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What Is Deconvolution?How Does Digital Imaging
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HB/MT software uses the nearest neighbor algorithm to mathematically calculate and remove out-of-focus haze from microscope images. It is designed to replace or supplement pinhole-based confocal microscopes.
Minski (1961) was the first to propose the technique of confocal microscopy used by laser scanning confocal microscopes. The principle is quite simple and is illustrated in the light paths in Figure 1.
The image seen through a microscope includes the in-focus portion and the out-of-focus portion above and below the plane of focus. The smear or blur produced by the out-of-focus planes is a natural consequence of the optics of the microscope.
Confocal microscopy removes out-of-focus haze by passing the light through one or more small apertures, leaving only a thin, highly focused plane. The light from this focused plane can be digitized and stored on a computer.
The distance between the specimen and the microscope objective is then changed producing a new focal plane. The new focal plane is digitized and stored. After a series of planes has been collected, individual slices can be examined or the whole specimen can be digitally reconstructed by a computer as a three-dimensional volume.
A confocal microscope consists of a standard microscope with a number of complex attachments to direct and process the beam of light. Most confocal microscopes use an intense laser light to scan the specimen. This intense light source is needed to compensate for the light loss which occurs as the light passes through small apertures.
HB/MT does in software what the confocal microscope does by virtue of hardware (i.e. the pinhole). Both systems use image processing but HB/MT is more flexible.
HB/MT, as illustrated in Figure 2, uses a standard white-light microscope and requires no special attachments. A video camera captures and digitizes the images from the microscope, which are then stored by a computer. Image enhancement algorithms are used to deconvolve the image, i.e. remove the blur or haze contributed by the out-of-focus image planes. The algorithms used by HB/MT have the same function as the apertures in the laser scanning confocal microscopes - removing the out-of-focus portion of the image.
You can transform your standard microscope and your computer by simply adding VayTek's HB/MT package.
A confocal image has the out-of-focus haze removed. This can theoretically increase image resolution. The increase in resolution, by as much as 1.4 times (Brackenhoff, 1989), results in improved measurements (Yelamarty, 1990) and visualization. In addition, the optical sectioning is non-invasive and can be performed on living specimens. Also, it is possible to acquire images with multiple wavelengths of light and merge the results for greater information.
Besides increasing the resolution of the image, the deconvolved slices can be stacked to produce a three-dimensional representation of the specimen. Visualization of a three-dimensional data set can lead to new insights.
There are a number of advantages in using HB/MT. First, it costs less than a laser scanning confocal microscope because the microscope you currently have can be used with the digital deconvolution approach. New optical equipment is not required. Prices for laser scanning confocal microscopes typically range between $75,000 and $300,000. The price of HB/MT is a fraction of this cost.
Depending upon components already in place, you may need to add a framegrabber, camera, and stepper motor in addition to HB/MT. For a complete description of products, turnkey systems and current pricng please refer to VayTek's Product Guide or call directly (515) 472-2227).
Second, the high intensity laser light, required by the LSM's, can harm living specimens. The digital approach is less harmful to living material since it typically uses a small fraction of the light used by the laser scanning microscopes.
Third, many fluorescence preparations bleach easily, even with standard light sources. These dyes cannot be used with laser scanning confocal microscopes. Even robust preparations can fade after many scans producing a brightness gradient along the vertical axis. HB/MT will result in less photobleaching.
Fourth, HB/MT is more flexible. When using a laser scanning confocal microscope, the amount of haze removed is set by the aperture size and thus cannot be adjusted after the image is captured. HB/MT, on the other hand, allows the user to set the amount of haze to be removed as a part of the deconvolution process after the image has been captured. Thus, the HB/MT user can explore the same data set multiple times with different degrees of haze removal.
And Fifth, data acquisition for HB/MT can be faster than confocal microscopes. Video cameras, used by HB/MT, can average or integrate several slices per second. Including the time to move the stage, three images can be captured in about 3 seconds. Each image is then deconvolved, requiring no more than four seconds (MicroTome version) per 512 x 512 slice.
Confocal microscopes can be slower. Each slice is often scanned and integrated multiple times with reduced laser power in an attempt to attenuate photobleaching effects.
The principal limitation of the digital deconvolution approach has been the amount of computer time required to deconvolve a single slice. Until now, a single slice could require several minutes to deconvolve on a personal computer. A large data set could take an entire day to process.
With the introduction of HB/MT/MicroTome, however, processing time has been reduced to no more than a few seconds per slice on a PC or Power Mac. These speeds are possible because of VayTek's unique, efficient implementations of the algorithms.
There is an additional limitation with HB/MT. Slices must be relatively close to each other to achieve the proper resolution after deconvolution. The exact distance will vary from sample to sample, but experience has shown that it should be between .1 micron and 10 microns.
The word "deconvolve" means to "untangle or unwind". A deconvolution algorithm is a systematic procedure for removing noise or haze from an image.
There are several well known deconvolution algorithms that can be applied to microscope images to remove the out-of-focus haze. The easiest to use is the nearest neighbor algorithm. This approach has the advantage of being very fast and yielding very good results. The nearest neighbor algorithm requires a minimum of three slices. Other algorithms include the inverse filter and the constrained iterative. These algorithms will yield slightly more precise results, but require many more slices and more computation time (Agard, 1989). For more information refer to the Technical Note.
The laser scanning confocal microscope varies the amount of haze removal by altering the size of the aperture. With HB/MT you vary the amount of haze that is removed after a data set has been collected by adjusting the haze removal parameter used during deconvolution.
With HB/MT you specify the amount of haze to be removed at the time of deconvolution, giving you more flexibility while working with your data. The ability to set this parameter, however, raises the issue of what the optimal haze removal setting should be. This setting will vary from data set to data set, but experience has shown that 90% removal is optimal for most data.
Most experts in the field of digital deconvolution agree that deconvolution technology and pinhole based microscopes complement each other. In fact, many believe that the two technologies should be available on the same system so the researcher can choose which to use. In fact, digital deconvolution can be used to further enhance images captured with a confocal microscope (Shaw, 1991).
The relationship between the two technologies is illustrated in Figure 3. The smaller circle in Figure 3 represents the collection of all images that can be acquired with the laser scanning confocal microscope. The larger circle represents the collection of all images that can be successfully acquired with HB/MT. The intersection of the two circles represents those images that can be successfully produced on either system.
Most experts on digital deconvolution agree that at least 90% of the images that can be created with the laser scanning confocal microscope can be produced equally well with digital deconvolution.
However, there are some images that can only be produced with a laser scanning confocal microscope. These images include those in which the distance between slices is, by necessity, quite large. Also, thick or semi-transparent non-living specimens that require powerful laser light to penetrate into the material will be best imaged by a laser scanning confocal microscope.
Figure 3. Images possible with both systems.
Conversely, there are some images that can only be produced by the digital deconvolution approach. For example, those specimens with sensitive fluorescent dyes.
The nearest neighbor algorithm needs a minimum of three optical slices. There is no theoretical limit to the maximum number of slices that could be used, although, as a practical matter, a researcher would seldom acquire more than 100 slices.
Based on the data in the slices, HB/MT computes a characteristic optical point spread function (PSF) for the lens in the microscope that was used to acquire the data. The PSF is then used to deconvolve each image using the images above and below the image being processed. This process identifies the haze which is subtracted from the slice of interest, resulting in the deconvolved image. See our technical note on algorithms
A point spread function is a mathematical term for the impulse response of a system. When the term "point spread function" is used in connection with an optical system it means the impulse or point response of an optical system to a point input.
A single point of light is focused by the lens into a complex shape known as a point spread function (PSF). The shape of the PSF depends upon light wavelength, lens numerical aperture (NA), and the optical aberration of the lens. By knowing the shape of the PSF the operator can remove the excess light from any image plane thus producing a high resolution image. See our technical note on algorithms
The PSF is calculated using diffraction theory. The parameters needed to calculate the PSF are light wavelength, numerical aperture of the lens, the distance between pixels within a plane and the distance between the acquired image planes. The user must supply HB/MT with these parameters. See our technical note on algorithms.
Yes. The nearest neighbor algorithm is tolerant of the difference between a theoretically and experimentally obtained PSF. HB/MT allows the user to input an experimental PSF, if desired, however.
There are two ways to assess the reliability and validity of the images produced by HB/MT . The first means of verification is mathematical. The deconvolution algorithms have been published, reviewed and accepted. The reader is invited to read the articles listed in the Bibliography.
The second means of verification is empirical. The algorithms work properly if 1) they image known structures correctly and 2) they produce images similar to those produced by laser scanning confocal microscopes. The reader is directed to the accompanying material illustrating images produced by HB/MT. In addition, readers may send blurred images to VayTek. We will deconvolve them and return the results.
Please refer to technical specifications for the latest deconvolution speeds. Times will vary from a few seconds to several minutes depending on the computer platform.
Yes. HB/MT has a friendly, point-and-click interface. HB/MT was designed to make the deconvolution algorithms easy to use and give you feedback of the results as quickly as possible.
It is very important to use high quality raw images for deconvolution; otherwise garbage-in, garbage-out. Good raw images mean using a good microscope, an appropriate camera, a good framegrabber, and acquisition software that lets you average and integrate during image capture. VayTek can provide the necessary components for data acquisition. Please consult a VayTek salesperson, the HB/MT manual, or the HB/MT demo program, for a more detailed discussion of data acquisition issues. See our technical papers on data acquisition.
HB/MT will support most file formats. You specify the header length, height and width and file type. The image data must be 8 bit integer, binary, raster scan format.
Yes. Technical support is available at no extra charge for the first year after purchase. After the first year, additional support and new releases are available for a maintenance fee.
HB/MT lets you view the 2D slices as you deconvolve them. VayTek also sells a 3D reconstruction program for the Windows, Macintosh and UNIX based workstations called VoxBlast.
Yes. There are a number of options for printing images. For more information on printers, please consult a VayTek sales representative. It is now possible to also print a 3D Lenticular Panel of an image processed with VayTek Software. For more information, see Eric Rayboy's "3D Hardcopy" site.
