LecturesPart22 - Computational Biology, Part 22 Biological...

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Unformatted text preview: Computational Biology, Part 22 Biological Imaging I G. Steven Vanni Meel Velliste Meel Robert F. Murphy Robert Copyright © 1998, 2000-2006. Copyright All rights reserved. Biological imaging s Significant advances in the fields of optics Significant and electronics in the past two decades have greatly increased the utility of imaging for addressing biological questions. addressing s These advances permit These x more diverse types of information to be more extracted from biological specimens x with greater accuracy with x and under more demanding conditions. Chemical and molecular biological probes Chemical may be targeted within a specimen may s s Imaging relies on generating a detectable signal Imaging which can be used as a measure of a property of interest in the specimen. This property of interest is the initial signal, but it This must be transduced or changed through several transduced forms before it becomes detectable. forms Chemical and molecular biological probes Chemical may be targeted within a specimen may s For example: A protein may be modified so that For when it enters a cell and bumps into another protein involved in a specific activity, it fluoresces. The original activity was probably not detectable, but this newly generated fluorescence signal is detectable. signal Front end of imaging system and detector Specimen Specimen may be difficult to see Specimen except where labeled by probe. except Image Formation and Acquisition s s Having an understanding of the specimen, the next Having step is the formation and acquisition of a digital image image A two dimensional image plane consists of a two rectangular grid of points, or pixels pixels Pixel Specimen Grid Image Formation and Acquisition s s A digital image plane is acquired by recording a digital digital value proportional to the intensity of light (or other form of energy) impinging on each pixel pixel of a detector detector This intensity usually corresponds to the amount This of light emitted by or reflected from a corresponding point on a specimen corresponding 0 0 0 0 0 0 Specimen Projection of specimen onto dectector grid 0 3 6 2 0 0 0 7 8 8 4 0 1 8 8 8 8 2 0 3 8 8 8 1 Image 0 0 2 4 3 0 0 0 0 0 0 0 “Pixel” is used interchangeably to mean: is s s s One of multiple regions on a detector, each One corresponding to the smallest area from which a signal can be distinguished signal The numerical value associated with each such The region in a digital image A region on display device, such as a monitor or region display such printer printer Pixel 0 0 0 0 0 0 Dectector grid 0 3 6 2 0 0 0 7 8 8 4 0 1 8 8 8 8 2 0 3 8 8 8 1 0 0 2 4 3 0 Pixel values 0 0 0 0 0 0 7-8 4-6 1-3 0 Display device Display of pixel values s s A pixel value is just a number in the data set pixel representing a digital image. representing Pixel values may be displayed in different ways, Pixel determined by a look up table (LUT). look 0 0 0 0 0 0 LUT 7-8 0 3 6 2 0 0 0 7 8 8 4 0 1 8 8 8 8 2 0 3 8 8 8 1 Pixel values 0 0 0 0 0 0 4-6 1-3 0 Hot to cold LUT 1-8 4-6 0 1-3 0 0 0 2 4 3 0 LUT 7-8 Arbitrary Binary Image Formation s Biological images may be acquired via a Biological variety of imaging modes or modalities modes modalities s Each mode is a combination of an image Each formation system and a detector detector Image formation system Sample Image formation system Detector Detector and image types While the examples so far have dealt with light microscope images, we will now back up for a microscope few minutes to consider many different types few of images before concentrating again on light of microscopy. microscopy. Detector and image types In general, images may be classified according to In what is being detected: what s (Visible) light transmission, scattering or emission 3 s s s s s single wavelength, 3 color, or full spectrum Electron transmission or scattering X-ray transmission Radioactive particle emission Magnetic field perturbation Physical displacement from “atomic force” Comparing types of imaging Method Light Electron Medical X-ray X-ray Diffraction Autoradiography Functional MRI/NMR Structural NMR AFM Resolution Living (nanometer) specimen? 200 or better Yes 10 No 1000 or Yes better 0. 1 No 10 5000 No Yes 1 No 1 No Light microscopy s Three primary types of detectors x human eye 3 no digital image x CCD or charge coupled device 3 “work horse” of modern biological imaging 3 acquires digital image directly x PMT or photomultiplier tube 3 scans to produce digital image CCD chips Penny A CCD chip is the actual detector within a CCD CCD camera. camera. Light sources in the object s Consider a fluorescent specimen made of Consider individual molecules of fluorescent dye. individual x Each molecule can emit light. x Each dye molecule may be seen as a vanishingly Each small emitter. small x Such an emitter is called a point source. Such point x The concept of a point source is useful because a The point is simple to model, and if we know how a point source is imaged, then we can easily model a complex specimen as a combination of many points and predict how it will be imaged. points Arc Lamp Excitation Diaphragm Excitation Filter Fluorescence Microscope Ocular Objective Emission Filter Light sources in the object s A specific example might be a microscope specific slide containing cells stained with fluorescent dye. fluorescent s In an ideal image, a point source would In show intensity in only one pixel show Point-spread function s In reality, the light from each point in the In specimen is seen to spread out and affect many pixels in the image. many s This “spreading” is an inescapable result of This the optical properties of the image formation system. s The mathematical description of this The spreading or blurring process is called a point-spread function (PSF) point-spread Point-spread function s The point-spread function (PSF) is The determined by the optics of the image formation system, including factors such as the refractive index, diameter and magnification of its components magnification s The resulting blurred region in the image The can be approximated by a 2D Gaussian distribution distribution Realistic Image of a Point Source s s This graph shows intensity on the z-axis for a PSF This defined in the X-Y plane. defined Later we will consider a PSF defined in three Later dimensions. 0.16 0.14 0.12 0.10 0.08 2 Intensity 0.06 1 0.04 0 0.02 0.00 3 ­1 ­3 ­2 ­2 X ­1 0 1 2 3 ­3 Y Light sources in the object s Thus, when a 2D image is acquired, each point in Thus, the specimen will be blurred in all directions and will contribute to the recording in many pixels around that pixel to which it directly corresponds around Introduction to 3D Microscopy s The spreading of light from a point source The actually occurs in three dimensions as will be shown. be s First, however, it is necessary to understand First, the three dimensional (3D) nature of the object and image as acquired via 3D microscopy. microscopy. 3D Microscopy s When a microscope is focused on When a specimen, the detector records an image from a plane. x This is the focal plane. This focal x Parts of the specimen in the Parts focal plane are in the best focus. focus. Detector Focal plane s s 3D data is acquired by combining data from several 3D different focal planes into a stack of images. stack This is accomplished by changing the distance This between the specimen and the microscope’s objective lens from one image acquisition to the next. next. Image stack Objective Real 3D image data s The next slide shows a real 3D image stack. s The specimen is a HeLa cell labeled with a The antibody against the cytoskeletal protein tubulin and a secondary antibody conjugated to a fluorescent dye. conjugated s The images were acquired using a confocal The fluorescence microscope. fluorescence s The image stack is presented here as a The movie with one acquired image plane per movie frame. movie Microtubules in a human cell QuickTimeª and a decompressor are needed to see this picture. Courtesy of Meel Velliste Real 3D image of a point source s s Now, with a better understanding of what makes Now, up a 3D image stack, we can better consider how light from a point source spreads out and is imaged in three dimensions. in On the following slide, intensity is shown by On variation in color. x Warm colors indicate greater intensity. x All axes indicate real spatial dimensions as All indicated. indicated. Real 3D image of a point source 3D Reconstruction 3D of Point Spread Function (PSF) from 0.2 Micron Bead y z x x NOTE: Spreading along the Z-axis is more pronounced. Courtesy of Image & Graphics Inc.: Courtesy Increasing intensity Image Formation s Image formation can be described as: x the convolution of an array describing the convolution original specimen or object x with a function describing the image formation with function system x to yield an acquired image. to The concept of a convolution s A convolution may be written in somewhat convolution simplified mathematical form as follows: simplified i(x,y,z) defines the image in its 3D space i(x,y,z) according to the form of the equation above. according s PSF(x-x’,y-y’,z-z’) defines the amount of light PSF(x-x’,y-y’,z-z’) from a point source at x’,y’,z’ that will be observed at x,y,z observed s o(x’,y’,z’) describes the specimen or object. s Image Formation s The mathematical view of convolution The emphasizes that each point in the sample can contribute to each point in the image can Widefield Fluorescence Microscopy •This type of fluorescence microscope collects light emitted from all points in the specimen (with varying efficiencies depending on position relative to focal plane) •The result for specimens that are thick relative to the depth of focus of the objective is a blurred image Confocal Microscopy s One way to obtain images that better One represent the fluorescence distribution just in the focal plane is to use a confocal confocal microscope microscope Laser Excitation Pinhole Confocal Microscope Principle Excitation Filter PMT Objective Emission Filter Emission Pinhole http://micro.magnet.fsu.edu/primer/confocal/index.html http://micro.magnet.fsu.edu/primer/confocal/index.html Benefits of Confocal Microscopy s s s s s s s Reduced blurring of the image from light Reduced scattering scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically Drawbacks of Confocal Microscopy s Slower acquisition - need to collect one Slower pixel at a time pixel s Increased photodamage (photobleaching) Increased due to longer exposure to exciting light due Spinning Disk Confocal Microscopy s To allow To faster acquisition, do confocal imaging “in parallel” parallel” Image Formats s An bit map image normally consists of an 8bit or 16-bit value for each pixel. s These values are stored as computer files in These various formats. various s Pixel values are normally stored linearly in Pixel a file with the values for the first row of pixels followed immediately by the values for the second row (etc.). for Image Formats s At a minimum, an image format contains: x Image size (# of rows and columns) x Number of bits per pixel x Order in which bytes within words are stored x Number of bytes to skip at the beginning of the Number image (the offset) offset 3 The beginning of image files often has a text header The header that can be skipped if the above values are known. that 3 This header may contain additional descriptive This information about the image such as: information • subject of image • name of person and/or application creating the image Common Image File Formats s TIFF (Tag Image File Format) x Originally for scanners and frame grabbers x x Used extensively on many platforms Can be read/written by NIH Image x Supports lossless compression Reference: www.shortcourses.com/chapter07.htm Common Image File Formats s JPEG (“jay-peg” Joint Photographic Experts Group) x x x x x Originally referred to a compression method but now Originally refers to the associated file format with or without compression compression Most common World Wide Web file format 3 Supports progressive display where an image is first Supports progressive displayed at low resolution and then at higher resolution. resolution. Uses a lossy compression technique Optimized for storing photographs and not as good for Optimized line art line Supports 24-bit color Reference: www.shortcourses.com/chapter07.htm Common Image File Formats s GIF (“jiff” Graphics Interchange Format) x Also widely used on the Web 3 Supports progressive display x Mostly used for line art as opposed to Mostly photographs photographs x Only supports 8-bit color Reference: www.shortcourses.com/chapter07.htm ...
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