Leica Moticam Stereo Microscope

Digital Crystal Documentation Procedure

Document Version: 1.2 (March 17, 2005); 1.1 (07162004); 1.0 (05102004)

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 (5.0.2195 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 412.

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 (5.0.2195 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 (128.186.103.107) 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 Set-up @ KLB 412

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 …
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” sub-menu …
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.
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
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
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
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
Login using username ‘xrayuser’ and password from Soma
The user will see a folder named “CrystalImages”
Clicking on “CrystalImages” will lead to user’s sub-folder 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 (5.0.2195 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

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 mm 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:

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.