Zeiss LSM 510 Inverted Manuel utilisateur

Iron Man

Zeiss LSM 510 Laser Scanning Confocal Microscope

User Guide

v. 1.4 (11/2016)

Objectives

5x/0.15NA EC Plan Neofluar

10x/0.3NA EC Plan Neofluar

20x/0.8NA Plan Apo Chromat

40x/1.3NA oil DIC EC Plan Neofluar

63x/1.4NA oil DIC Plan Apo Chromat

100x/1.4NA oil DIC Plan Apo Chromat

Standard coverslip thickness 0.17

(#1.5 coverslip)

Mercury Arc lamp (ocular)

Dapi, Fitc, Rhod, DIC II, DIC III, Brightfield

Laser lines

Argon/2 –458, 477, 488, 514

HeNe –543

HeNe –633

Diode –405/30

Iron Man User Guide v. 1.4 2

Contents:

Quick Guide 3

Starting up the system 4

The software will open to the ocular tab 5

To look at your slide 5

The Acquisition Tab 6

At the top

In the Setup Manager window

In the Imaging Setup window

In the Channels window

In the Online Acquisition window

Adjusting PMTs

Snap

Z series

The Information tab next to the image 11

When you’re done 11

APPENDIX A –Saving to your lab’s folder on MCDB Dept Server 12

APPENDIX B –Parts of the microscope 13

APPENDIX C –Configuring the Light Path 14

Iron Man User Guide v. 1.4 3

Quick Guide:

1) Turn on components in order. Don’t go too fast – PC & monitor won’t connect.

2) Log into Windows with IdentiKey and start Zen software.

3) Put sample on scope coverslip down. If using oil immersion, do not switch back to air

objective. No oil on air objectives!

4) Load or set up appropriate Channels/Tracks for imaging your sample.

5) Turn on necessary lasers. Do not turn on lasers you won’t be using. Argon/2 and Diode go to

Standby, then On. HeNe turn on. Argon/2: adjust % Output in Laser Properties window to

40% (do not go higher than 40%). Adjust once, then leave it alone.

6) Adjust imaging parameters –

a. Laser attenuation % (3-5)

b. Frame size (512 x 512 or 1024x1024)

c. Scan Speed (~4-5 is good starting point, faster for live samples)

d. Averaging (1)

e. Zoom (1)

f. Pinhole (1 AU)

g. Offset (few to no blue understaturated pixels)

h. Gain (Master) (few to no red oversaturated pixels)

7) Snap for single frame image or set up parameters for Z-series or timelapse. Start Experiment.

8) Note the details in Information tab next to your image –these are useful to write in your

imaging notes.

9) Save and copy your data. The LMCF is not responsible for long-term data storage. See

Appendix A for information on how to transfer data to MCDB server.

10) Lower stage away from sample, and remove sample from stage. Properly clean oil off of any

immersion objectives you may have used with lens paper. If you are at all unsure of this

process, ask for help!

11) Leave the microscope on a low power objective for next user.

If someone is signed up within the next two hours (Check the MCDBCal!):

12) Switch lasers to Standby (if that option is available) or Off.

13) Close Zen.

14) Log use in Excel Sheet, save and close.

15) Log off Windows.

If no one is signed up within two hours or you are the last of the day:

13) Switch lasers to Standby, then Off.

14) Close Zen, confirming that lasers are all Off.

15) Log use in Excel Sheet, save and close.

16) Shut down system: Microscope base (#4), PC (#3, Start Shutdown), Mercury lamp (#2)

17) WAIT FOR THE FANS ON THE LASERS TO SLOW/COOL DOWN. This takes a couple minutes, and

the room should sound like it did when you walked in. Then flip the two switches (#1a & 1b).

Iron Man User Guide v. 1.4 4

Starting Up the System:

Turn on components in numerical order.

1a) Power to the PC

1b) Power to microscope components (including lasers)

2) Mercury Arc lamp located underneath floating air table

3) PC

4) The microscope –green switch on right side of microscope base

If you only need to access the PC, only turn on #1a and #3.

Open Zen 2009 software. Choose appropriate option –Start System (to take new images) or Image Processing.

Iron Man User Guide v. 1.4 5

The software will open to the ocular tab:

Online mode will allow you to view your sample through the oculars and offline mode allows you to view the

sample on the PC screen.

This window will allow you to control/view the following

microscope positions:

1) Halogen bulb for Brightfield viewing

2) Aperture

3) Specimen / stage

4) Objective –You can change the objective here or using

the buttons on the side of the microscope

USE CAUTION WHEN SWITCHING BETWEEN AIR AND

OIL OBJECTIVES

5) Reflector cubes –These should be changed using the

shortcut configurations above

6) Hg bulb shutter

7) Hg bulb

8) Lens

To minimize photobleaching of your sample, close the

Fluorescence Shutter whenever you are not looking

through the eyepieces.

To look at your slide:

1) Load the slide, coverslip down, onto the slide holders on the stage (these can be adjusted manually).

Please be sure that your slide is clean and dry –do not get wet mounting media or condensation on

the stage.

2) Position the slide over the desired objective.

-If you are using an air objective, start at the lowest focus position.

-If you are using oil immersion, place a small drop of oil on the slide before placing it on the

microscope. Do not switch back to an air objective. Turn the focus knob up until the objective

lens is just touching the oil.

3) Click into online mode, and choose the appropriate reflector cube configuration (#5) and open the

fluorescence shutter.

4) Looking through oculars, carefully focus on your sample and center on the structure you want to

image. Done? Close the fluorescence shutter and switch to offline mode.

Iron Man User Guide v. 1.4 6

The Acquisition Tab:

1) At the top –select any options you want for your

experiment (Z-series, time series)

2) In the Setup Manager window –turn on the appropriate

lasers for your experiment. You do not need to turn on

lasers you will not be using.

-Argon/2: Select Standby from dropdown to warm up the

laser. The status will read “Ready” when it is done.

Choose On. Open the Laser Properties window by

clicking on the arrow, and adjust laser output [%] to

40%. Do not go over 40% output or over 6.1 A. Do not

keep changing this Output % - set it for your imaging

session and leave it alone. Keeping the laser at 6.0 A is

done to assure that the laser output is at a stable and

reproducible intensity when using the confocal

scanhead.

-Diode: Select “Standby” from dropdown to warm up

the laser. The status will read “Ready” when it is

done, and only then can you choose “On”

-HeNe –choose “on” if you need these lasers

Iron Man User Guide v. 1.4 7

3) In the Imaging Setup window –choose and set up the appropriate track(s) for your experiment.

-You can open a previous configuration (little folder icon button at the bottom of the Imaging

Setup window) and edit this as

needed, you can reuse settings

from a previous .lsm file (open the

image and click on Reuse button

at the bottom of the Display

window), or you can set it up

yourself (advice at the end of this

guide).

-Two major options –you can

image all fluorophores in multiple

channels on a single track or split

this into multiple tracks. Single

track will be faster, but may lead

to bleedthrough of your signal

across channels.

-An example of both a single track,

multi-channel acquisition and

multi-track acquisition are shown.

-To add or remove tracks, click the

“+” or “-“ buttons next to Track.

-If you will be imaging with

multiple tracks, set the “Switch

tracks every” option to “frame” to

avoid very slow imaging

4) In the Channels window - Make sure the correct lasers are

checked on for each track/channel you want to image. In

the example to the right, Track 3 utilizes the 488nm laser.

To adjust, select the channel above and check on or off the

appropriate lasers. You can also adjust the laser intensity

% at this point. For the Argon and Diode lasers, 3-5% is

usually sufficient, and for the HeNe lasers, 25-35% is

usually a good starting point.

Iron Man User Guide v. 1.4 8

5) In the Online Acquisition window –

-The objective should be the one you’ve selected (don’t recommend changing this here, go back to

the ocular tab to change objectives and refocus)

-The frame size indicates how many pixels in each

dimension you want your image to be –more

pixels = more scanning time and larger file. For

most applications, 512x512 or 1024x1024 is

sufficient.

-Speed is the scan speed. Faster scan speed will

expose the sample to less light (as it does not

dwell as long per pixel) and a shorter total scan

time, but may produce a less bright image. For

fixed samples, you can try dropping this to 3-5 as

a starting point. If you find your signal bleaching

during Z series, increase scan speed to reduce

pixel dwell time.

-Averaging –number of times the sample will be

scanned to produce an average image.

Increasing this number can help clean up noisy

images and can allow you to reduce your scan

speed.

-Bit Depth –the number of grey levels the image will contain. Higher bit depth will result in greater

signal dynamic range (greater discrimination between pixel intensity values). 12 or 16 bit.

-Scan Area –you can control the region to scan and zoom here. This is true optical zoom and

should be used to optimize sampling/pixel size to correctly gather required data. Ideally, you want

2.5-3 pixels per smallest resolvable object. So the Abbe limit of resolution is about 200-250 nm, so

the pixels should be about 65-100 nm each. Larger pixel size might allow you to collect the image

faster, but will not allow you to measure small features with high accuracy. Be consistent in your

choice of zoom to ensure your image pixel sizes are the same for multiple days and samples.

-Other features in this area that I would not mess with: scan mode (leave at frame, you want the

whole frame not just a single line); line step (leave at 1, changing this causes the system to skip

scanning lines to go faster and interpolate the data in between); direction (leave at -->).

Iron Man User Guide v. 1.4 9

6) Adjusting PMTs

These guidelines assume a fixed sample. For live samples, you should try to avoid Auto Expose or

Continuous modes as these will expose your sample to large amounts of excitation light prior to image

acquisition. Instead use Live mode or optimize in one region of slide

and move to another for data acquisition.

This step depends on your experimental design and question. To

ensure that your images are quantitative, you must avoid over- or

understaurating pixels, as you will not be able to properly measure

intensity in this case. It is also important to remember that your

brightest sample may be a particular Z plane, later slide, alternate

timepoint, and these may be oversaturated when you get to them

later.

-Select the first channel to optimize (check on/off in Imaging

Setup area). I usually prefer to optimize one channel/track

at a time, but if you plan to use multiple lasers on a single

track, make sure these will all be on during optimization.

-Ensure that your sample is on the brightest Z plane –go to

live mode and adjust using focus knob.

-Set lasers to appropriate starting values - for the Argon

and Diode lasers, 3-5% is usually sufficient, and for the

HeNe lasers, 25-35% is usually a good starting point.

-Set the pinhole size to 1AU. Unless you have a deliberate

reason to change this, 1AU is good for most experiments.

(Bigger pinhole = more light = less “confocal”). Don’t go

smaller. Bigger is useful for if you need to image a really

thick sample and do not want to image many 1AU confocal

slices.

-Click Auto Expose. The software will now attempt to set

the gain and offset for that channel.

-Click Continuous, and select the Range indicator Look Up

table beneath the image. Oversaturated pixels will appear

red and undersaturated pixels will appear blue. You can

manually adjust the gain and offset to achieve a good

balance between over- and undersaturation for your

experiment. You should adjust the offset first. This will

adjust the background. Be aware that if you change this too much you

will also remove signal form your sample. A little goes a LONG way, go

down to add undersaturated pixels, go up if you have too many

undersaturated pixels. After adjusting the offset, adjust the gain

(master). This will adjust the foreground and background signals. If you

are at the high end of gain, bump up laser power; if you are at low end,

reduce laser power.

-Repeat these steps for all channels in your experiment.

Iron Man User Guide v. 1.4 10

7) Snap –If you just want a single plane, click snap and save your

image. The PC does not have a lot of RAM –save and close

regularly.

8) Z series –If you selected Z-stack in the top left, you can now

control the Multidimensional Acquisition Z-stack window. You

can choose to either set the first and last positions, or you can

choose to center the stack around a given position.

-First/Last (most common choice)

oClick Live

oTurn focus knob on microscope in one

direction until desired distance is

reached, click set last

oRepeat in the other direction, click set

first

oEither choose number of slices or step

size. The software will suggest the

optimal step size. This is for Nyquist

sampling - ~2 Z planes per confocal

volume, depends on pinhole size and

wavelengths being used.

-Center

oClick Live

oTurn focus knob to get to center plane

of sample

oEither choose number of slices or step

size. The software will suggest the

optimal step size.

oClick Range Select.

oMove green line to approximate center of sample and red

lines to desired top and bottom of sample.

-Click Start Experiment. Time remaining will be shown at the

bottom of the screen.

-Save results.

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