Notes for Zeiss LSM 510 Version 3.2

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					                            Notes for Zeiss LSM 510 Version 3.2
             Ru Yi × 4-3474

Lasers (excitation wavelengths): Argon (458nm and 488nm) HeNe1(543nm) HeNe2 (633nm)

Inverted microscope; objective lenses in the rotation set:
10× dry; 20× dry available but not in the rotation set.
25× immersion—set correction factor to oil, glycerol, or water.
40× water
63× water; 63× oil available.
100× oil

Before turning on anything, turn on mercury arc lamp (HBO) underneath the microscope float table.
If HBO is not on, turn off computer first before turning the power on to prevent a power surge.
Turn on remote control box behind the microscope, computer should turns on automatically.
If the monitor screens say ‘invalid sync’, push power button to switch on the computer.

§ Login (Logon to ‘CDIF-server’ not ‘Monster NT’)
Start LSM 510 (shortcut available on computer desktop)
Select ‘Scan New Images’, and ‘Start Expert Mode’
§ File > New > Create a database for today’s session (file type *.mdb)
Make sure that you are saving to your personal folder in ‘Home’ directory on CDIF server (Z:/userid/…)
and not locally on the computer ‘Monster NT’.
File for future reference: load an old database and click on Reuse button to bring up previous settings.

§ Acquire
§ Laser
Turn on the laser lines required for your experiment; Argon 488 for FITC, GFP; Helium Neon 543 for
Rhodamine Red, Cy3; HeNe 633 for Cy5.
The Ar laser is switch on by clicking on ‘Standby’ for a mandatory warm up.
Set Ar laser output to ~50% (which corresponds to tube current of 5.9A~6.1A)
Point of reference: excitation wavelength is shorter than the emission wavelength of the fluorophore.

§ Microscope
Top part of scope flips back out of the way for putting sample onto the stage.
Two swing-out filters (polarizer for DIC) near top of scope should be swung out.
If switching between slide or dish holder, align the red dot on slide/dish holder with red dot on the stage.
Choose objective lens.
If using 25× immersion lens, remember to adjust for correction factor on the objective lens itself; line up
symbol on with the type of immersion you’re using, e.g. W|| is for H2 O immersion with coverslip
(inverted microscope) vs. W| is for H2 O immersion without coverslip on an upright microscope.
If it’s not a dry lens, add a tiny drop of H2 O or glycerol to objective before loading sample onto holder.
A useful tool: Go to Stage tab at the end of the submenu; hit Load to lower the objective, then put sample
on stage; hit Work to let the objective to work its way up to where it was.

Turn on transmitted light to align slide. Power box to right of scope should be switched on (green button).
Back to the LSM 510 software, hit ‘Light Remote’ and ‘On’; set light intensity to lowest setting, 0% is
fine—anything higher may be too bright for your eyes.
To find sample under fluorescence, set reflector turret to FSet09 for FITC/GFP, FSet15 for Rhodamine.

Sliders on either side of oculars have to be pulled out to be able to see through the eyepieces.
Sliders at bottom right of the microscope base have to be set for VISible mode > top in bottom out
                                           to send signal to the detector for LSM > top out bottom in

§ Configuration
Select single track or multi track (for two or more fluorophores).
In Single Track, to select a preset beam path setting from the pull down list, click on ‘Config’ floppy disk
button on the side panel.
In Multi Track, click on ‘Config’ button and choose one of the preset configurations from the list, then
click ‘Apply’; one can also choose to store/apply ‘Single Track’ settings to each of the beam paths.
User can check or uncheck the buttons to turn on/off the detectors in each channel. Clicking on the ‘Ch’
button allows user to apply a different false-color for that channel; black is designated as ‘off’.

§ Scan
Select mode, ‘frame’ or ‘line’
Frame size: 512×512 pixels is typical, 1024×1024 pixels or higher may be suitable for some experiments
Faster scan speed = lower resolution so 8 is good to start, then 7 or 6 might be better, it’s a slower scan
but may it gives brighter images at this particular size of pinhole.
If images are noisy, turn on averaging, method = mean > number = 2 (or higher); scan time would be
doubled likewise; averaging is an option for fixed slides and not for experiment using live samples.
8-bit is fine, but 12-bit is better, it yields higher degree of signal in each pixel. 2^8=256 vs. 2^12=4096
Scan direction  single direction; usually not loop or bi-directional.

Find the interested focal plane in specimen and adjust parameters:
Go to Channels, either hit Single to capture a snapshot of the specimen; or, hit Find (black and white
icon) which will shows specimen on screen at the specified pinhole size with all sort of auto-adjustments
to digital gain, amplitude offset, etc.

To align specimen in field of view, hit XY Continuous and move stage or focus in/out, then hit Stop
when it’s adjusted to minimize photo-bleaching. Fast XY gives real-time response to changes, but it
yields dimmer images.
Set the parameters for each channel independently; use XY Continuous to adjust
(1) Detector gain, typically ~800.
(2) Pinhole Ø should be minimized, but anything smaller than 1 Airy unit will significantly restricts light;
smaller pinhole Ø = more Z slices.
In Multi Track: pinholes should be aligned so that the size of optical slices match in each channel—not by
matching the airy units—an important consideration for quantitative analyses later.
(3) Amplitude offset (when necessary, see notes below).

To image in full range, use the color palette > range indicator to help adjust detector gain and/or
amplitude offset. Run XY Continuous to see the effect of adjustment to gain or offset.
Red dots = upper limit > lower detector gain to reduce saturation or overexposure.
Blue dots = lower limit > move amplitude offset scale bar further right to reduce darkness.
XY Continuous is recommended over Fast XY because what you see (on screen) is what you get (in final
results). With the scan speed maxed out, images inadvertently appear to be dimmer using Fast XY.
Range indicator changes image to grayscale (with red/blue) contrasting expression vs. background.
In an image with optimized range, there would be little red dots in the brightest cells, and some blue dots
in the background.
Turn off color palette when finished adjusting.

There is a Crop tool to resize the images, and fine-tuning on x or y-axis is possible through controls in
bottom of Mode panel. The Reset button is useful to resize the cropped or zoomed-in image.

§ Z stack
Under Z stack tab, select the Mark First/Last tab to mark the first slice and the last slice:
Hit XY Continuous and focus away from specimen to find bottom slice. Hit stop and mark as first slice.
Hit XY Continuous and focus back into specimen to find top slice, stop and mark as last.

Optimize # of slices > Hit the Z slice button
Select ‘Optimal interval’ that will yield the right # of slices to scan; ‘Optimal # of slices’ will changes
with different pinhole sizes. A bigger pinhole Ø = thicker optical slices = fewer # of slices.
Can check on ‘mid’ (the middle slice)—this will help to check intensity/brightness; or activate auto Z
correction [bottom quarter in Z stack tab] if working with fixed slides, not with live samples, and not for
quantitative analyses afterwards.
Merging slices in a z-stack makes final picture much brighter (× # of slices, e.g. 10 slices can be up to 10
times brighter on a flattened image).

When ready, hit the Start button, and it will scan the optical slices.
While it’s scanning, follow along through the Gallery view (shows the slices as they come up) or via the
Split view (shows each channel separately, plus the composite view).

Save  Always save acquired image(s) before you do anything else; the mantra is save to be safe.

Exporting images can be done later on the workstation—a cheaper option.
Export ‘Raw data’ as Tagged Image File (*.tif) as single or a series.
If adding other graphics, i.e. scale bar, export images as *.tif with ‘contents of image’.

When you’re done…
Switch Argon laser to ‘standby’ if there is another user after you. Switch off lasers if yours is the last
session of the day; remember to let Ar laser to cool off (it takes ~3min). HeNe lasers do not require a
cooling period. Clean the objective lens with lens paper (do not use Kimwipes).

§ Exit program. Logoff. Stop here if someone else is coming after you; check with CDIF calendar on the
Macintosh computer, remember to refresh the Internet browser for the latest information.
If you are the last one, let the computer shut down; when the "ready to be shut down" screen shows up, do
not to turn off the computer's power button, instead just switch off the remote power control (behind the
scope). Also, switch off the HBO lamp; the sequence is not as critical as when turning on the system.

Access your data from Mac OS X: In ‘Finder’, choose ‘Go’…‘Connect to server’; type
smb:// ; login with your username and password; logon to ‘Home’ directory on server; a
‘Home’ icon will appear on desktop; find your folder.
Problem connecting to CDIF-server after upgraded to  OS 10.4? Use Spotlight to find Keychain; delete
all previous entries in Keychain; restart/login again before connecting to the server as mentioned above.
To connect from Windows PC, Start > Run > type in “CDIF-server” and login with your user ID and
password. Or, locate CDIF-server through Network Neighborhood.
Backup and remove your data; please keep your folder under 1 gigabytes.

Some Rights Reserved. Creative Commons Attribution-NonCommercial-ShareAlike 2.5 License. These notes were adapted from notes courtesy
of Mary Kinkel and Vicky Prince at Univ of Chicago (Revised 2007.04.10).

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