Huygens Professional User Guide for version 15.05 Scientific Volume Imaging B.V. Huygens Professional User Guide for version 15.05 Scientific Volume Imaging B.V. Copyright © 1995-2015 by User Guide for version 15.05 All rights reserved Mailing Address Scientific Volume Imaging B.V. Laapersveld 63 1213 VB Hilversum The Netherlands Phone +31 35 6421626 Fax +31 35 6837971 E-mail [email protected] URL http://www.svi.nl/ Cover illustration: Macrophage recorded by Dr. James Evans (White-head Institute, MIT, Boston MA, USA) using widefield microscopy, as deconvolved with Huygens®. Stained for tubulin (yellow/green), actin (red) and the nucleus (DAPI, blue). Contents CHAPTER 1 Introduction. 1 What is Huygens Professional?. .1 Customizing Huygens Professional . .3 CHAPTER 2 Installation . 5 Microsoft Windows . .5 Mac OS X . .5 Linux (Debian) . .5 Linux (RPM) . .5 After the Installation . .6 The License String . .6 Updating the Software. .8 Removing the Software . .8 System Requirements for Huygens Professional. .9 Support on Installation . .9 Measuring the software usage . 10 CHAPTER 3 Getting Started . 11 Step 1: Start Huygens Professional . 11 Step 2: Load an Image . 12 Step 3: Inspect your Image . 12 Step 4: Generate a Point Spread Function (PSF). 18 Step 5: Estimate the Average Background Value. 18 Step 6: Estimate the Signal to Noise Ratio (SNR) . 19 Step 7: Perform a Deconvolution Run . 19 Step 8: Saving your Image . 21 Huygens Professional User Guide for version 15.05 i CHAPTER 4 Titan . 23 Titan as Freeware . 23 Titan does not edit or move your valuable image data. 24 Starting Titan . 24 Searching images . 25 Annotations and Projects . 26 Viewing and forwarding images. 26 CHAPTER 5 Deconvolution Wizard. 29 The Processing Stages in the Wizard . 29 Loading an Image . 30 Verifying Microscopy Parameters . 31 Starting the Deconvolution Wizard . 33 Using a Measured PSF. 34 The Intelligent Cropper. 34 Stabilizing STED data . 36 The Image Histogram . 37 Estimating the Average Background . 37 The Deconvolution Stage . 38 Finishing or Restarting a Deconvolution Run . 40 The Final Stage. 40 The Comparison View . 40 Multi-channel Images . 41 Z-drift Correcting for Time Series . 42 Saving the Result . 42 CHAPTER 6 The PSF Distiller . 43 Beads suited for PSF Distillation . 43 The PSF Distiller Window . 44 The Processing Stages in the Wizard . 45 Averaging Stage . 46 Distillation Stage . 47 Finalizing the result . 47 Full-width at half-maximum estimator . 47 CHAPTER 7 The Batch Processor. 49 The Batch Processor Window. 49 Usage. 50 Menus . 53 ii Huygens Professional User Guide for version 15.05 CHAPTER 8 Huygens STED Deconvolution . 55 Introduction. 55 STED principle . 55 STED parameters. 56 Example settings . 57 Estimating STED parameters . 57 STED Deconvolution . 59 Summary: deconvolution procedure . 60 CHAPTER 9 The Slicer. 61 Color. 61 Contrast . 62 Time . ..