Nuance MULTISPECTRAL IMAGING SYSTEM User's Manual for Nuance 3.0.2 Notice The information in this document is subject to change without notice and should not be construed as a commitment by PerkinElmer, Inc. PerkinElmer assumes no responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall PerkinElmer be liable for incidental or consequential damages in connection with or arising from the use of this manual. This manual describes system operation using Nuance version 3.0.2 software. For more information contact: PerknElmer, Inc. 68 Elm Street, Hopkinton, MA, 01748, USA Phone: 800-762-4000 or +1 203-925-4602 Fax: +1 203-944-4904 Email: [email protected] Web site: http://www.perkinelmer.com US Patent 5,892,612; 5,953,087; 7,655,898; and patents pending. Document Part No. 130765 Rev. 01 Table of Contents Part I Welcome To Nuance 6 Part II Introduction to Multispectral Imaging 7 1 Light...................................................................................................................................... 7 2 Human...................................................................................................................................... Perception of Light Intensity and of Color 8 3 Light ......................................................................................................................................Absorbance and Reflection 9 4 Fluorescence...................................................................................................................................... 10 5 The ......................................................................................................................................Multispectral Solution 12 6 Nuance...................................................................................................................................... FLEX Multispectral Imaging 15 Part III Getting Started with Nuance 17 1 Operator...................................................................................................................................... and Equipment Safety 17 2 About...................................................................................................................................... this Manual 18 3 CE,...................................................................................................................................... CSA, and UL Testing and Certification 19 4 Nuance...................................................................................................................................... Imaging Module 19 5 Installing...................................................................................................................................... & Starting Nuance 22 6 Understanding...................................................................................................................................... the Nuance Work Area 23 7 Window...................................................................................................................................... Layout 25 8 Specifying...................................................................................................................................... Nuance Hardware Settings 27 9 Calibrating...................................................................................................................................... Pixel Size 29 10 Using...................................................................................................................................... Low Screen Resolution Mode 30 11 Reinitializing...................................................................................................................................... Nuance Hardware 30 Part IV Method Development 31 1 Methods...................................................................................................................................... for Building Spectral Libraries 31 2 Saving...................................................................................................................................... Spectral Libraries 35 3 Saving...................................................................................................................................... Protocols 35 4 Importing...................................................................................................................................... Spectra Into a Library 36 5 Practice...................................................................................................................................... Exercise 37 Part V Brightfield and Fluorescence Imaging 40 1 Setting...................................................................................................................................... Up Your Microscope for Brightfield Imaging 40 2 Setting...................................................................................................................................... Up Your Microscope for Fluorescence Imaging 41 3 About...................................................................................................................................... Optical Density Images 42 4 Tips...................................................................................................................................... for Obtaining Quantitative Results 43 5 Acquiring...................................................................................................................................... Images Using a Saved Nuance Protocol 43 6 Viewing...................................................................................................................................... a Live Image Stream 44 7 Selecting...................................................................................................................................... Camera Binning and a Region of Interest 44 Contents 3 8 Specifying...................................................................................................................................... the Current Wavelength and Exposure 45 9 Acquiring...................................................................................................................................... Cubes in Brightfield 47 10 Acquiring...................................................................................................................................... RGB (Color) Images 48 11 Acquiring...................................................................................................................................... Fluorescence Cubes 49 12 Acquiring...................................................................................................................................... a Mono Image (Snapshot) 52 13 Assigning...................................................................................................................................... Sample IDs and Notes 52 14 Acquiring...................................................................................................................................... Timed Sequences of Image Cubes 53 15 Saving...................................................................................................................................... Images and Image Cubes 54 16 Viewing...................................................................................................................................... Cube Information 55 17 Extracting...................................................................................................................................... an Image Plane from a Cube 56 Part VI Unmixing Spectral Images 57 1 Tips...................................................................................................................................... for Accurate Unmixing 57 2 Opening...................................................................................................................................... a Spectral Library 57 3 Opening...................................................................................................................................... an Image Cube 58 4 Computing...................................................................................................................................... and Unmixing Spectra Automatically 58 5 Computing...................................................................................................................................... and Unmixing Spectra Manually 61 6 Saving...................................................................................................................................... an Unmixed Result Set 65 7 Working...................................................................................................................................... With Saved Result Sets 65 8 Checking...................................................................................................................................... Your Spectral Library 66 9 Subtracting...................................................................................................................................... Spectra from a Cube 67 10 Using...................................................................................................................................... Line Profiles to Analyze Signals 67 11 Comparing.....................................................................................................................................