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Michael Hooker Microscopy Facility (MHMF.ORG)

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Leica SP2 Laser Scanning Confocal Microscope

 Location: 6123 Thurston Bowles

  

Index

0.   Notices -  New computer installed September 25, 2013
1.   Operating the system (a step by step guide to basic operation of the confocal)
2.   The System
3.   First time use (new user)
4.   Known Bugs
5.   Viewing Leica Files - getting viewing software for Windows
6.   Leica confocal file analysis and processing
7.   Links
8.   Reporting Problems
9.   Password problems/German key board
10. Dealing with software crashes and microscope stand lockups

0.  Notices

  • A new computer was installed on Sep 24, 2013.  Most functions will work as they did on the old computer.  Some points worth noting are:
    • Please let us know if you would like assistance or have questions or find anything odd
    • During LCS initialization (while the drosophila embryo image is rotating) the process will stop at initializing at "Fluorescence Module".  To continue move the mouse or press shift to proceed.
    • There is no E: drive anymore.  Use D:\USERS\your-login-name (which should contain a LCS subdirectory - see/contact Michael if a LCS directory is not there for your directory)
    • D:\Users is shared as \\Laplace\users1 on the network
    • New/modified/added files at the D:\Users directory are copied to the main file server \\Minsky\laplace\users1 every evening at about 2 AM. 
    • USB ports are available on the computer and may be used to transfer data (as of Sept 27 we have not tested this).  Windows XP does not support USB 3, so transfers will be at USB 2.0 speeds.  Do not use a USB drive if you are not absolutely sure that it is free of viruses, trojans and malware. 
    • The display screens may look brighter. This is due to characteristics of the video card.  This has no effect on the acquired images.  Hopefully you are using the glow over & under scale while imaging to set gains and offsets.  Use the Q-Lut button.
    • Do not push the computer off the table!  Be very careful, deliberate and mindful around the computer and the objectives.
  • Also worth noting:
    • Mappings, (M: drive) to the file server \\Minsky will need to be hand recreated for each users (ask Michael for help or instructions)
    • UK to German keyboard mappings no longer apply
    • Security restrictions which were imposed on the old computer are lifted (until ~April 2014 when all Windows XP machines will be banned on the UNC SOM network)
    • Passwords can be changed at the new \\Laplace computer (No need to go to a computer in room 6129)
    • Around April 2014 direct access to \\Laplace\users1 may be blocked.  Image files will need to be transferred to \\Minsky for network access from your lab/office.
  • Old Data:
    • Files from the old computer at D:\users are at \\Minsky\laplace\users and files that were on the E:\ drive are at \\Minsky\laplace\Users2 (this applies to files back to June 2012)
    • Files older than June 2012 and a few more years back (may be up to ~4 years ago) are on the backup servers and can be made available by arrangement with Michael or Neal
  • General:
    • Some operations are faster with the new computer.  e.g. Average projections.   Most operations occur at about the same speed as the old computer.
    • Please help us out and let us know if you notice any quirks on the system
    • Please let us know if you would like assistance or have questions

1.  Operating the system

Power up, setting up microscope, starting software, scanning, saving, shutting down, special scanning modes

2.  The System

2.1  Lasers:

2.2  Objectives:

Objectives *
Turret position Mag. NA type WD corrections cover slip Immersion part no. For DIC (Nomarski)
Objective Wollaston *** Condenser Wollaston 
  10x 0.4   3.6 mm  

#1.5

 air      
  16x 0.5 PL Fluotar 150 um  

#1.5

 oil/glycerol/water 506012 C 2
  20x 0.7 PL Apo 590 um  

#1.5

 air 506513 C 2
In drawer 40x 0.85 PL Apo 240 um corr

0.14-0.18

 water      
  40x 1.25 to 0.75 Apochromat 240 um aperture

#1.5

 oil   E 5
  63x 1.4 to 0.6 PlanApo 90 um aperture

#1.5

 oil   E 4
In drawer 63x 1.2 Apo 220 um corr 0.14-0.18 water   D 5
In drawer L40x 0.8 HCX Apo 3 mm U-V-I none water ** 506155 C 3
In drawer L63x 0.9 HCX Apo 2 mm U-V-I none water ** 506148    

* Note: exact objective installed should be confirmed by looking at the microscope
** dipping lenses use no cover slip.  All other lenses prefer a number 1.5 (170 um) glass cover slip.
*** When not using DIC the objective Wollaston should be in the blank (BF) position and the Analyser should be out
Use only Leica immersion oil

*Counter-clockwise= increasing position number ↑
clockwise= decreasing position number ↓

2.3  Detection:

2.4  Analysis:

Epifluorescent cubes used for viewing by eye (not confocal scanning)

LCD display

Carousel Position #

Leica cube ID

Leica part

Excitation Color of light

Emission Color

Typical fluorophores

 

1

(Empty)

-

-

-

-

 

2

(Empty)

-

-

-

-

A

3

A

513824

Light blue  BP360/40

Blue  LP425

DAPI

N21

4

N2.1

513832

Green  BP539/45

Red  LP590

Texas Red/Rhodamine/CY3

Displays I3

5

L5

513849

Blue  BP480/40

Green  BP527/30

FITC, GFP, CY2

Displays L5

6

I3

513828

Blue  BP470/40

Yellow  LP515

YFP, FM 1-43

(SCAN)

7

(Empty)

-

-

-

-s

JST

8

(unlabeled)

-

(A 3% reflective mirror cube for alignment of arc lamp)

Carousel counter-clockwise= increasing position numbers ▲
Carousel clockwise = decreasing position numbers ▼
Leica fluorescent cube information

3.  First time use (new user)

Copy LCS parameter files from d:\users\_typical\lcs to d:\users\username\lcs
Do not use factory default settings since most parameters are not appropriate for our system

4.  Known Bugs

5.  Viewing Files

 

Note: Read/Write permission may be required required to open a Leica Image Database: Image data base files must be in a location with read & write permissions.  Since CD/DVDs are read only, copy the whole database to the hard drive, and open it from the hard drive if necessary.  Depending on the operating system permissions may need resetting on the hard drive after copying from CD/DVDs. 

8.  Leica confocal Image analysis and processing

7.  Links

      www.leica-microsystems.com
       LeicaLCS-lite software FTP site - ftp://ftp.llt.de

8.  Reporting Problems

     Please contact facility personnel

     Leica Confocal Service and Support
     866-830-0735    8:00 A.M. to 5:00 P.M. EST/EDT
     confocal@leica-microsystems.com
     serial number 195353
     Note: if you contact Leica directly please inform facility personnel as well so that we can follow up.
      Installation date: 23 April 2002 

9.  German Keyboard mappings can prevent passwords from being recognized

German keyboard mappings translates some keyboard keys to other characters.  Basically some keys have a different meaning to the label embossed into the key cap.  Characters to avoid include: !  @  #  '   there are others.  These characters may be found in other unusual positions on the keyboard. 

 

10.  Dealing with software crashes and Microscope Stand Lock Ups

11. Applications:

 
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Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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Questions/comments/problems: Michael Chua

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Last Updated: 2014-07-23