Zeiss Laser Scanning Microscope 510 Zuidhorst 178

Zeiss Laser Scanning Microscope 510 Zuidhorst 178
Zeiss Laser Scanning Microscope 510
Zuidhorst 178
For more information contact
Tom Groothuis
ZH153, t.a.m.groothuis@utwente.nl
Start Hardware
Remove the blue dust cap from the microscope.
If needed, turn on the mercury short arc lamp light
switch (will not start before next step)
Note: Whenever the mercury lamp is turned ON, it
should be left on for at least 30 minutes. Once the lamp
has been turned OFF, it should not be turned on again
for 30 minutes.
Turn on the whole microscope system with the
remote control
Turn on the PC power
(Note: if the computer was already on before the rest of
the system, the computer might not recognize the
system and you cannot make images)
Start Software
Log on to windows XP with the account: user and
password Confocal
Start the Zen software by clicking the Zen icon
The Zen program will start and show a selection menu
were you can choose between Start System and Image
Processing. Choose the first one, after which the
hardware will be initialized. This may take up to 1
minute.
Tool groups with tools:
I.Setup Manager
I.i.Laser
I.ii.Imaging Setup
I.iii.Light Path
II.Online Acquisition
II.i.Acquisition Mode
II.ii.Channels
II.iii.Focus
II.iv.Regions
III.Multidimensional Acquisition
III.i.Z-Stack
III.ii.Bleaching
III.iii.Time Series
III.iv.Information On Experiment
Choosing and Changing Objectives
Corrections
Magnifi
cation
N.A.
Spec.
Optical
Immer
sion
Cov.Gl.
(mm)
Work dist.
(mm)
Plan Neofluar
10x
0.3
DIC
-
0.17
5.5
Plan Neofluar
20x
0.5
DIC/Ph2
-
0.17
2
LD-Achroplan
40x
0.6
DIC
-
0-2
1.8 @ 1.0
C-Apochromat
63x
1.2
DIC
Water
0.15 - 0.17
0.28 @ 0.17
LD-Achroplan
63x
0.75
Ph2
-
0 - 1.5
1.5 @ 1.0
Plan-Apochromat
63x
1.4
DIC
Oil
0.17
0.19
Select a proper
objective.
The objective should
suite your desired
MAGNIFICATION and
THICKNESS of the
material between the
specimen and the
objective.
Change objectives:
I. In the Light Path
tool - Ocular tab
II. With the Objective
buttons on the
microscope
Select a proper sample
holder from the 4 holders
available.
(e.g. cell culture plate holder,
object glass holder, heated
holder).
Note: make sure the red dot
of the holder (blue arrows) is
in the same corner as the
red dot on the microscope
stage (white arrow) and
clamp it.
Mount and Find the Specimen
Change the microscope-light path to direct the light
(white or fluorescence) to the eyepieces by clicking
on the Ocular button in the Zen toolbar.
The mercury-lamp-light can be shuttered ON
and OFF by the FL button on the
body of the microscope on the right
hand-side.
Bright-field, halogen light can be
switched on and off HAL button on
the body of the microscope.
FL
HAL
Reflector
The fluorescence filter block (a.k.a. reflector) carousel
can be switched back and
forward using the two buttons marked Reflector on the
body of the microscope,
behind the right hand focus knob.
The reflector contains 3 filter blocks:
Name
Ex.
D.M.
Em.
DAPI (01)
BP 365/12
FT 395
LP 397
FITC (09)
BP 450-490
FT 510
LP 515
Rhod. (15)
BP 546/12
FT 580
LP 590
Once you have found your specimen, redirect the
light-path to in the confocal by clicking the LSM
button.
The microscope will automatically change the filterposition and shutter the light sources
Starting the lasers
Open the Laser Tool and activate the lasers you
need for your experiment.
Remember, the argon multi-line laser has to first be put
to standby for a 5 minute warm-up before it changes to
on.
The activated lasers are indicated with a color icon of a
laser warning sign.
When all the power statuses of the desired lasers
read On, close the Laser Tool.
Configure the Lasers and the Light-Path
Open the Light Path tool - Acquisition tab.
The Light Path tool displays the selected track
configuration which is used for the scan procedure.
You can change the settings of this panel using the
following function elements:
Activation / deactivation of the excitation
wavelengths (check box) and setting of
excitation intensities (slider). If necessary
open the Laser Control tool (see above).
Selection of the main dichroic beam
splitter (HFT) or secondary dichroic beam
splitter (NFT) position through selection
from the relevant list box.
Selection of an emission filter through
selection from the relevant list box.
Activation / deactivation of the selected
channel (check box in front to icon) for the
scanning procedure and assigning a color
(Look Up Table, LUT) to the channel.
Open the Imaging Setup tool.
The Detection Bands & Laser Lines assigned in the
previous step are displayed in a spectral panel to
visualize the activated laser lines for excitation (vertical
lines) and activated detection channels (colored
horizontal bars).
You can save the settings of a Track at the bottom of
the tool in pro mode (click ʻproʼ to activate/deactivate).
Setting the Parameters for Scanning
Open the Acquisition Mode tool (pro-mode)
Set the image size to the size you want your images to
have (512 x 512 is a good start setting).
Set the scan speed (faster scanning gives smaller time
interval between images, but lower signal-to-noise ratio;
slower scanning gives longer time intervals, but higher
signal-to-noise ratio).
Averaging will increase the signal-to-noise ratio, but
longer time intervals. You can select to average frameby-frame or line-by-line in pro-mode.
For quantification of your images, select a bit depth
(dynamic range) of either 8-bit (256 intensity-values) or
12-bit (4096 intensity-values).
Set the Scan Area by changing the zoom and the xyrange with the sliders or by dragging the square to the
site of interest; you can rotate your image with the
rotation slider.
To reset a setting, push the respective button on the
right hand side of the setting: C, 0 or 1.
Acquire Preliminary Confocal Image
Open the Channels tool
Set your preferred pinhole size for each channel:
• click 1 AU for high z-resolution (confocal) images,
but lower fluorescence signal;
• click Max for low z-resolution, but maximum
fluorescence signal.
Click the Find button of the action buttons in the
upper left corner.
This will start the laser scanning for automatic preadjustment of detector gain and offset in the Channels
tool. If applicable, Zen will also generate a new tab for
the image.
NOTE: Zen will only open a new tab if there is no image
open or if the previous image has been saved manually.
The preview image will look something like the image
shown on the left.
For multi-label experiments, it is best to have all single
channels displayed separately. To do this: click the Split
tab next to the image.
Optimizing the Settings
Expose (if not already visible) the View |
Dimensions section of the screen.
To toggle between the ascribed LUT and the range
indicator LUT simply click on the color field in the
button under the channel button (blue circle)
Note: To get an optimal image that you can use for
quantification it is necessary to use the full dynamic
range you selected above (8- or 12-bit). The dynamic
range can be shown with the Range Indicator LUT
(Look Up Table; the microscopeʼs photomultipliers
(PMTs) catch photons, which donʼt have a color,
therefore you gave them a false color representation (a
LUT) in the Light Path configuration above).
The split view of a Green, Red and Merged image
(check box in lower left corner of the image) with range
indicator LUTs will appear as shown below.
Toggle a channel On - Off with the respective button
(blue arrow) and select a different LUT with the pulldown menu (white arrow).
Optimizing the Settings (2)
Expose (if not already visible) the Channels tool.
For continuos scanning during optimization click
either the Fast button or the Continuous button of
the action buttons
Note: Continuous differs from Fast in that it is already
using the Parameters for Scanning you assigned above,
and Fast does not. So continuous will give a more
representative image of the end result, while Fast allows
fast optimization.
Adjust the Gain and Amplifier Offset for each channel
such that you get an optimal image. You may also
decrease the scanning speed or increase the laserpower to get more signal, but beware of photobleaching and sample heating in the latter case.
After you optimized the channels, stop scanning by
pressing the Stop button.
Acquire the final image by pressing the Start
button.
Your image will look like shown below.
Saving and exporting the image(s)
To Save an image click the respective icon in the
Open Images section.
Save an image as an LSM 5 file to preserve all
metadata (objective, zoom, light path etc. used). This
metadata is important for calibration (x,y,z dimensions of
an image in µm) and Reuse of the settings.
Saving an image can also be done via the File menu.
Store your images on the D:\ partition of the hard-drive.
The folders are being shared, you can find the computer
in the TNW directory: BFT0151
You can Reuse the settings of an image by opening the
image and then click the Reuse button in the bottom of
the View | Dimensions section (Note: this will NOT turn
the lasers to the ON state, do this manually).
Use the View | Overlay section to overlay the image
with text / scalebar / arrows etc.
Once your image is complete, use Export in the File
menu to export the image as you like. There are several
options, please check them to see if the result is the
same as you intended.
Acquiring a Z-series
Open and activate the Z-Stack tool in the
Multidimensional Acquisition.
Note: To activate the tool, click the check-mark of the
tool (blue arrow), this will activate z-stack imaging as
shown by the little icon (blue circle) below the action
buttons.
I. Make sure you have a proper pinhole setting for zstacks.
II. Activate scanning by using the Fast or
Continuous action button.
III. Change the z-level manually at the microscope to
the top most position you want to image and click
the Set First button.
IV.Change the z-level manually at the microscope to
the bottom most position you want to image and
click Set Last button.
V. Click the Optimal Interval button so the software
can determine the optimal number of slices for the
chosen Range (First and Last) and Pinhole
thickness (indicated on the Optimal interval
button).
VI.Activate the scanning procedure with the Start
button.
The result can be displayed in Gallery view (below) and
some other views as indicated by the view-tabs.
Multitrack imaging (= sequential imaging)
When you are using multiple dyes, fluorescence of
one fluorophore may also be detected in a ʻwrongʼ
channel. This phenomenon is named cross-talk,
leak-through or bleed-through.
Fluorescence from one channel detected in another is
shown below (blue circles).
You can test for leak-through by setting the transmission
of a laser to 0% in the Channels tool during Fast or
Continuous scanning. This will be so if there is still
fluorescence detected in e.g. the red channel when the
543 nm laser is set to 0%.
To overcome the problem of leak-through you can make
use of multitrack scanning, this will make a scan of each
label one after the other.
Open the Imaging Setup tool
• Use the Track + and - buttons to add or remove a
track.
• Configure a Light Path for each track as above.
• Optimize the settings (as in Optimizing Settings)
for each channel separately by un-checking the
check-box in front of the other track.
• Push either the Fast or Continuous action button
to start the scanning.
• You can Save the Configuration in the top of the
Imaging Setup tool.
Shutting the System Down
Turn Off the lasers in the Laser tool
In the Laser control window turn each laser Off or to
Standby if somebody is using the system within the
next hour (check if the person shows up!!!).
Remove Specimen and clean microscope
• Wipe-off water or oil from objective and specimen.
• Move to a low power objective (10× or 20x).
• Lower the objective nosepiece using the lower
focus button on the right hand side of the
microscope base.
• Turn off the epifluorescence lamp if nobody is
using the system within the next hour (check if
the person shows up!!!).
• Cover the microscope avoiding the hot mercurylamp-housing.
Exit the software
• Exit the Zen software.
• A message will come up reminding you not to
power down the system until the laser is cool
(about 5 minutes). Click OK.
• If you have left the lasers on for the next user, you
will also be asked whether you want the lasers
switched off. Click No.
• Burn your data to CD or copy across network.
• Once you have finished with the computer,
LOGOUT of SHUT-DOWN.
Power down the system
If nobody has booked for the next hour, please
shutdown the system If the next person is the last
booking of the day, please call them and confirm
that they will be using it.
Shutting down requires that the remote control be
switched off.
Offline Image Analysis and Manipulation
Opening your images offline
Zeiss Zen
A downloaded file of Zen is ready for installation in
D:\Software\ and can be used for Off-line use, no license
needed.
You can also download the newest version yourself at
http://www.zeiss.com/zen
After the animation you can find a link in the lower left
corner. This does require to fill in a registration form.
Requires Microsoft Windows XP
ImageJ
An open source Java application for multiple platforms:
http://rsb.info.nih.gov/ij/
Has a lot of plugins available, including Zeiss plugin:
http://rsb.info.nih.gov/ij/plugins/lsm-reader.html
Troubleshooting: Error messages
The Error Log pops-up with a COM2 Error
The 2-photon, Chameleon laser can also be in use on
another microscope next-door, therefor you have to
book the Chameleon separately from the LSM.
If the Chameleon is in use on the other system the LSM
computer is not able to communicate with the laser,
which will generate COM2 Errors. This is NO problem
for using the rest of the features of the microscope.
Leave the Error menu in the background or minimize it
with the _ button in the upper right corner.
If you encounter other COM errors, please contact
the administrator.
When you are loading a configuration (Track or
Reused) you might get the warning displayed on the left.
Loading a configuration can change objectives, filters,
dichroic mirrors etc. but CANNOT turn a laser On, so do
this manually.
Troubleshooting: Strange circles
My images have strange fluorescent rings
If you have multiple colors, please make sure you are
not detecting one of the laser-lines, which are partially
reflected by the coverglass. The reflected laser-light may
end up in one of the detectors, but will be relatively
strong compared to the fluorescence signal.
A detected laser-line (543 nm) will appear as shown in
the green channel below.
Solutions:
• Use the BP 500-530 nm filter for channel 2, so the
543 nm line will not be detected.
• Use a multitrack procedure.
Light Path Hardware
Main dichroic beam splitter (HFT)
The main dichroic mirror deflects the indicated laser
lines onto the specimen and allows the emitted
fluorescent light to pass through.
A HFT KP XXX/YYY is a combination of a HFT YYY and
HFT KP XXX used for simultaneous IR multi photon and
single photon excitation.
Example: HFT KP 700/488.
A HFT KP XXX (KP = Short Pass) is a special type of a
main dichroic used for IR multi photon excitation
(Chameleon). The HFT KP 650, deflects laser light with
a wavelength longer as 650 nm onto the
specimen and allows fluorescent emission light in the
visible range below 650 nm to pass through.
NT XX/YY = Neutral Density Filter, works as an
attenuation filter. NT 80/20 passes 80% of the light, cuts
20% of light.
Secondary dichroic beam splitters (NFTs)
A secondary beam splitter is used to split the emitted
light which will be guided into separate channels. A
cascade of NFTs (NFT 1,2,3) allows distribution of
the emission light to more than two channels/
detectors.
A None or Plate will let all light pass.
A Mirror will reflect all light.
A NFT XXX will deflect light with shorter
wavelength than XXX nm, while light with
longer wavelength passes the NFT.
A BG 39 (Blue Green glass) blocks infrared
excitation light by absorption.
Light Path Hardware
Channel 1:
KP 685
LP 475
LP 505
Remark:
BP = Band Pass
LP = Long Pass
KP = Short Pass
BP–IR = a band pass
filter suitable for detection
of IR excited dyes, which
blocks the IR light.
LP 530
LP 560
LP 650
BP 565-615 IR
Channel 2:
KP 685
BP 390-465 IR
BP 435-485 IR
BP 480-520 IR
BP 500-530
BP 500-550 IR
LP 505
Channel 3:
LP 560
BP 510-520 IR
BP 500-550 IR
BP 535-590 IR
BP 565-615 IR
Laser
Lines:
458 477 488
514
543
633
Chameleon
680 to 1080 nm
1.
2.
3.
4.
5.
6.
7.
Check the microscope if everything looks clean and normal. If not report it in the logbook.
Switch on the fluorescent lamp (white box above the laser module, marked “1.”)
Switch on the remote control box (small box on the PC desk, marked “2.”)
Switch on the PC
Login to the computer with your account
Wait 1 min so the system is fully booted
Click on the ZEN software icon on the desktop and click on the start system button in the
pop-up window inside the ZEN software
8. If you are the first of the day there will be an error message when ZEN software is started, just
click OK to all the error messages, shut down the software, wait 2-3 minutes (the program is
still running in the background for a while) and start it again. For the second time it would start
properly.
9. Switch on those lasers that you want to use in the ZEN software under the acquisition menu.
Note that the 405 laser needs at least 20 minutes warm up time for proper use.
Matyas Molnar
tel: 0701679083
When you are finished with your study do the following:
1. Burn your data files to a data CD/DVD with the Nero software (You can reach it from the start menu.
Don’t forget to push back the CD/DVD drive after you finished the burning.)
2. Clean all the objectives, the condenser and the stage
3. Swing in the lowest magnification objective (or empty slot)
4. Close ZEN software and log-out from your windows account (if you are not the last one of the day, leave all
the lasers ON)
If you are the last user of the day (check it in the booking homepage), follow with these steps to shut
down the microscope:
5. Switch off all lasers in the ZEN software before close the software.
6. Wait 5 min while the lasers cool down. You can hear when the ventilation switches off automatically.
7. Shut down the computer
8. Switch off the remote control (small box on the PC desk, marked “2.”)
9. Switch off the fluorescent lamp button (white box above the laser module, marked “1.”)
10. Cover the microscope with its blue covering textile
11. Use the logbook and write in any problems and comments occurred during your microscope session! If you
signed the logbook and did not report any problem, it means you left the machine in perfect condition.
12. Switch off all the lamps in the room and close the door
Matyas Molnar
tel: 0701679083
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertising