1. No category

LSM 510 Meta Confocal Microscope
and LSM 510 Meta 3.2 Software
Room A503b
User Guide
Molecular Imaging Unit
University of Helsinki
www.miu.helsinki.fi
7.12.2007
1.
2.
3.
4.
GENERAL ....................................................................................................................4
TURNING ON THE SYSTEM.....................................................................................6
TURNING ON LASERS (ACQUIRE/LASER) ...........................................................7
VIEWING SAMPLES THROUGH OCULARS (ACQUIRE/MICRO)......................9
4.1 Fluorescence imaging through the oculars............................................................................... 11
4.2 Transmitted light imaging through the oculars......................................................................... 11
5. CREATING CONFIGURATIONS FOR LSM 510 META.......................................12
5.1 Creating a basic configuration (Acquire/Config) ..................................................................... 12
5.1.2 Main dichroic beam splitter (HFT n)................................................................................ 14
5.1.3 Secondary dichroic beam splitter (NFT n)........................................................................ 14
5.1.4 Emission filters ............................................................................................................... 15
5.1.5 Detectors ........................................................................................................................ 15
5.1.6 Transmitted light imaging ............................................................................................... 15
5.2 Saving and loading configurations .......................................................................................... 15
5.3 Meta detector ......................................................................................................................... 16
5.3.1 Lambda mode ................................................................................................................. 16
5.3.2 Linear unmixing.............................................................................................................. 16
5.3.3 Online fingerprinting....................................................................................................... 17
6. SCANNING IMAGES WITH LSM 510 META (ACQUIRE/SCAN) .......................17
6.1 General settings (Scan control/Mode/Frame)........................................................................... 17
6.1.1 Objective Lens, Image Size, and Line Step Factor............................................................ 18
6.1.2 Speed.............................................................................................................................. 18
6.1.3 Pixel Depth, Scan Direction and Scan Average................................................................ 18
6.1.4 Zoom, Rotation, and Offset ............................................................................................. 19
6.2 Channel settings (Scan control/Channels)................................................................................ 19
6.2.1 Channels......................................................................................................................... 20
6.2.2 Pinhole ........................................................................................................................... 20
6.2.3 Detector and amplifier..................................................................................................... 20
6.2.4 Excitation ....................................................................................................................... 21
6.2.5 Buttons in the Scan control window................................................................................. 21
6.3 Optimal settings for DETECTOR GAIN and AMPLIFIER OFFSET....................................... 21
6.4 Pinhole XY-settings ............................................................................................................... 22
6.4.1 Pinhole ........................................................................................................................... 22
6.4.2 Position X and Y............................................................................................................. 23
6.4.3 Store Current Position ..................................................................................................... 23
6.5 Acquisition of a Z-stack.......................................................................................................... 23
6.5.1 Optical slice settings ....................................................................................................... 23
6.5.2 Defining first and last slice.............................................................................................. 23
7. IMAGE WINDOW MENU.........................................................................................24
7.1 CHAN.................................................................................................................................... 24
7.2 ZOOM ................................................................................................................................... 24
7.3 SLICE.................................................................................................................................... 24
7.4 OVERLAY ............................................................................................................................ 24
7.5 CONTR ................................................................................................................................. 25
7.6 PALETTE.............................................................................................................................. 25
7.7 ANIM .................................................................................................................................... 25
7.8 REUSE .................................................................................................................................. 25
7.9 CROP .................................................................................................................................... 25
7.10 COPY .................................................................................................................................. 25
7.11 SAVE................................................................................................................................... 25
7.12 SAVE AS............................................................................................................................. 26
7.13 XY....................................................................................................................................... 26
7.14 SPLIT XY............................................................................................................................ 26
7.15 ORTHO ............................................................................................................................... 26
7.16 CUT..................................................................................................................................... 26
7.17 GALLERY........................................................................................................................... 26
7.18 HISTO ................................................................................................................................. 27
7.18.1 Histogram ..................................................................................................................... 27
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7.18.2 Area measurements ....................................................................................................... 27
7.18.3 Colocalization measurements ........................................................................................ 28
7.19 PROFILE ............................................................................................................................. 29
7. 20 2.5D.................................................................................................................................... 30
7.21 3D........................................................................................................................................ 30
7.22 Topo .................................................................................................................................... 30
7.23 Prev ..................................................................................................................................... 30
7.24 Info ...................................................................................................................................... 31
8. SAVING AND EXPORTING IMAGES.....................................................................31
8.1 Saving images ........................................................................................................................ 31
8.2 Exporting images ................................................................................................................... 32
8.2.1 File formats..................................................................................................................... 32
8.2.2 Image formats................................................................................................................. 32
9. ENDING YOUR SESSION.........................................................................................33
9.1 There are users after you on the same day ............................................................................... 33
9.2 You are the last user of the day ............................................................................................... 33
10. ADDITIONAL FUNCTIONS FOR LIVE SAMPLES...............................................34
10.1 Acquisition of time series ..................................................................................................... 34
10.2 FRAP (fluorescence recovery after photobleaching).............................................................. 34
11. TROUBLESHOOTING..............................................................................................35
11.1 Starting the system ............................................................................................................... 35
11.2 Using the microscope ........................................................................................................... 35
11.3 Scanning .............................................................................................................................. 36
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1. General
1.1 Requirements for use
A 2-hour training session given by MIU staff is a requirement for independent use of
Meta. You need to bring your own samples for the training and this user guide. After
the training, you can make online reservations for Meta. You are also expected to
have an LTDK user ID (IT user permit center, 1st floor in Biomedicum), and get an
access application form from Elisa Lehtonen (room A501a, tel. 191 25575).
1.2 Reservations
Instructions for Scheduler online reservation system can be found on the MIU web
page: http://www.miu.helsinki.fi/reservations.htm. You can make your Meta
reservation three weeks in advance. If you need to cancel your reservation, you have
to do it two hours before your reservation starts.Unused reservations will also be
charged. Make sure you are familiar with the use of Scheduler and the MIU fees and
user policy (http://www.miu.helsinki.fi/fees.htm).
When you make reservations, please always remember to indicate in the Event name
field which lasers you will use during your imaging session. This enables the users
before you to turn off those lasers that are no longer needed on that day, thus
extending the lifetime of the very expensive lasers.
You have 15 minutes to start your session. If you are late more than that, your
reservation is no longer valid and anybody else can use your time. If you want to
extend your session and there are no reservations after you, you can use the "extend"
option in Scheduler (click first on your reservation). If your reserved time is already
up, you cannot extend your present reservation any more.
1.3 Billing
The billing is based ONLY on the reservations made in Scheduler - the logbook
markings do not count. The fee for the training session is twice the regular hourly fee.
Any time after the initial training, you can ask MIU staff to give you additional
customized training (charged the same way as the initial training).
1.4 Unauthorized use
MIU periodically checks the loggins of the Meta computer. If your logon/off times
exceed your reserved time in Scheduler, MIU will consider that as unauthorized use
of the instrument and ask for an explanation. For unauthorized use of the instrument,
MIU may issue a warning. After three warnings, MIU may revoke the user's license.
Also, misuse/neglect of the instrument may lead either to a warning or cancellation of
the user license, and the repair costs may be charged.
If there are no reservations for Meta and you only want to use the computer for data
transfer, mark in the logbook "data transfer". When the computer logins are checked,
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the logbook marking will tell MIU staff that you did not use the confocal without
reservation.
1.5 Meta-users mailing list
All registered Meta users are automatically added to the Meta users mailing list.
Please read those e-mails to know what's going on! MIU web site
(www.miu.helsinki.fi) contains lots of useful information on microscopy and imaging,
as well as descriptions of other MIU instruments available.
1.6 Meta manual and user guide
In the Meta room, there is a complete manual of the LSM 510 Meta. Chapter 5 in the
manual covers all the functions there are in the software. Please do not remove the
manual from the Meta room. MIU has a copy of the manual that you can borrow.
This user guide will be continuously updated by the MIU personnel. Check that you
have
the
latest
version
of
the
user
guide:
go
to
http://www.miu.helsinki.fi/instruments/zeiss510.htm and compare the date of the
latest update with the date on the first page of your printed user guide. This is the only
place for update announcements; no e-mails about updates will be sent.
The user guide can also be accessed in Meta computer through the Internet Explorer
browser: http://www.miu.helsinki.fi/instruments/zeiss510.htm.
1.7 Acknowledging MIU
Whenever you are using instruments and/or services maintained/provided by MIU,
you are expected to acknowledge MIU in your publications and inform MIU about the
papers.
1.8 Sample preparation
A confocal microscope can remove out-of-focus haze that decreases the signal-tonoise ratio. However, it cannot compensate for bad sample preparation. Garbage in,
garbage out, is also true for confocal microscopy!
1.8.1 Selection of fluorochromes
If you want to achieve the best possible resolution, then use a fluorochrome whose
emission spectrum covers as short wavelengths as possible. This is because the
resolution of the microscope is determined by the numerical aperture of the objective
used and the wavelength of the light detected.
For thick tissues, image acquisition deeper in the tissue is often a problem because of
light scattering in the tissue. The longer wavelength the light has, the less it scatters,
and the better its penetration into tissues. Thus, use fluorochromes whose emission
spectra are in the far-red region if you need to image deep inside tissue.
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For multilabel imaging, select fluorochromes that are spectrally wide apart. E.g.
Alexa488 and Alexa594 usually have cross-talk between channels if scanned
simultaneously, so they should be scanned sequentially. If sequential scanning is not
an option because of longer scan time required, then you should use fluorochromes
that do not have any cross-talk.
1.8.2 Mounting samples
You should either use a mounting medium that will harden or, in the case of
nonhardening one, seal the edges of the cover glass with nail polish or some other
sealant. Let the sealant dry well before you come to the confocal - nail polish is one of
the most harmful substances for optics. Also, unsettled mounting medium, or one
containing too much water (slides not dry enough when mounted), or too much
mounting medium, can affect imaging by causing shadows and/or stripes in the
image.
If you are using oil immersion objectives, the refractive index of the mounting
medium used should be close to the refractive index of the immersion oil (1.518). For
more information on mounting media and antifade reagents, visit:
http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf.
Most of the objectives are designed for coverslip thickness 0.17 mm, which
corresponds to number 1.5 coverslips. Do not mount the coverslip so that it will be
pressed down by the clips in the sample holder.
1.9 Image processing and analysis
MIU has two Imaging Workstations that have software for further image processing
and analysis, such as deconvolution, cell counting, and more. Visit the MIU web site
or contact MIU for more information. Use of Imaging Workstations is free of charge,
and MIU staff gives training and help with the software. MIU also provides
customized image analysis tools in case the commercial and free softwares do not
satisfy your image analysis needs.
2. Turning on the system
If you are the first user you should turn the computer and monitors on - computer by
pressing the round button in the top right corner of the front panel, monitors by
pressing the buttons below the screen. Write down in the log book your name and
other information.
Log on to the computer using your LTDK user ID (START/LOG ON). Switch
REMOTE CONTROL ON (Fig. 1). This will turn on the microscope. Please notice
that after turning the system on, you have to wait at least 30 min before turning it off
because of the mercury bulb. Warming of the lasers will take about 2 hrs for maximal
stability but you can start using them sooner unless the stability is critical for your
imaging.
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Figure 1. Remote control button.
Initiate LSM 510 Program through a shortcut at the desktop (Fig. 2).
Figure 2. Shortcut for LSM 510 software.
Choose SCAN NEW IMAGES and then click START EXPERT MODE (Fig. 3).
Figure 3. The start dialog of LSM
510 software.
3. Turning on lasers (Acquire/Laser)
Go to ACQUIRE / LASER (Fig. 4).
Figure 4. The buttons for
laser settings.
The six laserlines available are shown in Table 1.
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Laser
Diode
Argon
Argon
Argon
HeNe1
Laserline
405 nm
458 nm
488 nm
514 nm
543 nm
HeNe2
633 nm
Examples of fluorochromes that can be used
DAPI, Hoechst
CFP
Alexa Fluor 488, GFP, Cy2, SytoxGreen, Acr.Orange/DNA, FITC
YFP, Cy3
Alexa Fluor 546, Alexa Fluor 594,Texas Red, HcRed1, DsRed2, PI,
TRITC/Rhodamine, Mitotracker Orange
Alexa 633, Alexa 647, Alexa 660, Cy3, APC
Table 1. Laserlines available.
Turning on Diode-, HeNe1- and HeNe2-laser:
Turn on the lasers you are going to need by selecting one laser at a time and clicking
ON (Fig. 5).
Figure 5. Laser control window.
Turning on the Argon-laser:
For the argon laser, click STANDBY to warm up the laser (Fig. 6). Let the laser warm
up for as long as possible, this will extend the lifetime of the laser. Only when you are
ready to start scanning, click ON (Status has to be Ready). Gradually (stepwise)
increase the output to about 50% with a slider. For images taken during separate
sessions, the output values should be the same, otherwise you cannot directly compare
the images.
Figure 6. Laser control for Argon-laser.
Note: Do not click lasers on/off during your session. Select Standby for the Argon if
you will take a longer break. Drag the slider all the way to the left, into the minimum
output value.
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4. Viewing samples through oculars (Acquire/Micro)
First check that the stage holder is firmly in place. If not, take it out and reinsert it
correctly (red dot in the stage holder indicates the corner that should go where there is
another red dot marked on the stage). As there are two springs in that corner holding
the stage holder, you have to press the holder in from the opposite corner (requires
some patience and practice).
Put your specimen on the stage so that it fits snugly the indentions – remember
inverted position (coverslip down)! Use the metal clips to secure the slide (Fig. 7).
Figure 7. Inserting the
specimen on the stage.
Make sure that the coverslip thickness is about 0.17 mm (No. 1.5 cover glass) and that
the clips do not press down the coverslip, as that creates tension on the coverslip that
may affect your scanning. Use smaller coverslips if that is a problem.
To view your sample through oculars without laser scanning, click VIS-button on the
right (Fig. 8). This will allow only the light from halogen or mercury bulb to pass
through the oculars.
Figure 8. The button for
VIS-mode.
Go to ACQUIRE / MICRO (Fig.9).
Figure 9. The button
for microscope
settings.
The Microscope Settings window pops up (Fig. 10).
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Figure 10. Microscope settings.
Here you can change objectives (click on the objective button) and make adjustments
for halogen and mercury light sources. There are six objectives in the microscope, and
a 63x, NA 1.2 (korr.) C-Apochromat water immersion objective is available upon
request (Table 2). The oil objectives have protective felt rings.
Objective
5x Plan-Neofluar
10x Fluar
20x LD Achroplan
40x Plan-Neofluar
63x Plan- Apochromat
63x Plan-Neofluar
WD
13.6
2
10.2
0.2
0.18
0.1
NA
0.15
0.5
0.4 korr.
1.3
1.4
1.25
63x C-Apochromat
0.28
1.2 korr.
CG
0.17
0.17
0-1.5
0.17
0.17
0.17
0.140.19
Immersion
None (air)
None (air)
None (air)
Oil (Immersol 518F)
Oil (Immersol 518F)
Oil (Immersol 518F)
Comments
Water
DIC
No phase or DIC
DIC
DIC. Ph2
DIC
DIC
DIC, Ph3
Table 2. Objectives available. Working distance (WD) is shown in mm. CG: Cover
glass thickness that should be used, shown in mm.
Numerical aperture (NA), together with the wavelength of the light used, determines
resolution of the microscope; the higher the NA, the better the resolution. 20x LD
Achroplan and 63x C-Apochromat objectives have correction collars, which should be
adjusted according to the cover glass thickness.
Obviously, objectives differ in magnification and numerical aperture but they also
differ in other respects. Some properties of the Meta objectives are shown in Table 3.
Flatness: Flatness of the imaging field = How well objects that are around the edges
of the image are at the same focal plane with objects that are in the middle of the
image.
Color correction (CC): How well different color channels are in register; indicates
how well the objective suites for multichannel work
UVIR transmission: The better the transmission, the less ultraviolet/infrared light gets
lost on its way through the objective.
Multichannel/DIC: How suitable the objective is for multichannel/DIC imaging.
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Objective
5x Plan-Neofluar*
10x Fluar
20x LD Achroplan
40x Plan-Neofluar*
63x Plan-Apochromat
63x Plan-Neofluar*
63x C-Apochromat
Flatness
4
1
3
4
5
4
5
CC
4
1
3
4
5
4
5
UV trans.
4
5
3
4
3
4
4
IR trans.
3
4
3
3
4
3
5
Multich.
4
1
2
4
5
4
5
DIC
4
1
1
4
5
2
5
Table 3. Properties of Meta objectives. Scale is from 1 (worst) to 5 (best). For
objectives marked with an asterisk (*), properties shown are not for the actual
objective but for the more recent version (EC Plan-Neofluar). For Plan-Neofluar type
objectives, information cannot be found on the Zeiss web site. Thus, for PlanNeofluar objectives the actual rating is either equal or lower than the one shown.
You do not necessarily need to change the objective when you want to change
magnification; you can also zoom.
4.1 Fluorescence imaging through the oculars
For fluorescence imaging, all microscope manipulation is done through this panel; do
not change anything through the microscope itself except focusing and the stage
position through the joystick. Use the joystick for large movements and the black
round wheel for fine movements. Select first if you want to move along X or Y axis
by pressing the corresponding X/Y button.
For viewing of your sample with mercury light source (HBO), select the filter from
REFLECTOR appropriate for your staining and click REFLECTED LIGHT on. The
four fluorescence filters available are shown in Table 4.
FSet
00
02
15
16
Fuorochromes
Alexa Fluor 594, TexasRed
DAPI, Hoechst
Alexa Fluor 546, TRITC, Cy3
Alexa Fluor 488, FITC
Table 4. Fluorescence filters available.
When you end viewing your sample through oculars, choose NONE for the
REFLECTOR and click again on REFLECTED LIGHT to turn it off. Also check that
TRANSMITTED LIGHT is off.
4.2 Transmitted light imaging through the oculars
To look at your sample with halogen (visible) light, click TRANSMITTED LIGHT.
The reflector has to be in position NONE. Select the correct PHASE/DIC optics from
CONDENSOR. Bright-field imaging is mainly for specimen with histological
staining. Phase contrast imaging is used for thin, unstained specimen, whereas
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differential interference contrast (DIC) imaging is meant for thick, unstained
specimen. DIC can also be used if phase contrast imaging shows disturbing haloes
around objects.
Make sure that the oculars are correctly adjusted for your eyes. If you have normal
vision, the zero mark on the sides of the oculars should match the white dot. If you
wear eyeglasses but want to use the microscope without them, you must adjust the
oculars according to your vision. First look at your specimen with your right eye
closed (through the left ocular). Slowly turn the eyepiece scale ring until the image is
optimally in focus. Repeat with the other eye. The scale on the oculars is the diopter
scale.
Make the Koehler illumination adjustments (if you are not familiar with them, MIU
staff will help you). Control the brightness with the INTENSITY slider. When you
want to turn off transmitted light click TRANSMITTED LIGHT button and click
again ON. That will toggle between on and off states. Remember to turn off the light
when you are not viewing your sample, otherwise you may unnecessarily bleach your
specimen.
For phase contrast imaging, you need to use an objective that has a phase ring (see
Table 2), and a matching condenser position. Leave the condenser aperture fully open.
For DIC (differential interference contrast) imaging, the polarizer, analyzer, and
modified Wollaston prisms have to be on the light path, and correctly adjusted. Ask
MIU staff to show you how to do that.
5. Creating configurations for LSM 510 Meta
5.1 Creating a basic configuration (Acquire/Config)
To scan your sample using LSM 510 META, click LSM on the right (Fig. 11). This
will NOT allow anything to pass through the oculars.
Figure 11. The button for
LSM-mode.
To set the beam path configuration go to ACQUIRE / CONFIG (Fig. 12).
Figure 12. The button for
configuration control.
The Configuration Control window pops up (Fig. 13).
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Figure 13. The configuration control window.
If you only use one fluorochrome, click CHANNEL MODE / SINGLE TRACK,
otherwise CHANNEL MODE/MULTI TRACK. In MULTI TRACK mode, you add
more tracks by clicking ADD TRACK and adjust settings separately for each channel.
Whenever you are using the MULTI TRACK mode, make sure that there is no crosstalk between the channels that are scanned at the same time; you should have control
slides stained only with one of the fluorochromes. Scan these controls using a
configuration in which only the "wrong" channel is enabled - you should not see any
signal.
A track can be enabled/disabled by ticking the box on the left side of the track name.
Only enabled tracks will be acquired. It is often useful to enable only one track at the
time when making adjustments.
If you are using e.g. four fluorochromes (A = emission at shortest wavelengths, B =
emission at 2nd shortest wavelengths, C = emission at 3rd shortest wavelengths, D =
emission at longest wavelengths), make a MULTI TRACK configuration as follows:
1. track: A and C, 2. track: B and D. In this way, the detected wavelengths in each
track are as far apart from each other as possible. Again, check if there is any crosstalk between A and C. Also check cross-talk for B and D. If there is too much crosstalk, you either need to make separate tracks for those channels, or use Meta-detector
and linear unmixing (see chapter 5.4).
A beam path can be controlled with filters and mirrors starting from the lasers and
ending in the detectors. Ch2 and Ch3 are standard PMT detectors and ChS is a socalled Meta-detector for spectral detection. ChD is for transmitted light. Note that:
Mirror: light deflects from line
None: light passes through
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Create a configuration by following the light path from lasers to the detectors (Fig.
14). When you click on any button in the configuration control window, a list of
options available will open. If the configuration control window is positioned near the
bottom of the screen, you may not be able to see the whole list. In that case, you first
need to move the configuration control window upwards.
Figure 14. Setting laserlines, filters and
beam splitters for a basic configuration.
5.1.1 Excitation
Select the laserline and adjust the transmission efficiency with a slider. Recommended
transmission for each laserline is shown in Table 5.
Laser
Diode
Argon
Argon
Argon
HeNe1
HeNe2
Laserline
405 nm
458 nm
488 nm
514 nm
543 nm
633 nm
Transmission
5 - 10 %
5 - 10 %
5 - 15 %
5 - 15 %
50 - 100 %
50 - 100 %
Table 5. Recommended transmission efficiency for each laser line.
5.1.2 Main dichroic beam splitter (HFT n)
Select excitation wavelengths that will pass to the specimen (these should match with
the laserline you use). Use as few wavelengths as possible and do not use wavelengths
that are within the emission spectrum as they are cut off.
5.1.3 Secondary dichroic beam splitter (NFT n)
Select the splitting of the emission wavelengths between two detectors. The
wavelengths longer than n will pass through to the Ch3 and the wavelengths shorter
than n are reflected to the Ch2. With the mirror all wavelengths are directed to the
Ch2.
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5.1.4 Emission filters
Choose the wavelengths that are detected with each detector.
LP x = Long pass filter = wavelengths longer than x are passed through
BP x-y = Band pass filter = wavelengths from x to y are passed through
5.1.5 Detectors
Enable a detector by selecting the check box. Select the pseudocolor for the channel
by clicking the button Ch2 or Ch3.
You can check that your configuration is correctly made by clicking SPECTRA (Fig.
15). This shows you the laser line(s) used and emission wavelengths that will be
detected.
Figure 15. Spectra-button.
5.1.6 Transmitted light imaging
You can use the ChD detector for transmitted light (bright-field, phase contrast, or
DIC) imaging. Check the ChD box and use e.g. the argon laser as a light source.
Check that you have selected proper condenser position and that all other transmitted
light settings are correct (see chapter 4.2). Make sure that the pseudocolor selected for
the ChD channel is white.
As laser light is polarized, it is not necessary to use the polarizer for DIC imaging.
Remember that the transmitted light image is not confocal, as there is no pinhole in
front of the ChD detector.
There are some dust particles inside the ChD detector that often appear in the images.
As there is no way to get rid of the dust, you should try to crop your image so that the
particles will be excluded. If that is not possible, use background subtraction.
5.2 Saving and loading configurations
The configuration can be saved with a CONFIG button on the right side of the
configuration window (Fig. 16). Click the button, name the configuration, and click
STORE. Note that configurations are saved separately for SINGLE TRACK and
MULTI TRACK modes.
Figure 16. CONFIG-button.
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To load a saved configuration, click CONFIG and select the configuration from the
pull-down list. Click APPLY. This will automatically switch on the desired filters and
mirrors. Note that this will also adjust the laser lines, you must check these later on.
Alternatively, you can open a previously stored image. In the main menu, select FILE
/ OPEN, then an image, and click REUSE (Fig. 17). This will adjust microscope
settings (not including the objective), configuration, and scan control options
according to the ones used for the image loaded (as shown in INFO in the image
window).
Figure 17. REUSE-button.
5.3 Meta detector
META detector (ChS) is a spectral detector which means that you can freely select the
range of emission wavelengths that you want to use for detection. It consists of 32
photomultipliers, each of which covers a spectral range of 10.7 nm (total range about
340 nm).
5.3.1 Lambda mode
You may want to use META detector in case the emission filters available are not
optimal for your fluorochrome. Additionally, you will get the spectral emission
(intensity for each wavelenght within your selected range). When you scan in this socalled lambda mode, the result will be a lambda stack that shows the signal over the
spectral range selected. It is also possible to combine lambda mode with Z mode
and/or time series.
However, META detector is less sensitive compared to the other detectors, so you
should have a strong signal if you want to use the META detector. Also, you may
need to open the pinhole more than usually (try using e.g. 3 Airy units).
To use lambda-mode, select LAMBDA MODE in the configuration control window.
Set the excitation and main dichroic beam splitter (HFT n) as in the CHANNEL
MODE. For selecting the detected wavelenghts, either use the slider or write the
starting/ending wavelengths in the proper input boxes. When you click on the
CONFIG button, you can save and load lambda configuration. NUMBER OF
PASSES will tell you how many scan are needed to cover the required spectral range.
Proceed to SCAN as usually.
For separation of spectral signals, you have two options: either LINEAR UNMIXING
or ONLINE FINGERPRINTING.
5.3.2 Linear unmixing
META detector and linear unmixing can be used to separate signals from two or more
fluorochromes with overlapping emission spectra. Also, autofluorescence can be
separated from the true signal.
16
In linear unmixing, the software will calculate for each pixel how much of the signal
is derived from each spectral component that needs to be separated from the others,
based on the reference spectrum for each component. In order to make a reference
spectrum, you need to have control specimen that are each stained with only one of
the fluorochromes whose spectra need to be separated. In case of autofluorescence,
the control is simply an unstained specimen.
LINEAR UNMIXING is used after you have acquired a Lambda stack (using META
detector). In the MAIN menu, select PROCESS, and then UNMIX. Insert in the stage
the specimen from which you want to acquire a reference spectrum. It is essential that
the specimen is only stained with the fluorochrome for which you want to get a
reference spectrum. Select the channel for the reference spectrum and name it. Press
the LOAD FROM IMAGE button. Continue until you have acquired reference spectra
for all the fluorochromes whose spectra you want to separate from each other.
In the Source panel of the UNMIX window, click on the arrow button and select the
image you want to use for unmixing by clicking on it. In the Parameters panel of the
UNMIX window, select all options. The RESIDUALS channel will show you the
amount of signal that could not be assigned for any of the unmix channels. ASK FOR
THE BACKGROUND ROI option will ask you to mark an area in the image that
represents background. This background channel should be defined for optimal linear
unmixing.
After clicking on APPLY, a new window that shows the result of the unmixing will
open.
5.3.3 Online fingerprinting
ONLINE FINGERPRINTING does the unmixing during scanning, and the lambda
stack will not be shown or stored. If you want to use this option, you must have saved
the reference spectra in the PROCESS/UNMIX window in advance (see previous
chapter). Select ONLINE FINGERPRINTING in CONFIGURATION CONTROL
window. Load the reference spectra using the RS1 ... 8 control buttons. Set the
excitation and main dichroic beam splitter (HFT n) as in the CHANNEL MODE. For
selecting the detected wavelenghts, either use the slider or write the starting/ending
wavelengths in the proper input boxes. When you click on the CONFIG button, you
can save and load configurations. NUMBER OF PASSES will tell you how many
scan are needed to cover the required spectral range. Proceed to SCAN as usually.
6. Scanning images with LSM 510 Meta (Acquire/Scan)
6.1 General settings (Scan control/Mode/Frame)
Go to ACQUIRE / SCAN (Fig. 18).
17
Figure 18. The button
for scan control window.
SCAN CONTROL window pops up. Choose MODE and FRAME (Fig. 19).
Figure 19. Scan Control window.
6.1.1 Objective Lens, Image Size, and Line Step Factor
Select FRAME SIZE. OPTIMAL shows the minimal sampling frequency (optimal
number of pixels) that should be used. If you use smaller frame size than suggested by
OPTIMAL, you will lose information when you scan your image. It is often desirable
to use an even larger frame size than suggested by OPTIMAL, especially if you are
going to deconvolve your images. LINE STEP indicates how many lines are scanned.
Always use setting 1 (every line will be scanned).
6.1.2 Speed
The OPTIMAL button (see previous paragraph) will also optimize the scanning
speed. If the speed is too high, the quality of the image is low. On the other hand,
setting the speed lower than the optimal speed will not improve the image quality.
6.1.3 Pixel Depth, Scan Direction and Scan Average
DATA DEPTH: 8 Bit means that for each pixel, there are 256 possible intensity
values available. In a 12-bit image, the gray values can range from 0 to 4095 for each
pixel. For quantative imaging, 12-bit images are often the preferred choice. However,
12-bit image files are much larger than 8-bit ones, so if you only use the images for
visual inspection and you cannot see any difference between 8- and 12-bit ones, use
the former one.
18
SCAN DIRECTION: Default is unidirectional. If you use bidirectional scanning
(twice as fast), you have to correct the pixel shift either automatically by using the
AUTO button, or manually by adjusting the Scan Corr X (for zero rotation) or both
Scan Corr X and Y (for images rotated 90 degrees). Bidirectional scanning should
always be used for live samples.
SCAN AVERAGE: used to get rid of random noise. In MODE, select Line (averaged
after each line) or Frame (averaged after each frame). Use Line for live samples. In
METHOD, Mean is used for averaging. SUM adds up pixel values, so it also
amplifies noise. In NUMBER, select the number of scans used for averaging.
CONTINUOUS option will continuously scan the sample and at the same time
average the scans. It is useful for estimating how many times the sample should be
scanned for averaging.
6.1.4 Zoom, Rotation, and Offset
You can zoom to your sample and rotate it in the indicated field. The new settings will
be used for the coming scans. Zooming will not increase resolution when the zooming
factor is displayed in red. The area to be zoomed can also be selected from a taken
image with CROP in the image windows. When the CROP command is active, you
can increase the size of the zoom window by clicking on one of the corners of the
window and dragging the window to a desired size. The position of the zoom window
can be similarly adjusted by clicking on the zoom window somewhere else than the
corners and dragging the window.
Rotation of the zoom window is done by clicking on one end of the crosslines and
dragging the crossline until the required rotation angle is reached. The blue line
indicates the top of the new scan area.
If you want to modify the shape of the scan area, click on the intersection of any
crossline and zoom window outline, and drag with mouse. Remember that even if you
decrease the scan area along X, the whole length of each line will be scanned,
meaning that the scan time will not decrease even if you decrease the horizontal size
of the zoom window (horizontal in relation to the scan direction).
6.2 Channel settings (Scan control/Channels)
Select ACQUIRE/SCAN in the main menu. Click CHANNELS in the SCAN
CONTROL window (Fig. 20).
19
Figure 20. Channel settings.
6.2.1 Channels
Select which channel you want to adjust by pressing the desired channel button.
6.2.2 Pinhole
For optimal resolution and depth discrimination, the pinhole size should be close to 1
Airy unit. However, if the signal level is not satisfactory, it is possible to increase the
pinhole size to 2-3 AU. Before using a larger pinhole size, make sure you have
optimized your staining and other scanning parameters. Opening the pinhole more
than that will significantly decrease the signal-to-noise ratio and is not recommended.
Always start imaging using the pinhole size 1 and only use larger size if necessary.
Using pinhole sizes smaller than 1 often leads to greatly reduced signal levels.
The pinhole diameter is adjusted with a slider. Corresponding optical slice thickness is
shown below the pinhole diameter value. To set pinhole to 1 Airy unit press button
marked "1". Note that for multiple channels, you have to adjust the pinhole sizes so
that the optical slice thickness is the same for all channels; if you adjust the pinhole of
the channel with longest wavelengths to 1, then the pinhole size for the other channels
will be larger than 1. Accordingly, if you adjust the pinhole size for the channel with
shortest wavelengths to 1, then for the other channels the size will be smaller than 1.
6.2.3 Detector and amplifier
AMPLIFIER OFFSET adjusts general intensity levels of pixel values, DETECTOR
GAIN adjusts dynamic range of pixel values (Fig. 21). AMLIFIER GAIN amplifies
signal afterwards and therefore amplifies noise as well. It is recommended to set it to
1. See the next chapter for information about optimal offset and gain settings.
20
Figure 21. Adjusting intensity
range of pixel values with
amplifier offset and detector gain.
6.2.4 Excitation
In EXCITATION window you can indicate which laser lines to use and adjust the
percentage of transmission (see 5.1.1)
6.2.5 Buttons in the Scan control window
Important safety issue: avoid looking at the laser light - Meta's lasers may be harmful
for your eyes.
During scanning, it is important to eliminate vibrations: Do not touch the air table (e.g
do not lean on it or press your feet against it); make sure the arm rests of the table do
not touch the computer table; avoid banging the door or anything else in the room.
NEW: opens a new image window (if not selected, the new image will be
scanned over the previous scanned image, which will be lost unless saved)
FIND: the software tries to find the optimal adjustment of DETECTOR GAIN
and AMPLIFIER OFFSET while scanning a single image
FAST XY: continuous scanning with maximum speed
SINGLE: scans a single image
STOP: stops scanning
CONT: continuous scanning (beware of photobleaching)
6.3 Optimal settings for DETECTOR GAIN and AMPLIFIER
OFFSET
This needs to be done for each channel separately. It is recommended to first let the
software try to adjust settings for DETECTOR GAIN and AMPIFIER OFFSET with
FIND button. After that, you may tune the settings if needed. There is an aid for
finding optimal settings for DETECTOR GAIN and AMPLIFIER OFFSET manually:
In the image window, click PALETTE and select RANGE INDICATOR in
the drop-down menu.
For fast continuous scanning, press FAST XY on the right panel of the scan
control window. If you are using multiple channels, you may want to
inactivate the other channels for the scan to be faster.
21
Adjust AMPLIFIER OFFSET so that there are only very few blue pixels
visible (minimum intensity value = 0) –look at the image for the channel you
are adjusting.
Then set DETECTOR GAIN so that there are almost no red pixels (intensity
value = maximum (255 for an 8-bit image, 4095 for a 12-bit image).
Make the adjustments for each channel; select the channel by clicking on its
button.
After all adjustments, click PALETTE and select NO PALETTE.
6.4 Pinhole XY-settings
Usually the pinhole position adjustment is not necessary. You should only need to
adjust the pinhole position if you are using a configuration that has never been used
before. Be aware that if you change the pinhole XY-settings, they will be changed for
all users. Thus, you should make this adjustment only if you are sure it is needed. If in
doubt, ask MIU staff.
This adjustment will position the pinhole in the middle of the light path. First set the
diameter of the pinhole to 1 Airy unit. Then adjust X- and Y-positions of the pinhole
by choosing MAINTAIN / PINHOLE (Fig. 22).
Figure 22. The button
for pinhole XY-settings.
The Pinhole settings window pops up (Fig. 23).
Figure 23. Pinhole settings window.
6.4.1 Pinhole
Choose the pinhole to be adjusted according to the detector: Ch2 = PH2, Ch3 = PH3.
22
6.4.2 Position X and Y
Adjust POSITION X slowly back and forth until you get the best image. Do same
with POSITION Y.
6.4.3 Store Current Position
By clicking STORE CURRENT POSITION pinhole settings for the current
configuration will be saved.
6.5 Acquisition of a Z-stack
Z-stacks are usually acquired when you need information on the 3D expression
pattern of your labeled molecule, or you want to analyze if two fluorochromes that
seem to colocalize in xy will also show similar distributions along z - something you
should always check if you claim that two signals colocalize.
For optical sectioning through your specimen, select ACQUIRE/SCAN in the main
menu. In SCAN CONTROL, press Z-STACK (Fig. 24).
Figure 24. Settings for a Z-stack.
6.5.1 Optical slice settings
Click on Z SLICE. OPTICAL SLICE window pops up. Select by clicking OPTIMAL
INTERVAL if you use multitrack. OPTIMAL INTERVAL will set the interval so that
adjacent slices overlap by 50 %. For single track, you can also select OPTIMAL
PINHOLE DIAMETER.
6.5.2 Defining first and last slice
23
Click on MARK FIRST/LAST button. Press on XYcont scan. While scanning, use the
manual focusing knobs in Axiovert to reach to the focus edge of your sample (away
from you = up, towards you = down). While at each edge, click the MARK FIRST
and MARK LAST buttons, respectively. This sets the boundaries of your samples.
Click STOP to stop the XYcont scan and click START to acquire the Z-stack.
7. Image window menu
All image window tools can be closed by pressing the corresponding button again.
Depending on your current image (single/multichannel/Z stack etc.), some tools may
not be active.
7.1 CHAN
Clicking on the CHAN shows the Channels toolbar. When you click on a channel
button in this toolbar, you can then either turn off the selected channel by clicking the
OFF button, or change the pseudocolor used for that channel by clicking on another
color.
More color selection buttons can be created by pressing COLORS button. First define
the hue of the new color in the larger window, and then select the saturation of the
color by moving the arrow along the smaller window. Clicking the ADD button adds
the new color button in the color selection window.
7.2 ZOOM
Zooms the image (does not affect scanning); after clicking on the ZOOM button,
ZOOM toolbar becomes visible. ZOOM-AUTO: Automatically fits the image to the
size of the Image window. ZOOM-RESIZE and ZOOM 1:1: both restore the image
into its original size. ZOOM-+: Enlarges the image 200%. ZOOM--: Reduces the
image into half of its original size. ZOOM-MOUSE: After selecting this button, the
left mouse button can be used for enlarging the image, the right button for reducing
the size (cursor has to be over the image). The underlined functions can only be used
when ZOOM-AUTO is inactive. The zoom factor can also be adjusted by using the
slider. The display box shows the zoom factor.
7.3 SLICE
Used for viewing individual images of a Z stack or time series. By dragging the slider,
you can view the images one by one. The display box shows the number of the current
image and the total number of images.
7.4 OVERLAY
By pressing OVERLAY, overlay toolbar becomes visible. SCALE button (1
micrometer scale bar shown in the button) allows you to draw a scale bar in your
24
image. You can change the color of the selected scale bar by clicking on one of the
color boxes. The length of the selected scale bar is adjusted by dragging.
You can measure area and lengths of objects drawn in the image: first draw an object
using the drawing tools. Then select the object by clicking on it, and also click on the
MEASURE tool (yellow ruler in the button). Depending on the object, either its
lenght or area will be shown.
OFF removes the selected object; the button becomes ON, and clicking it again will
show the object again. RECYCLE BIN tool deletes the selected object. Is is also
possible to drag objects into the RECYCLE BIN if you want to delete them.
7.5 CONTR
Changes the contrast and/or brightness either for each channel separately or all
channels simultaneously. Usually not a very useful feature, as the settings are
automatically optimized by the software. By clicking MORE, you can also adjust
gamma; just remember that this adjustment should be described in your manuscript.
7.6 PALETTE
Pseudocolor palettes can be selected: RANGE INDICATOR is useful for determining
the settings for DETECTOR GAIN and AMPLIFIER OFFSET. RAINBOW can be
used for indicating intensity values using a color scale instead of a brightness scale.
GLOW SCALE is a combination of color and intensity scales.
7.7 ANIM
Animation of frames of a Z stack or time series. For description of the buttons, see
LSM 510 manual, p. 5-259. Speed 1: very fast; speed 2: more slow.
7.8 REUSE
Allows you to load acquisition parameters of the selected image, provided that the
image is in LSM format in an MDB database.
7.9 CROP
Defines the zoom area for scanning (see chapter 6.1.4).
7.10 COPY
Copies the current image into the clipboard.
7.11 SAVE
25
Saves the current image into an MDB database without showing the dialog window.
7.12 SAVE AS
Saves the current image, shows a dialog window in which you can select the database
and name the image.
7.13 XY
Displays a single image; multiple channels are in superimposed mode.
7.14 SPLIT XY
Shows channels in separate windows; the last window shows the superimposed mode.
7.15 ORTHO
Shows an orthogonal view (= xy, xz, and yz planes of Z stacks). XY plane is in blue,
XZ in green, and YZ in red. The section plane can be positioned at any XYZ
coordinate of the Z stack by moving the sliders in the Orthogonal toolbar.
Alternatively, the number of the slice can be entered in the input box. The section
place can also be selected directly in the image as described in the LSM 510 manual
(p. 5-268).
By clicking on the DIST button it is possible to measure distance in 3D: Click on
MARK button to define the first XYZ point. Set the second point by either moving
the X-, Y-, and Z-sliders or the green, red, and blue lines in the image. The distance
between the two points is shown in micrometers at the bottom of the Orthogonal
toolbar. The projections of the spatial distances are shown in yellow in the image.
2D DVC button can be used for 2D deconvolution, which improves the axial (Z)
resolution.
7.16 CUT
From a Z stack volume, you can select a section plane, which can be of any
orientation, and the CUT function will create the corresponding Z stack. Clicking on
CUT will show the Cut toolbar. The cut plane is determined by adjusting the X-, Y-,
Z-, Pitch-, and Yaw-sliders. The selected cut plane is shown in red at the bottom of
the Cut toolbar, as well as in the image window.
RESET ALL restores the original settings. TRILINEAR INTERPOLATION
improves the image quality by applying 3D interpolation.
7.17 GALLERY
Displays all images of a Z stack, lambda stack, time series, or their combination side
by side. In the Gallery toolbar, clicking on the DATA button shows either the Z slice
26
distance, wavelength, time, or their combination, whichever is relevant. COLOR
button allows you to change the pseudocolor for all images at the same time. Clicking
on SUBSET, the Subset window opens, enabling you to select only a subset of the
images: select the START SLICE and END SLICE for the new subset by dragging
the sliders. Be aware that when you click on the OK button, the slices not included in
the new subset will be permanently lost.
7.18 HISTO
Displays a histogram (distribution of pixel intensities) of an image, gives size and
intensity measurements for a given area, as well as analyzes colocalization between
two channels.
7.18.1 Histogram
In the histogram toolbar, the following functions are for the histogram display:
SKIP BLACK: Pixels that have the gray (intensity) value of zero are not shown in
the histogram
SKIP 4%: Pixels that have the lowest 4% of the gray (intensity) values are not
shown in the histogram
SKIP WHITE: Pixels that have the highest gray (intensity) value (255 for an 8-bit
image and 4095 for a 12-bit image) are not shown in the histogram
STEP: Determines the number of intensity levels shown in the histogram. Step 1
corresponds to 256 intensity levels for an 8-bit image, step 64 to 256/64 = 4
intensity levels
SHOW IMAGE: Shows the image window next to the histogram
SHOW TABLE: Shows the numerical values of the histogram in a table
COPY TABLE: Copies the histogram table into the clipboard
SAVE TABLE: Saves the histogram table as a text file (.txt)
7.18.2 Area measurements
In the histogram toolbar, the following functions are for the area measurements:
AREA: Used for defining the area for size and intensity measurements by using
the Area tools at the bottom of the histogram toolbar. First, define the area using
the drawing tools. Then, make the necessary adjustments for the area histogram
using the following tools:
27
STEP: Determines the number of intensity levels shown in the area histogram.
Step 1 corresponds to 256 intensity levels for an 8-bit image, step 64 to 256/64
= 4 intensity levels
LOW: Sets the threshold intensity level; pixels with lower gray values than the
threshold value are masked with the color selected using the COLOR selection
button below the low threshold value box
HIGH: Pixels with higher gray values than the threshold value are masked
with the color selected using the COLOR selection button below the
high threshold value box
Mean intensity value, standard deviation of the intensity, and the size of the nonmasked area are shown below the COLOR selection buttons.
Instead of defining the range of intensity values for area measurements, you can
also use the Mask tools for masking areas - masked pixels are not included in
measurements:
MASK: Enables the Mask mode, in which masked areas can be defined by ink
FLOOD FILL: Fills the defined area with the color selected under MASK
COLOR SELECTION: Defines the color of a mask
CLEAR MASK: Removes the mask color
7.18.3 Colocalization measurements
In the histogram toolbar, the following functions are for the colocalization
measurements:
COLOCALIZATION: Shows a scatter plot in which the X coordinate of the pixel
Px presents the intensity value of that pixel in channel 1, and the Y coordinate
presents the intensity value of Px in channel 2. If there is complete colocalization
between the channels, the scatter plot should show a diagonal line from the bottom
left corner to the top right corner.
SHOW TABLE: Shows the numerical values of the scatter plot in a table
AREA: Shows the image window next to the scatter plot
CROSSHAIR: Displays movable crosshair that, when correctly adjusted, divides
the scatter plot into four regions: bottom left region: background intensities;
bottom right region: pixels that have intensity values above threshold only in
channel 1; top left region: pixels that have intensity values above threshold only in
channel 2; top right region: pixels that have intensity values above threshold both
in channel 1 and 2.
28
Note that because there are no stringent criteria how to set the crosshair (threshold
values for each channel), this is always a somewhat arbitrary method.
THRESHOLD: SET FROM IMAGE ROIS: Sets background threshold from ROI
(region of interest) drawn in the image
INVERT MASK: Inverts the mask or scatter plot
SOURCE 1: Selection of channel 1 and its color via the Color box
SOURCE 2: Selection of channel 2 and its color via the Color box
MASK: Select either RGB or OVERLAY for the mask; selection of mask color
via the Color box
Drawing tools can be used for making ROIs in the scatter plot and image, which
are interactively linked (selecting a ROI in the image shows a scatter plot for those
pixels; selecting a ROI in scatter plot shows the corresponding pixels in the
image).
In the table, the following measurements are shown:
- Number of pixels (in the whole image or ROI)
- Area (in the whole image or ROI)
- Mean intensities and SD (in the whole image or ROI)
- Colocalization coefficients
- Weighted colocalization coefficients
- Manders' overlap coefficient
- Pearson's correlation coefficient
Remember that overlap in distribution patterns does not indicate that the labeled
molecules interact with each other, or that their distribution patterns correlate with
each other.
7.19 PROFILE
Displays the intensity distribution for each channel along a line drawn on the image.
Also shows the numerical intensity values that can be saved. By using markers (red
and blue), you can measure intensity difference and distance between two points
along the line.
In the Profile toolbar, the following functions are available:
The drawing tools can be used for drawing either a straight or curved line along which
the intensity values will be shown. You can also select the thickness and color of the
line; these will not affect the measurements.
DIAGR. IN IMAGE: Shows the intensity diagram as an overlay on the image
29
MARKER 1 (red): Shows a red circle marker in the image, and a red line marker in
the intensity diagram
MARKER 2 (blue): Shows a blue circle marker in the image, and a blue line marker
in the intensity diagram
ZOOM: Zooms on the intensity diagram; click and drag a rectangle on the graph and
that area will be enlarged. Can be applied several times. Right-click will reset the
original size
RESET ZOOM: Resets the zoomed intensity diagram into the original size
SHOW TABLE: Shows the numerical intensity values in a table
COPY TABLE: Copies the table into the clipboard
SAVE TABLE: Saves the table as a text file (.txt)
7. 20 2.5D
Shows pseudo-3D intensity distribution for each channel. Handy for presentations.
For Z stacks, the slice selected using SLICE in the Image Window menu is displayed.
Scrollbars around the image are used for rotating the image around the horizontal and
vertical axes. The right scrollbar on the right side of the image controls the intensity
scale of the image.
In the Pseudo 3D toolbar, the following functions are available:
PROFILE: Vertical polygon display
GRID: Horizontal grid display
FILLED: Color display, in which MONO, RAINBOW, and SIX STEP options
CHANNEL: selection of the channel displayed in case of a multichannel image
7.21 3D
Not available.
7.22 Topo
Not available.
7.23 Prev
30
Composition of images, diagrams, tables, and text for printing. However, there is no
printer connected to the Meta computer.
7.24 Info
Displays the parameters that were used when the image was acquired. This
information will be stored together with the image in the MDB database. If you save
your image in TIFF format, none of this information will be stored, just the image.
8. Saving and exporting images
If there are no reservations for Meta and you only want to use the computer for data
transfer, mark in the logbook "data transfer". When the computer logins are checked,
the logbook marking will tell MIU staff that you did not use the confocal without
reservation.
Of course, you can use your reserved time for data transfer - just then you pay for that
time, even if you do not use Meta at all. This is because during your reservation,
nobody else can use Meta. If Meta is not on and you want to do data transfer, start the
LSM software in "Use Existing Images" mode instead of " Scan New Images ".
8.1 Saving images
When you save your images in LSM file format, all the information about scanning
parameters will be saved as well. LSM files can be opened either using the LSM
software or a free LSM Browser that can be downloaded for PC from Zeiss web site
(there
is
a
link
in
MIU
web
page).
http://www.zeiss.com/C12567BE0045ACF1/ContentsFrame/CAA2EF638EC5F0D3C1256ADF0050E2F1. The software is also installed in
the MIU Imaging Workstations. MIU recommends that you save all your images in
the original .lsm file format, even if you also save them in tif format.
When you save your images in LSM file format, they have to be saved in a database.
If you want to save your image in a new database, click on SAVE AS. Then, select
NEW MDB to create a database. Give a name for the database. After creating a
database, you can add images in it by using SAVE.
You can save your databases and images either over network, e.g. in the snapserver
(MCBserver), or in the local D drive in the folder having your name (D:\”your
name”). Each user folder has a disk space quota of 3 GB in Meta’s computer. If you
approach this limit, you should back-up and delete files from the computer
immediately to make more room for new files. Meta’s computer is not part of any
back-up system so it is up to you to take care of your data! Keep in mind that very
large databases are difficult to transfer (too big for a memory stick, CD, etc.). Thus, it
is often more convenient to create several smaller databases.
31
8.2 Exporting images
8.2.1 File formats
To save images in other file formats than LSM, use FILE/EXPORT. Export images in
D:\”your name”, directly to your USB memory stick, or over network. Images from
.lsm files are strongly advised to be exported as TIFFs, as this format does not lose
any information. AVI and QuickTime are used for movies. Click on SAVE.
When selecting FILE/EXPORT, there are several options for TIF. When an image has
been taken in an 8-bit mode, use the following format: TIF –Tagged Image File. This
is the “normal”(8-bit = 256 levels of gray per channel) tif format.
TIFF 12-bit: This will make a 12-bit tiff file (4096 gray levels). Note that you must
have acquired a 12-bit image to benefit from this option. Not usually used, as most
imaging software is not able to open 12-bit tiff files.
TIFF 16-bit: Used if you take 12-bit images and your imaging software does not
recognize 12-bit TIFFs. Be aware that if you open 16-bit images in Photoshop or
Image Pro Plus, they will appear black. To see the image and ensure that quantitative
analysis will be performed properly, you have to make some adjustments. In IPP, you
can select the proper range manually from “Enhance->Display Range… ” menu and
entering values 0 - 4095. You will also find good instructions about 12-bit images
and Photoshop in 2004 issue of Microscopy Today (p. 24) http://microscopytoday.com/PDFFiles/MT-2004-02-small.pdf.
Brief explanation what bit depth means: In an 8-bit image, each pixel can have
intensity values from 0 to 255, whereas in a 12-bit image, each pixel can have
intensity values from 0 to 4095. 256 different intensity values are more than a human
can usually distinguish, so in most cases, they are enough for viewing images.
However, for quantative analysis, 12-bit images are better than 8-bit ones.
8.2.2 Image formats
In the Export Images and Data window, you have several options how to export your
images (Single option is for a single image, Series is for a Z-stack):
Raw Data Single/Series: Use if you want to control the separation of labels into
different RGB channels (e.g. if you want to have green channel as an own image file).
Exports only the original scanned data, i.e. brightness/contrast or pseudo-color
changes are not applied to the exported image. Also, no overlay information is shown
in a raw data image. This is the choice if you are going to analyze your image. Note
that an image can only have three different channels (RGB), so if you have taken an
image with four fluorochromes, you will have to export two images (one with three
channels and another with one channel).
Contents of Image Window Single/Series: Image resolution is not determined by the
frame size selected for scanning but by the image window size on the screen. Because
the resolution may change when this option is used, it is not recommended.
32
Full Resolution Image Window Single/Series: Exports a full-resolution image as
determined by the frame size before scan (in contrast to the “Contents of Image
Window single” option). No control over channels as in “Raw data single/series“.
Will include scale bars and other overlays. Changes made in the image after scanning
(e.g. brightness/contrast or pseudo-color changes) are also included - so be aware that
even if you took care not to have any saturated pixels in your original scanned image,
you can still create saturated pixels by making post-acquisitional changes. Those
changes will remain in the image, if you use this exporting option.
9. Ending your session
9.1 There are users after you on the same day
Leave the computer, monitor and REMOTE CONTROL on.
windows by either clicking X in the top right corner of
Alternatively, in the main menu, select WINDOW/CLOSE
WINDOWS. A dialog box will ask you for each image if you
or not.
Close all open
each window.
ALL IMAGE
want to save it
Go to ACQUIRE/LASER in main menu. Check if the users after you on the
same day will need the lasers that are on (the default page of Internet Explorer
shows reservations). If not, turn them off. If Argon laser will still be needed,
leave it in STANDBY and drag the output slider all the way to the left
(minimum value). If 405 Diode and HeNe lasers will be used on the same day,
leave them on.
Click on FILE/EXIT to terminate LSM program (click OK in the pop-up
window, and again click on EXIT in the second window). Select START/LOG
OFF. Write down the time in the logbook.
Clean up the objectives; have the lights turned on so that you can see properly.
Use cotton swabs immersed with ethanol (take from the correct bottle) to clean
oil from objectives. After this, dry with a clean swab. Start the wiping in the
middle of the lens and move outwards in a spiral way. You can also use lens
paper to first wipe up most of the oil. If you use lens paper, do not rub it
against the top lens of the objective. There are two ethanol bottles in the Meta
room; one is only for cleaning objectives, the other for cleaning tables, the
stage etc. in case there are oil spills.
9.2 You are the last user of the day
Go to LASER control panel (main menu). Switch all lasers OFF. Close all
windows and EXIT the program. Shut down the computer. The lasers will
need about 5 min for cooling!!!
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Do not turn off REMOTE CONTROL until the fan has stopped. Once the fan
has stopped, turn OFF the REMOTE CONTROL (Fig. 1).
Write down the time in the logbook.
Clean up your mess, tables, stages, and especially the objectives; have the
lights turned on so that you can see properly. Use cotton swabs immersed with
ethanol (take from the correct bottle) to clean oil from objectives. After this,
dry with a clean swab. Start the wiping in the middle of the lens and move
outwards in a spiral way. You can also use lens paper to first wipe up most of
the oil. If you use lens paper, do not rub it against the top lens of the objective.
There are two ethanol bottles in the Meta room; one is only for cleaning
objectives, the other for cleaning tables, the stage etc. in case there are oil
spills.
Cover the front part of the microscope with a dust cover but do not cover lamp
housings.
10.
Additional functions for live samples
If you want to image living samples with Meta, the cells have to be in a suitable
chamber, for example Nunc LabTec chamber. There is a heated stage available for
living samples but no CO2. For more information on imaging live cells, contact MIU
personnel. For very fast live cell imaging, the other confocal (LSM DUO) is
recommended. In DUO, there is an incubation chamber attached to the microscope,
and both temperature and CO2-concentration can be controlled.
10.1 Acquisition of time series
You can make a time series collection of both single images and a Z-stack.
1.
2.
3.
4.
In ACQUIRE, press TIME SERIES.
Under END SERIES, fill in number of time points.
Under TIME DELAY, choose min/sec, then Time (in selected units), press
APPLY.
PRESS START.
10.2 FRAP (fluorescence recovery after photobleaching)
This is a time series collection with bleach. FRAP is used for studying molecular
dynamics: when GFP-labeled molecules are bleached in a certain area, the nonbleached GFP-labeled molecules outside that region will enter the treated area and
replace the bleached molecules. The time it takes for the new fluorescent molecules to
replace the signal depends on the rate of diffusion of the labeled molecules, as well as
whether active transport mechanisms are involved
1.
Set desired TIME SERIES parameters.
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2.
3.
4.
5.
6.
7.
11.
In ACQUIRE/EDIT BLEACH, choose number of iterations = number of
bleach scans.
Define REGION; Bleach Regions window opens. In INTERACTIVE ROI
DEFINITION, use the drawing tools for defining the region you want to
bleach. Save the ROI and close the window.
For excitation of the bleach track, increase transmission to 100% on
relevant lasers for bleach step only.
Press START B in Time series control. Take some images before the
bleaching routine so that you can count the initial intensity levels.
Also select one ROI for background measurement (in an area with no
signal), and one ROI for general photobleaching (an area that has signal
but will not be treated with the bleach routine).
Open HISTO for intensity measurements.
Troubleshooting
11.1 Starting the system
Cannot find the light switch in the Meta room
To turn on the lights, pull the string hanging from the ceiling light.
Cannot turn on the table lamp
The table lamp is connected to an extension cord that has power only when Meta is
on. If the confocal is off, use ceiling lights.
Cannot control the microscope through the software
Perhaps you selected "Use Existing Images" in the start-up window instead of "Scan
New Images"; in that mode, the software does not communicate with the hardware.
Solution: Exit the software and restart it.
11.2 Using the microscope
Cannot see anything through the oculars
Check that the VIS mode is selected in the software and not the LSM. Also, make
sure that you are using the correct fluorescence filter to view your sample and that the
light is on (can you see the light)? If you cannot see any light, it is possible that the
mercury bulb is blown. In that case, contact MIU staff.
If there is light but you cannot see anything through the oculars, check that the oculars
are correctly adjusted for your eyes (for normal vision, zero mark on the side of the
oculars should match the white dot marked in the ocular tube). Also check the
following: the slide should be upside down on the stage; the stage should be firmly in
place; there should be cells/tissues in the field of view and they should be in focus. If
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it is difficult to find the correct focus, use an objective with a lower magnification
first. If you are using an oil objective, make sure there is enough oil in between the
objective and the cover glass.
Cannot focus on the sample
See the previous answer for Cannot see anything through the oculars.
11.3 Scanning
There are stripes in the image
These may be caused by vibration during scanning. Do not lean at the Meta table and
do not let anything else touch it. Check that the sample is properly made and securely
placed on the holder (see Paragraph 4).
The stripes may also be caused because unwanted laser light reaches the recording
channel (e.g. when 488 and 543 laser lines are being used in the same configuration,
together with a LP505 filter, some of the 543 laser line light is detected in channel 1,
even when the 545 beam splitter is being used).
How to do background subtraction with BF/DIC/Ph images on LSM 510 Meta
Take a normal image with any number of channels you wish.
Take another image, but focus off and preferably of an area outside the cover
slip glass (i.e. a place with no signal of any kind).
Go to: Process Subtract.
o source1 is the normal image.
o source2 is the off focus image.
o choose only channel chD for both.
o The image sizes have to be the same.
o You may add some brightness by changing the "+ ___" setting (default
is 128). Test the effect by viewing the Preview image.
o Create a new image with only chD.
Go to: Process
Copy.
o source is the subtracted image.
o target is the normal image.
o Copy the subtracted image on to the normal image's channel chD.
Change the pseudo color on the resulting image on chD to white.
Here is what the off focus image, the normal image and the resulting corrected image
should look like:
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