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Video Microscopy, Video Cameras, and Image Enhancement Masafumi Oshiro, Lowell A. Moomaw and H. Ernst Keller Cold Spring Harb Protoc; doi: 10.1101/pdb.top081448 Email Alerting Service Subject Categories
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Topic Introduction
Video Microscopy, Video Cameras, and Image Enhancement Masafumi Oshiro, Lowell A. Moomaw, and H. Ernst Keller
Video microscopy is the application of video technology to microscopy, resulting in two fields of microscopy called video-enhanced contrast microscopy (VEC) and video-intensified microscopy (VIM). VEC involves the production of an image from a specimen that is invisible to the eye, either because of a lack of contrast or because of its spectral characteristics (UV or infrared). VIM involves imaging a specimen when the light levels are too low for standard cameras or, in some cases, even for the eye. Images are produced by VIM using image analysis computers.
INTRODUCTION
The major components for video-enhanced contrast microscopy (VEC) are a differential interference contrast (DIC) or phase-contrast microscope, a standard video rate high-resolution camera, a realtime image processor, and a high-resolution video monitor. The use of a video rate camera in conjunction with a real-time image processor greatly improves the contrast of the image and enables the use of the maximum aperture of the optical system. As a result, maximum resolution of the microscope is achieved with sufficient contrast to render the information perceivable. The video-intensified microscopy (VIM) method usually uses a fluorescence microscope, a lowlight-level video camera, a real-time image processor, and a high-resolution video monitor. The lowlight-level video camera and real-time image processor act together to integrate the image. As a result, detailed structure within the specimen is revealed, and photo damage to the sample is minimized by reducing illumination intensity. Even in ultralow-light situations, such as bioluminescence and chemiluminescence, a photon-counting camera detects single photon events, and an image processor integrates the image to generate gray-level information based on photon counting per pixel. This rapidly growing field is well on its way to replacing conventional photomicroscopy. Technical advances in cameras, camera electronics, and image processors, as well as printers, have reached a point where the resolution of the video image becomes indistinguishable from that of photographic fine-grain film. Although it is substantially more expensive, the added technical benefits of video microscopy are considerable. These include:
• • • •
Direct display and optional recording of dynamic events, including time-lapse studies of live cells. Availability of a wide range of analog and digital cameras for every budget and to suit many specific applications. Electronic contrast enhancement via gain and black-level control, or digital image processing, reveals image information inaccessible to eye or film. It is possible to record exceptionally low light levels with time integrating or intensified (e.g., ICCD) cameras. This is important in studies involving weak fluorescence emissions in
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081448
359
Downloaded from http://cshprotocols.cshlp.org/ at University of Southern California - Norris Med Library on April 2, 2014 - Published by Cold Spring Harbor Laboratory Press
M. Oshiro et al.
vivo (e.g., following microinjection of fluorochrome-labeled proteins or transfection of cells with green fluorescent protein [GFP] constructs) (see Spector et al. 1998a) or for minimizing the phototoxic effects of illuminating cells with high light intensities.
• • •
Electronic color balancing with single, three-tube, or chip color cameras. Electronic image transfer to other locations. Optional image storage for further processing or image analysis.
To take full advantage of video-enhanced microscopy, it is important to know a few basic facts about video cameras and image processors. There are many versions of each, and each version has certain characteristics that must be considered for different applications.
DEFINITIONS
Several terms used throughout this topic introduction that may be unfamiliar to the reader are defined below. Analog and digital cameras: An analog video signal is generated from the detector itself. This video signal is processed and converted to certain formats to interface to display monitors, image storage devices, and image processors. If this interface signal is analog, the camera is called an analog camera. If the interface signal is digital, the camera is called a digital camera. Analog cameras normally generate a standard video signal such as RS-170. Integration camera: The standard video camera’s frame rate is 30 frames/sec. This means that incoming light is integrated in a detector for 1/30 sec, and the signal is read out after the integration. To realize low-light sensitivity, integration cameras integrate incoming light for more than one frame, and the integrated signal is then read out. The sensitivity is increased in proportion with the number of integrating frames. However, maximum integration frames are limited by the dark noise of the camera itself. Video rate camera: A camera that generates a standard analog video signal, such as RS-170, is called a video rate camera. Image processor: An image processor modifies, analyzes, displays, and stores images. Readout speed: The speed at which each pixel is read out of a charge-coupled device (CCD) camera. The unit is megahertz per pixel or kilohertz per pixel. Linearity versus lag: Linearity is the relationship between incoming light to a video camera and the output signal from the video camera. If some signal generated in a detector remains in the detector after the reading, the leftover signal appears as the lag. Frame buffer: A frame buffer (or memory plane) stores one or more complete images for further image processing. Horizontal versus vertical resolution: The horizontal and vertical spatial resolutions are not always the same. In the case of video rate cameras, the horizontal resolution is higher than the vertical. Normally, slow-scan cooled CCDs have square pixels, and the resolution for both directions is the same. Geometric distortion: Tube-type cameras and intensified cameras have geometric distortion. The distortion of tube cameras is caused by the mechanism of the beam scanning. The distortion of intensified cameras is caused by the intensifiers and relay optics, such as relay lens and tapered fiber plates. Spectral range: The spectral sensitivity range of detectors. Temporal resolution: The speed at which one complete image is acquired determines the temporal resolution. The temporal resolution of video rate is 33 msec. The temporal resolution of slow-scan cooled CCD cameras is low because of the slow readout speed and the time of signal integration. Camera nonuniformity: Detectors do not have uniform sensitivity over an entire area. The camera nonuniformity of tube cameras and intensified cameras is higher than that of CCD cameras. 360
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081448
Downloaded from http://cshprotocols.cshlp.org/ at University of Southern California - Norris Med Library on April 2, 2014 - Published by Cold Spring Harbor Laboratory Press
Video Microscopy
VIDEO CAMERAS
The following are two basic classifications of cameras: High-light-level cameras and low-light-level cameras. A common component to the two classifications is a CCD camera. This is an electronic device that incorporates a two-dimensional (2D) photodetector, an amplifier, and a timing device that produces a signal that conforms to one of the industry standards. These standards are indicated by the terms RS-170, RS-330, RS-343, NTSC, RGB, and others. This signal transfers an image from the detector to an image processor, a storage device, or display device. The details of this operation can be found in various sources. High-Light-Level Cameras
Although most low-light-level cameras work in high light levels, these cameras are expensive. If highlight-level images are to be recorded, the use of a high-light-level camera offers benefits in both cost and convenience. The following are characteristics of high-light/CCD cameras.
• • • • • • • • •
Detectors are available in 1/3, 1/2, 2/3-in, and larger formats. The size of the detector does not determine the number of horizontal pixels. Horizontal resolution is a function of the number of horizontal pixels. Spectral sensitivity ranges from
Video Microscopy, Video Cameras, and Image Enhancement Masafumi Oshiro, Lowell A. Moomaw and H. Ernst Keller Cold Spring Harb Protoc; doi: 10.1101/pdb.top081448 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Imaging/Microscopy, general (537 articles) Video Imaging / Time Lapse Imaging (164 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at University of Southern California - Norris Med Library on April 2, 2014 - Published by Cold Spring Harbor Laboratory Press
Topic Introduction
Video Microscopy, Video Cameras, and Image Enhancement Masafumi Oshiro, Lowell A. Moomaw, and H. Ernst Keller
Video microscopy is the application of video technology to microscopy, resulting in two fields of microscopy called video-enhanced contrast microscopy (VEC) and video-intensified microscopy (VIM). VEC involves the production of an image from a specimen that is invisible to the eye, either because of a lack of contrast or because of its spectral characteristics (UV or infrared). VIM involves imaging a specimen when the light levels are too low for standard cameras or, in some cases, even for the eye. Images are produced by VIM using image analysis computers.
INTRODUCTION
The major components for video-enhanced contrast microscopy (VEC) are a differential interference contrast (DIC) or phase-contrast microscope, a standard video rate high-resolution camera, a realtime image processor, and a high-resolution video monitor. The use of a video rate camera in conjunction with a real-time image processor greatly improves the contrast of the image and enables the use of the maximum aperture of the optical system. As a result, maximum resolution of the microscope is achieved with sufficient contrast to render the information perceivable. The video-intensified microscopy (VIM) method usually uses a fluorescence microscope, a lowlight-level video camera, a real-time image processor, and a high-resolution video monitor. The lowlight-level video camera and real-time image processor act together to integrate the image. As a result, detailed structure within the specimen is revealed, and photo damage to the sample is minimized by reducing illumination intensity. Even in ultralow-light situations, such as bioluminescence and chemiluminescence, a photon-counting camera detects single photon events, and an image processor integrates the image to generate gray-level information based on photon counting per pixel. This rapidly growing field is well on its way to replacing conventional photomicroscopy. Technical advances in cameras, camera electronics, and image processors, as well as printers, have reached a point where the resolution of the video image becomes indistinguishable from that of photographic fine-grain film. Although it is substantially more expensive, the added technical benefits of video microscopy are considerable. These include:
• • • •
Direct display and optional recording of dynamic events, including time-lapse studies of live cells. Availability of a wide range of analog and digital cameras for every budget and to suit many specific applications. Electronic contrast enhancement via gain and black-level control, or digital image processing, reveals image information inaccessible to eye or film. It is possible to record exceptionally low light levels with time integrating or intensified (e.g., ICCD) cameras. This is important in studies involving weak fluorescence emissions in
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081448
359
Downloaded from http://cshprotocols.cshlp.org/ at University of Southern California - Norris Med Library on April 2, 2014 - Published by Cold Spring Harbor Laboratory Press
M. Oshiro et al.
vivo (e.g., following microinjection of fluorochrome-labeled proteins or transfection of cells with green fluorescent protein [GFP] constructs) (see Spector et al. 1998a) or for minimizing the phototoxic effects of illuminating cells with high light intensities.
• • •
Electronic color balancing with single, three-tube, or chip color cameras. Electronic image transfer to other locations. Optional image storage for further processing or image analysis.
To take full advantage of video-enhanced microscopy, it is important to know a few basic facts about video cameras and image processors. There are many versions of each, and each version has certain characteristics that must be considered for different applications.
DEFINITIONS
Several terms used throughout this topic introduction that may be unfamiliar to the reader are defined below. Analog and digital cameras: An analog video signal is generated from the detector itself. This video signal is processed and converted to certain formats to interface to display monitors, image storage devices, and image processors. If this interface signal is analog, the camera is called an analog camera. If the interface signal is digital, the camera is called a digital camera. Analog cameras normally generate a standard video signal such as RS-170. Integration camera: The standard video camera’s frame rate is 30 frames/sec. This means that incoming light is integrated in a detector for 1/30 sec, and the signal is read out after the integration. To realize low-light sensitivity, integration cameras integrate incoming light for more than one frame, and the integrated signal is then read out. The sensitivity is increased in proportion with the number of integrating frames. However, maximum integration frames are limited by the dark noise of the camera itself. Video rate camera: A camera that generates a standard analog video signal, such as RS-170, is called a video rate camera. Image processor: An image processor modifies, analyzes, displays, and stores images. Readout speed: The speed at which each pixel is read out of a charge-coupled device (CCD) camera. The unit is megahertz per pixel or kilohertz per pixel. Linearity versus lag: Linearity is the relationship between incoming light to a video camera and the output signal from the video camera. If some signal generated in a detector remains in the detector after the reading, the leftover signal appears as the lag. Frame buffer: A frame buffer (or memory plane) stores one or more complete images for further image processing. Horizontal versus vertical resolution: The horizontal and vertical spatial resolutions are not always the same. In the case of video rate cameras, the horizontal resolution is higher than the vertical. Normally, slow-scan cooled CCDs have square pixels, and the resolution for both directions is the same. Geometric distortion: Tube-type cameras and intensified cameras have geometric distortion. The distortion of tube cameras is caused by the mechanism of the beam scanning. The distortion of intensified cameras is caused by the intensifiers and relay optics, such as relay lens and tapered fiber plates. Spectral range: The spectral sensitivity range of detectors. Temporal resolution: The speed at which one complete image is acquired determines the temporal resolution. The temporal resolution of video rate is 33 msec. The temporal resolution of slow-scan cooled CCD cameras is low because of the slow readout speed and the time of signal integration. Camera nonuniformity: Detectors do not have uniform sensitivity over an entire area. The camera nonuniformity of tube cameras and intensified cameras is higher than that of CCD cameras. 360
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top081448
Downloaded from http://cshprotocols.cshlp.org/ at University of Southern California - Norris Med Library on April 2, 2014 - Published by Cold Spring Harbor Laboratory Press
Video Microscopy
VIDEO CAMERAS
The following are two basic classifications of cameras: High-light-level cameras and low-light-level cameras. A common component to the two classifications is a CCD camera. This is an electronic device that incorporates a two-dimensional (2D) photodetector, an amplifier, and a timing device that produces a signal that conforms to one of the industry standards. These standards are indicated by the terms RS-170, RS-330, RS-343, NTSC, RGB, and others. This signal transfers an image from the detector to an image processor, a storage device, or display device. The details of this operation can be found in various sources. High-Light-Level Cameras
Although most low-light-level cameras work in high light levels, these cameras are expensive. If highlight-level images are to be recorded, the use of a high-light-level camera offers benefits in both cost and convenience. The following are characteristics of high-light/CCD cameras.
• • • • • • • • •
Detectors are available in 1/3, 1/2, 2/3-in, and larger formats. The size of the detector does not determine the number of horizontal pixels. Horizontal resolution is a function of the number of horizontal pixels. Spectral sensitivity ranges from
