What Is An Imaging System?

An imaging system is a tool for extracting additional information from an image. It can do this by measuring and reading out data about some aspect of an image. It can also augment the superb pattern recognition capability, which we all possess, by exaggerating some detail of the image.

Components Of An Imaging System

1. a monochrome video camera or digital camera
2. a frame grabber/ digitizing board
3. a computer
4. an image analysis program

Video Camera

The video camera converts light into an electrical signal. Conventional cameras employ a vidicon vacuum tube to make this transformation. CCD cameras use an array of solid state light sensitive charge coupled cells. In either case, the video signal is output as a voltage level corresponding to the light intensity of the image starting from the top left and going to the bottom right in a series of horizontal scan lines. Synchronization pulses are added to the output. The resolving power and output characteristics of monochrome video cameras is set, in the U.S., by the Electronic Industries Association (EIA). The most common EIA standard is RS-170. These cameras have a format that is essentially compatible with black and white closed circuit television. This standard and the slightly higher resolution European CCIR standard are the most common ones used for imaging. Video cameras are available with various light sensitivities and spectral responses.

Digital Camera

With a digital camera you have a direct digital signal from the camera to the computer. In most cases a digitizing board is still required within the computer to convert the signal but since there are less or no analog to digital conversions the signal usually has a higher resolution.

Frame Grabber

The frame grabber is basically an analog-to-digital converter optimized for digitizing (capturing) video signals. It is usually a board that plugs into a desk-top computer. This board senses and locks to the synchronizing pulses imbedded in the video signal coming from the camera. On command, it digitizes one complete video image. It does this by making a number of conversions on each scan line, then repeating this process for each line scanned by the camera. A captured image usually occupies all or most of a 512x512x8 bit array in the frame grabber memory. The contents of this memory is transferred to the computer memory for processing. Frame grabbers designed for high speed applications have image enhancement circuitry built onto the board.

Computer

The computer running an image analysis program comprises the heart of an imaging system. Desk-top computers, primarily IBM-PC and Macintosh Power PC models, are the most common and cost effective platforms for these systems since they are inexpensive and are supported by the widest variety of frame grabber boards and image analysis software. The image analysis program controls the operation of the frame grabber board, provides the means for obtaining direct data from the image, enhances images through menus of functions, and generates and controls output. The user controls its operation using the keyboard and mouse. Increasingly, control is accomplished by using a virtual instrument control panel displayed on the computer monitor. This control panel, manipulated by a mouse, has the look-and-feel of an actual instrument. By using this approach novice users can quickly learn to do sophisticated image processing operations. Because the computer screen can be fully employed just to display the control panel, a second color monitor and associated display board can be added to display the images. Good image analysis programs have color enhancement and a rich repertoire of fast image processing functions. Better image analysis programs offer, in addition, a scripting type language whereby sequences of operations can be recorded for automatic playback later. The best image analysis programs, in addition, provide for direct and scripted control of other devices that could be used in a full-feature imaging system. Examples of these devices include devices for microscope automation, illumination, and image detection.

Image Processing

If you have not seen image processing performed, it is an amazing process to watch. Seemingly invisible or subtle characteristics of the original image can be transformed into crisp and colorful patterns, veritably leaping out at you from the screen. Alternatively, images filled with superfluous detail can be simplified until all that remains is just what you are looking for. The dynamic range of very dark or very light images can be expanded to full scale. Edges can be enhanced or blurred. Solids can be turned into outlines. Focus can be sharpened. Noise can be eliminated. An assortment of quantitative measurements can be made. Intensity can be directly measured and displayed as actual values or as a histogram. Spatial measurements can be made, objects can be counted. The image can be scaled, zoomed, panned, rotated and warped.

This "magic" is accomplished in several ways. Noise is reduced and focus sharpened by averaging several images. Look-up-tables (LUT's), logic operations, and simple arithmetic are used to expand or distort contrast and to colorize. Complex mathematical operations such as convolution and fast fourier transformation (FFT) are used for a wide variety of filtering operations. These are applied to either individual pixel values or to groups of pixels, depending on the desired result. FFT's allow filtering in the frequency domain. Histograms and line profiles are used to obtain statistical information about the image. Morphological measurements are made by using several of the above operations.

There are many applications and examples of imaging systems used in basic research. Some systems, such as calcium ratio systems, are designed to look at very fast events and quantify the amount of a given indicator. Other systems such as time-lapse imaging systems are used to look at events as they occur over a longer period of time. In the application outlined below fluorescently labeled cells are tracked over time to develop a lineage analysis.

Example - An imaging system for time lapse recording of live dye-filled cell activity able to follow activity in three dimensions.

This imaging system is comprised of:

1. Intensified video camera
2. Computer with a color display.
3. Frame grabber board installed in the above computer.
4. A secondary display board also installed in the above computer.
5. Secondary color or black & white video monitor (this is in addition to the computer color display).
6. Image analysis program.
7. Panasonic model TQ3031 optical memory disk recorder or other device for storing the acquired data.
8. Applied Scientific Instrumentation (ASI) SC-2000 dual shutter controller.
9. ASI MFC microscope focus controller (for optical serial-sectioning).

The system is set up with the camera and image intensifier mounted on the microscope. The camera's video signal is connected to the frame grabber board. The display board output is connected to the optical memory disk recorder and, through it, to the Mitsubishi color monitor. The computer is connected to the shutter controller, focus controller and optical memory disk recorder in a daisy chain fashion. This latter connection is the communications pathway between the imaging program and the time lapse and optical serial sectioning hardware. The electro-mechanical shutters that come with the SC-2000 dual shutter controller are installed between the UV and tungsten light sources and the microscope. The motor drive that comes with the MFC is mounted to the microscope and connected to the fine focus shaft in a closed -loop fashion to insure precise and repeatable focusing.

The system functions as follows:

1. using the lowest UV stimulation levels possible
2. picking up the subsequent very weak fluorescence using an image intensifier and ultra-low light level camera
3. exposing the cell to UV stimulation for the briefest period necessary to capture an image

Command parameters are selected via onscreen controls that do the following:

1. Opens the UV shutter.
2. Captures several images in quick succession, averaging them at the same time for noise reduction.
3. Closes the UV shutter.
4. Performs an assortment of image enhancement operations.
5. Sends the enhanced image to the optical memory disk recorder where it is written on the removable disk pack.
6. Steps the microscope focus to a new layer within the blastomere then repeats steps 1-5 until the depth of interest has been covered.
7. Returns to the top step, pauses until the next time-lapse sequence is due, then repeats steps 1-6 for the duration of the experiment. Periodically steps 1-6 are repeated with the tungsten light shutter open instead of the UV shutter in order to obtain images of the entire blastomere rather than just the fluorescing cells in a black background.

At the end of the experiment onscreen controls are selected to play back the stored images (in any order) for animation. Thus it is possible to follow the course of cell division, growth and differentiation over a period of time through a 3-dimensional area covered by the microscope. In addition, images can be recaptured for additional analysis. By superimposing the fluorescent and a tungsten light images, exact and relative locations of the dye filled cell and its progeny, with respect to the entire blastomere, can be easily observed and measured.

ASI's Unique Products

Applied Scientific Instrumentation (ASI) makes some of the finest and innovative accessories for microscope automation For instance, because the Macintosh computer has few ports, the ability to daisy chain these devices on one port with the optical memory disk recorder is essential. This is possible because ASI's products use a command protocol compatible with the optical memory disk recorder. Because our shutter controller has the unique ability to control two shutters simultaneously, substantially more information can be obtained during an experiment than if only a single shutter were used. Our focus controller offers resolution and repeatability that is among the best in the industry. Both these devices have features that are not only useful during a time-lapse experiment, but at anytime the microscope is used.