCs has been studied in situ. It was made possible only through the use of a fast CLSM using a resonant scanning mirror. To see movies of these responses the reader is referred to the web site of Dr. Sanderson. How to Build a CLSM Based on a Fast Resonant Scanner The schematic below shows the basic layout of the confocal microscope. The system is based on an inverted microscope, but the microscope type is not important. All imaging components are mounted on anti-vibration table with an optical bench top. The illumination is generated by a 20 mW solid-state laser (488 nM). The laser light (blue line), controlled with a shutter, is reflected through a neutal density filter to regulate the intensity and a plano-concave lens to expand the beam. The laser beam is selectively filtered and reflected through a dichroic mirror to the scanners to generate a raster scan that enters the microscope via the side camera port. The returning fluorescent light (orangeline) is de-scanned by the same mirror and passes through the dichroic mirror and long pass filter to reach the confocal aperture. Optional dichroic mirrors reflected selected wavelengths to 3 different photomultiplier tubes. A similar, but simpler design with the same control electronics and software can be used for 2-photon microscopy. In this case, the confocal aperture is not required and the PMTs are placed immediately below the objective with a dichroic mirror. (See below) The fast, flexible and relatively inexpensive custom CLSM described here has the potential to dramatically improve the ability of researchers to observe dynamic biological processes in real time. The CRS resonant scanner helps provide the fast frame rates that are the key to its performance. Complete details including all the information needed for the purchase of components and construct of the microscope can be found on the web pages of Dr. Michael Sanderson or Ian Parker. http://users.umassmed.edu/michael.sanderson/mjslab/confocal_microscopy_main.htm
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(First photo) An airway in a lung slice observed with phase-contrast microscopy is induced to contract by the application of the drug methacholine. When the drug is washed away the airway relaxes again. (Second photo) The wall of an airway in a lung slice viewed with confocal microscopy. The airway lumen is lined with cuboidal epithelial cells and is surrounded by thin, long smooth muscle cells. Methacholine increases the Ca2+ inside the SMCs to make the cell contract and this is detected by confocal microscopy as an increase in the brightness of the Ca2+ reporter dye loaded into the smooth muscle cells.
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