Zeiss user notes and responsibilites

Registered User Responsibilities:

Be precise in signing up online and in logbook (know the sign-up rules)
Be prompt for your reserved start time - it matters.
keep lenses and work area clean (if workstation is found messy, last user will be charged)
fill out sample ID form (follow all safety regulations)
do not store more than 50MB in your keeper file on drive D:
make sure to save your data on the E drive and back it up within 7 day
understand start-up and shutdown procedures!!!

START-UP PROCEDURE:

SHUTDOWN PROCEDURE:

The HeNe lasers should be turned off.
The Argon laser and Hg arc lamp should not be left ON unnecessarily (The reason for complicated rules.....)
Check online if anybody is using the instrument after you. (The reason that promptness is essential.)
if somebody is scheduled to use the confocal within 1/2 hour of your session, AND you can contact them, leave ON (Standby for Argon)

The next user is responsible for time beginning at their reserved start time (The reason that promptness is essential.)

If you cannot contact the next user with the info they placed on the Reservation, shut down.
if it is 5pm or later, and you have not been specifically contacted by the next user, shutdown.
you must cancel your session more than one hour before your reserved time, otherwise you are responsible to come and shut down/check.
if the instrument is left running because you failed to follow protocol, your account will be charged for the time.

to leave system on, just exit the software and log-off from your account (Pay attention to the cautions regarding Argon Laser!!!)

to turn system off, go to the laser icon, select each laser and click on "OFF" (Pay attention to the cautions regarding Argon Laser!!!)

Lower the objective lenses and clean them, leave in "empty" lens position
NOTE of Caution! You MUST wait until the Argon laser fan has stopped before turning off electronics via remote control switch
exit the program
turn the Hg arc lamp off
write your CD
shutdown the computer,
turn off the remote control switch (Main Switch)
cover the stand (not the Hg arc lamp in the back)
Make sure you signed the Logbook.

To SAVE images:

click on "save as" on the image display
click "New MDB" to make a new database (each day you work)
go to the E drive and choose the appropriate day you are working
once you are in that folder name your database (name_date) and click create
name your image
your files will be erased after 7 days
if you forgot a cd, transfer files to the computer next door - Imgpc5 and write your cd there later

you can sign up for the computer next door ONLINE - it is called the Imaging workstation
to transfer files to it go to My Network Places | Computers Near Me: \ Imgpc5\ImageTemp
<< Login information is provided to all users during training >>
make yourself a folder based on a recognizable form of your name and transfer images to this folder on ImagePC5
use Nero Burning Rom on the Imaging Workstation (ImagePC5) to write your CD or DVD

To scan an image:

warm up appropriate lasers: go to acquire and click on laser icon
warm up argon laser (if needed) click standby, when status ready, 50% output and "on"
if you need other lasers, select them and click on
go to "vis" mode
lower lenses with down focus and check they are clean with eyepiece lens
put sample on the stage (coverslip side down) and focus using desired objective lens
setup DIC optics for the laser scanning mode (optional)

go to the config icon to set your laser beam path (channel mode, single track)
click on the config disk to bring up a menu of default methods
choose the appropriate method (single track and multitrack configs have different menus)
if a default method does not work for you, we can customize one
go to the scan icon (here you will toggle between mode and channels)
under mode, choose 512x512 (default) or 1024x1024 pixel area (if you need higher res)
collect 8-bit images unless you will quantify fluorescence-then choose 12-bit
under channels (still in scan control) click on ch2, 3, or S and under pinhole click on "1"
to scan an image: ALWAYS CLICK ON FIND WHEN SCANNING A SAMPLE
clicking on FIND will automatically adjust the detector gain preventing damage
your image will appear as an overlay, you can crop your sample and hit "single"
you can click on split xy to separate your channels
on the image display choose palette and range indicator
open your stage icon and set your step size for focusing through your sample
choose cont scan and focus through your sample to find the brightest plane of interest
adjust the range with detector gain (bright=red) and amplifier offset (black level=blue)
under mode in scan control, max your scan speed and enter averages
perfect your ChannelD for DIC (gain and offset)
click on "single" to acquire a single scan

if you want to collect a z-series:

(keep range indicator on as you focus thru your sample to make sure you don't saturate)
in scan control, choose Z settings or Z stack and click "mark first/last"
click on xy cont scan and focus with the "down arrow" to identify your first xy plane of interest and mark first (zero stage if you wish)
focus thru your sample with the "up arrow" under the stage icon to identify your last section and mark this as last
decide on your sectioning interval (under z settings or z-stack) check lens resolution
make sure averages are set in "mode"
click START (this button will always change the focus and slice through preset values)
You can optionally manually focus, then click the "First" and "Last" button at those positions.
There is an advanced control to "pull" the gain during the Z-stack to compensate for brightness decreases as you move away from the coverglass: this is covered in the manual.