Digital Crystal Documentation Procedure

Transcription

1 X-Ray Crystallography Facility Kasha Laboratory Institute of Molecular Biophysics Florida State University, Tallahassee, FL , USA (850) (Off) (850) (Lab) Leica Moticam Stereo Microscope Digital Crystal Documentation Procedure Procedure for taking and viewing digital pictures of crystals

2 Thayumanasamy Somasundaram Page 2 3/18/2005 Table of contents Introduction... 3 Location... 3 Leica Motic Digital Documentation System (KLB 412)... 3 Viewing and Documenting Crystal Images... 5 Quick procedure for recording crystal images... 5 Accessing Crystal Images Remotely Procedure for viewing crystal images Hardware Information Dell Workstation Leica Stereo Zoom Microscope Moticam FireWire Camera Software Information Motic Software Remote Access (XRAY and Non-XRAY domain) Conclusion Thayumanasamy Somasundaram 414 Kasha Laboratory Institute of Molecular Biophysics Florida State University Tallahassee, FL soma@sb.fsu.edu URL: URL: Phone Fax March 18, 2005 Version 1.2 2

3 Thayumanasamy Somasundaram Page 3 3/18/2005 Leica Moticam Stereo Microscope Digital Crystal Documentation Procedure Document Version: 1.2 (March 17, 2005); 1.1 ( ); 1.0 ( ) Introduction In early 2004 X-Ray Crystallography Facility added a LeicaS8 APO stereo zoom microscope with 1.3 Mega pixel Moticam FireWire digital camera with Motic Advanced Images software. This stereo zoom microscope allows the routine examination and digital documentation of macromolecular crystals at ambient temperatures. The Leica Moticam Digital Documentation System is attached to an Intel 864 MHz Pentium III Dell Precision Workstation 220 (phe.sb.fsu.edu) running Windows 2000 Prof ( SP4) operating system. The system is located in KLB 412 (see Layout 1). The following note describes the procedure for recording images of macromolecular crystals and viewing the images from any computer in the Kasha Laboratory. A copy of this Note will be posted in XRF webpage shortly after receiving suggestions from the users. Location Leica Motic Digital Documentation System (KLB 412) Layout 1. Location of Leica Motic Digital Documentation System in KLB

4 Thayumanasamy Somasundaram Page 4 3/18/2005 Leica Motic Digital Documentation System is part of the X-Ray Crystallographic Facility and is located in KLB 412. The system consists of the following individual components: 1.3 Mega pixel Moticam digital FireWire (aka IEEE 1394 or I-Link ) camera Leica S8 APO stereo-zoom microscope with transmitted light-base Intel 864 MHz Intel Pentium Dell Precision Workstation (Phe.sb.fsu.edu) Microsoft Windows 2000 Professional ( SP4) operating system Motic Advanced Imaging software version 3.2 The Moticam digital camera is connected to the workstation via a FireWire cable. Focusing and magnification are done manually while recording and manipulation of images is controlled via the computer using the special software. The workstation Phe.sb.fsu.edu ( ) is part of the XRAY domain and can be accessed by all Facility users. Moticam 1300 FireWire Camera Leica S8 Stereo Zoom Microscope Moticam Leica Digital Documentation System Layout 2. Leica Motic Digital Documentation System Components and KLB 412 4

5 Thayumanasamy Somasundaram Page 5 3/18/2005 Viewing and Documenting Crystal Images Quick procedure for recording crystal images Log into phe.sb.fsu.edu using the username xrayuser and ask Soma for password Start Motic Images Advanced program either of the two ways: 1) By clicking on the shortcut shown on the left, or 2) From Start Menu > Programs > Motic > Motic Images Advanced 3.2 > Motic Images Advanced 3.2 Motic Images Advanced 3.2 Welcome screen comes up, followed by 5

6 Thayumanasamy Somasundaram Page 6 3/18/2005 The Graphical User Interface (GUI) of the program opens First set-up the default folder where images will be stored by: Clicking the menu: File > Album in the GUI, this open a dialog box shown to the left. Then select the Set default folder submenu 6

7 Thayumanasamy Somasundaram Page 7 3/18/2005 Then browse to D:\CrystalImages\ PIName\UserName folder and set it (If the folder doesn t exist, create one). The user s images will be stored in this folder. Now set-up image settings by Selecting the Setting>Setting menu, This will open a new window with image setting options. Under Capture tab select.jpg file extension and select Using current date and time as file names options. 7

8 Thayumanasamy Somasundaram Page 8 3/18/2005 Now open MoticTek camera either of the two ways: 1) following Modules > MoticTek menu, or 2) by clicking the video camera-like icon on the second row of menu bar This opens the MoticTek Camera Preview interface Now place your sample in the optical base on Leica S8 microscope and illuminate it Focus your crystal in an appropriate well using the Focus knob 8

9 Thayumanasamy Somasundaram Page 9 3/18/2005 Adjust the magnification using the Magnification knob Now switch from the Visual mode to Document mode using Switching Knob Now you will see image of your crystal in the MoticTek Preview in the computer You can play with various controls like Saturation Gamma and Exposure on the preview window to get a better picture 9

10 Thayumanasamy Somasundaram Page 10 3/18/2005 When an appropriate image is ready click the Camera button on the top menu bar or Capture button in Capture tab to record a single image. Click on Auto capture to series of images at a particular Frame Rate per second. Image will be captured and recorded as per the Settings condition Accessing Crystal Images Remotely Procedure for viewing crystal images Log into any Windows O/S personal computers in KLB 10

11 Thayumanasamy Somasundaram Page 11 3/18/2005 Select either of two procedures: 1) Start > Run button and enter: \\phe.sb.fsu.edu and hit return, or 2) In My Network Places Address section enter \\phe.sb.fsu.edu and hit return The user will see a login screen for phe.sb.fsu.edu 11

12 Thayumanasamy Somasundaram Page 12 3/18/2005 Login using username xrayuser and password from Soma The user will see a folder named CrystalImages Clicking on CrystalImages will lead to user s subfolder and images. Hardware Information Dell Workstation Dell Precision 220 Workstation The Dell Precision 220 workstation has a single Intel 864 MHz processor with 245 Mbytes of memory running Microsoft Windows 2000 Professional Edition ( SP4). The Moticam camera is connected to the workstation via a FireWire cable. The Motic Images Advanced Software is located at: c:\program Files \Motic\Motic Images Advanced 3.2 \MIAdvance.exe 12

13 Thayumanasamy Somasundaram Page 13 3/18/2005 Leica Stereo Zoom Microscope Leica S8 APO Microscope The Leica S8 APO stereo zoom microscope has a 1:8 zoom ratio, apochromatic (free of both chromatic and spherical aberration) optical system. This microscope has a polarizer and an analyzer attachment, an incident light base, a gliding sample stage, a 1x objective, a 10x eye piece and a c-mount photographic attachment for digital documentation. Moticam FireWire Camera Moticam 1300 The Moticam 1300 digital camera has a CMOS sensor and is capable of capturing 1.3 mega pixel (1280x 1024) color images of crystals. It has 7.5 µm pixel pitch and has a scan rate of 10 fps at 1280x1024 pixel size. The camera is attached to a workstation via a FireWire cable Software Information Motic Software Motic Images Advanced 3.2 The Motic Images Advanced 3.2 software allows the capture of a still image of a crystal as well as series of images at pre-set intervals of time. Analysis like measuring linear dimension, gamma correction, gray scale rendition, etc can be performed on the images. User s crystal images are stored in the following directory: D:\CrystalImages\PI_Name\ Project_Name\ Date_of_Image_Capture\Time.jpg Remote Access (XRAY and Non-XRAY domain) The top level directory of CrystalImages is a shared folder and anyone with an account in phe.sb.fsu.edu will be able to access it by simply clicking on the link below: \\phe.sb.fsu.edu\crystalimages (only under IMB Windows Environment). However, if you are not logged into a computer that is not part of XRAY domain, say a laptop (MSHOME domain), or IMB computers (IMB2 domain), but you are still inside KLB firewall then the procedure may be different: 13

14 Thayumanasamy Somasundaram Page 14 3/18/2005 First browse to \\phe.sb.fsu.edu. You will see a login screen similar to the one shown on the right Remember to prefix the domain name XRAY\ before the user name xrayuser and type in the password You will see the CrystalImages Folder Conclusion Leica Moticam digital documentation system makes it convenient to record, compare and analyze digital images of crystals both during screening and crystal mounting. Detailed procedure for calibrating the microscope and setting up automated recording of images are available at the Motic Images Advanced User Manual. If you have any further questions or comments please contact Soma. 14