Living Image 3.1

More documents

Recommendations

Info

D. Image Data Display & Measurement D.3 Flat Fielding D.4 Cosmic Ray Corrections 202 Flat fielding refers to the uniformity of light collected across the field of view (FOV). A lens usually collects more light from the center of the FOV than at the edges. The Living Image ® 3.0 software provides a correction algorithm to compensate for the variation in the collection efficiency of the lens. This enables uniform quantitation of ROI measurements across the entire FOV. To apply the correction algorithm, choose the Flat Field Correction option in the Corrections/Filtering tools. The algorithm multiplies each pixel by a predetermined scale factor. The scale factor for each pixel depends on its distance from the center of the image. The scale factor near the center of the field of view is one, but can be up to two or three near the corners on the IVIS ® Imaging System 100 Series. (The IVIS Imaging System 200 Series has a larger lens with a smaller flat field correction.) You may notice an increase in noise near the edges and corners of the FOV when flat field correction is applied– this is normal. Cosmic rays are extraterrestrial high-energy particles that register a false signal on a CCD detector. Cosmic rays as well as other sources of ionizing radiation cause infrequent interactions (a few per minute) on the CCD. These interactions result in large signals that are usually isolated to a single pixel, making them easy to correct. The Living Image ® 3.0 software searches for isolated, high amplitude hot pixels and replaces them with a collective average of surrounding pixels. The Cosmic Correction option should always be selected for in vivo image data because hot pixels can significantly affect an ROI measurement. Cosmic ray correction is not recommended when imaging very small objects such as individual cells. An individual cell may only light up one or two pixels and can sometimes be misinterpreted as a cosmic ray. In this case, clear the Cosmic Correction option in the Corrections/Filtering tools to avoid filtering out single-cell images.
Living Image ® Software User’s Manual Appendix E Luminescent Background Sources & Corrections E.1 Electronic Background Electronic Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Background Light On the Sample . . . . . . . . . . . . . . . . . . . . . . . . 204 Background Light From the Sample . . . . . . . . . . . . . . . . . . . . . . . 206 The background sources of light from bioluminescent images are inherently very low. This appendix discusses sources of background and how to manage them. Due to the extreme sensitivity of the IVIS ® Imaging System, residual electronic background (dark current) and luminescent emission from live animals (autoluminescence) are measurable and must be taken into account. For information on fluorescent background, see Appendix F, page 215. The cooled CCD camera used in an IVIS Imaging System has electronic background that must be accurately measured and subtracted from the image data before the light intensity is quantified. Raw data that is not corrected for electronic background results in erroneous ROI measurements. Incorrect background subtraction may also result in serious errors. However, it is not necessary to subtract the electronic background when making a simple visual inspection of an image. The types of electronic background include: • Read bias - An electronic offset that exists on every pixel. This means that the zero photon level in the readout is not actually zero, but is typically a few hundred counts per pixel. The read bias offset is reproducible within errors defined by the read noise, another quantity that must be determined for quantitative image analysis. • Dark current - Electronic background generated by the thermal production of charge in the CCD. To minimize dark current, the CCD is cooled during use. Read Bias & Drift Prior to a luminescent image exposure, the Living Image ® software initiates a series of zero-time exposures (image readout) to determine a read bias measurement. If a dark charge background is available for the luminescent image, the average bias offset for the read bias image stored with the dark charge measurement is compared to the average bias offset determined with the read bias measurement made prior to the image. The difference, or drift correction, is stored with the luminescent image data, and is later used to correct minor drift (typically less than two counts/pixel) that may occur in the bias offset since measuring the dark charge background. If a dark charge background is not available at the time of the luminescent image exposure, the software checks to see if the selected image parameters warrant a dark charge measurement (large binning and long exposure time). If a dark charge image is not required, the read bias will be used. If a dark charge is recommended, the software provides the option of using the read bias measurement instead. Since the read bias is by far the largest component of background, using a read bias measurement instead of a dark charge measurement is often acceptable. If read bias is used instead of a dark charge background, 203
Page 1 and 2:
Living Image® Software User’s Ma
Page 3 and 4:
Living Image ® Software User’s M
Page 5 and 6:
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77 and 78:
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158: Living Image ® Software User’s M
Page 207: Living Image ® Software User’s M
show all

Living Image 3.1

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?