Computational Biology Part 22 Biological Imaging I E Glory G Steven Vanni Meel Velliste Robert F Murphy Copyright 1998 2000 2007 All rights reserved Biological imaging Significant advances in the fields of optics and electronics in the past two decades have greatly increased the utility of imaging for addressing biological questions These advances permit more diverse types of information to be extracted from biological specimens with greater accuracy and under more demanding conditions Chemical Chemical and and molecular molecular biological biological probes probes may may be be targeted targeted within within aa specimen specimen Imaging relies on generating a detectable signal 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 must be transduced or changed through several forms before it becomes detectable Chemical Chemical and and molecular molecular biological biological probes probes may may be be targeted targeted within within aa specimen specimen For example A protein may be modified so that 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 Front end of imaging system and detector Specimen Specimen may be difficult to see except where labeled by probe Image Formation and Acquisition A digital image plane is acquired by recording a digital value proportional to the intensity of light or other form of energy impinging on each pixel of a detector This intensity usually corresponds to the amount of light emitted by or reflected from a corresponding point on a specimen Specimen 0 0 0 0 0 0 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 Pixel 0 0 2 4 3 0 0 0 0 0 0 0 Image Formation Biological images may be acquired via a variety of imaging modes or modalities Each mode is a combination of an image formation system and a detector Sample Image formation system Detector While the examples so far have dealt with light microscope images we will now back up for a few minutes to consider many different types of images before concentrating on light microscopy Detector and image types In general images may be classified according to what is being detected Visible light transmission scattering or emission 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 Three primary types of detectors human eye CCD or charge coupled device no digital image work horse of modern biological imaging acquires digital image directly a CCD chip is the actual detector within a CCD camera 1 penny PMT or photomultiplier tube scans to produce digital image Arc Lamp Excitation Diaphragm Excitation Filter Fluorescence Microscope Ocular Objective Emission Filter Light sources in the object Consider a fluorescent specimen made of individual molecules of fluorescent dye Each molecule can emit light Each dye molecule may be seen as a vanishingly small emitter Such an emitter is called a point source The concept of a point source is useful because a 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 Light sources in the object A specific example might be a microscope slide containing cells stained with fluorescent dye In an ideal image a point source would show intensity in only one pixel Point spread function In reality the light from each point in the specimen is seen to spread out and affect many pixels in the image The mathematical description of this spreading or blurring process is called a point spread function PSF The point spread function PSF is determined by the optics of the image formation system including factors such as the refractive index diameter and magnification of its components Realistic Image of a Point Source The resulting blurred region in the image can be approximated by a 2D Gaussian distribution This graph shows intensity on the z axis for a PSF defined in the X Y plane Later we will consider a PSF defined in three dimensions Light sources in the object Thus when a 2D image is acquired each point in 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 Introduction to 3D Microscopy The spreading of light from a point source actually occurs in three dimensions as will be shown First however it is necessary to understand the three dimensional 3D nature of the object and image as acquired via 3D microscopy 3D Microscopy When a microscope is focused on a specimen the detector records an image from a plane This is the focal plane Parts of the specimen in the focal plane are in the best focus Detector Focal plane 3D data is acquired by combining data from several different focal planes into a stack of images This is accomplished by changing the distance between the specimen and the microscope s objective lens from one image acquisition to the next Image stack Objective Real 3D image data The next slide shows a real 3D image stack The specimen is a HeLa cell labeled with a antibody against the cytoskeletal protein tubulin and a secondary antibody conjugated to a fluorescent dye The images were acquired using a confocal fluorescence microscope The image stack is presented here as a movie with one acquired image plane per movie frame Microtubules in a human cell QuickTime and a decompressor are needed to see this picture Courtesy of Meel Velliste Real 3D image of a point source Now with a better understanding of what makes up a 3D image stack we can better consider how light from a point source spreads out and is imaged in three dimensions 3D Reconstruction of Point Spread Function PSF from 0 2 Micron Bead y z x Increasing intensity x Courtesy of Image Graphics Inc http www imagepro co kr NOTE Spreading along the Z axis is more pronounced Image Formation Image formation can be described as the