Zeiss
user notes and responsibilites
rtf version
Registered
User Responsibilities:
- be precise in signing up
online and in logbook (know the sign-up rules)
- keep lenses and work area
clean (if workstation is 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 days
- acknowledge NSF BBS
8714235 in your publications
- understand start-up and
shutdown procedures!!!
START-UP
PROCEDURE:
- Turn on Hg arc lamp
before other electronics
(if
you don't need fluorescent light to find your sample don't turn on)
- Turn on remote control
switch--this powers all other electronics
- Turn on computer
- Press control-alt-delete
to log into your account
- Double click on LSM 510
software
- Choose "scan new images"
and "start expert mode"
SHUTDOWN
PROCEDURE:
- Check online if anybody
is using the instrument after you:
- if somebody is to use the
confocal within one hour of your session, leave on
- if it is after 5pm, and
you have not been contacted by the next user, shutdown
- you must cancel your
session one hour or more before your sign-up time,
otherwise you are responsible to
shut down
- if the instrument is left
running, your account will be charged
to
leave system on,
just exit the software and log-off from your account
to
turn system off, go
to the laser icon, select each laser and click on "OFF"
- you MUST wait until the
Argon laser fan has stopped to turn off electronics
- exit the program (turn
the Hg arc lamp off)
- write your CD
- shutdown the computer
- turn off the remote
control switch
- lower the lenses and
clean them, store in "empty" lens position
- cover the stand (not the
Hg arc lamp in the back)
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
my network places/computers near me/Imgpc5/ImageTemp/your folder
- 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)
- close down field diaphragm
- focus and center condenser
- open field diaphragm just
enough to fill field of view
- set digital display to
DIC II
- open condenser aperture
to NA 0.45
- adjust prism below the
objective until optimal
- set polarizer to 70-75
degrees
- make sure analyzer or
"slider" below the objectives is out of the beam path
- 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)