Leica TCS SP8 Benutzerhandbuch

 Last Updated on Sep 2021 
BEGINNERS SAFETY MANUAL FOR LEICA TCS SP8 
CONFOCAL SYSTEM 
Produced by Graham Wright, Fiona Chia, Cris Barzaghi, Wang Jing Fang, Shaalini, Melvin 
Safety Guidelines ........................................................................................................................ 2 
1 Lasers .................................................................................................................................... 3 
2 Immersion Oil........................................................................................................................ 3 
3 Metal Halide Lamps (MHL) ................................................................................................... 4 
Training ....................................................................................................................................... 4 
1 To arrange a training session:............................................................................................... 4 
Online Booking and System Access............................................................................................. 5 
1 Acknowledgements............................................................................................................... 6 
Operation Procedures................................................................................................................. 6 
1 Switching ON Protocol .......................................................................................................... 7 
2 Objective Lens....................................................................................................................... 7 
3 Software initiation ................................................................................................................ 8 
4 Turning on the lasers ............................................................................................................ 8 
5 Using Dye Assistant tab......................................................................................................... 8 
6 Setting Laser & Detector configuration .............................................................................. 10 
7 Using the Acquisition tab.................................................................................................... 11 
8 Image Adjustments –Setting the Gain and Offset properly............................................... 12 
9 How to do a Z stack for a 3D Image.................................................................................... 13 
10 Saving images and reusing settings .................................................................................. 14 
11 Switching OFF Protocol..................................................................................................... 15 
Troubleshooting........................................................................................................................ 16 
1 Troubleshooting Appendix.................................................................................................. 18 
 
 

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Last Updated on Sep 2021

1. Safety Guidelines

Please adhere to the safety guidelines for your own safety and health. When in doubt, always

approach the bioimaging facility for assistance.

1.1 Lasers

Lasers in the confocal facility are class 3B lasers. This means that the lasers are strong enough

to cause serious damage to your eyes, including temporary to permanent blindness. Therefore,

please always follow the safety guidelines below when using the confocal microscopes:

•Before turning on the lasers, make sure the power connections are all connected

properly. If there are any disconnected wires, please reconnect them or inform the facility

staff. If you see any exposed wires, do not attempt to use the equipment and inform

facility staff immediately.

•For all confocal systems, there is a minimum time for lasers to be switched ON/OFF.

Please check the operation procedure for the respective microscopy system that you are

assigned to. This is to ensure the lasers have sufficient time to heat up and cool down

before running again.

•When using the lasers to image, NEVER look directly at the laser while imaging. If a safety

shutter is present, make sure it is in the correct position to better protect your eyes.

1.2 Immersion Oil

Modern immersion oil has no known hazards to human beings so far, yet they can cause

discomfort to a person if the immersion oil has been left on skin for too long or inhaled.

Therefore, please take note of the following safety guidelines for using immersion oil:

•Use the applicator to apply the immersion oil onto the slides for upright system.

•Do not touch the applicator oil directly on the objective lens as this will scratch or break

the lens. Instead, allow the drop of oil to contact the lens surface.

•If the bottle containing immersion oil has oil on the sides, wipe down with kimwipes and

ethanol. Wash hands immediately after with soap and water.

•Wipe immersion oil off objective lenses after use using lens paper and wash hands

immediately after with soap and water.

•Always clean up any spilled oil or residue from oily slides immediately after your session.

Last Updated on Sep 2021

1.3 Metal Halide Lamps (MHL)

Metal halide lamps are fluorescence lamps that gives a visible light range of 300 –650nm,

enabling us to observe fluorescence signals such as GFP, DAPI and RFP. Metal halide lamps

contain mercury vapours that are extremely toxic to the human body. Therefore, please take

note of the following safety guidelines when working with the metal halide lamp:

•Ensure that the metal halide lamp has been off for at least 30 minutes before switching it

on. Once the lamp has been turned on, it must remain on for a minimum of 30 minutes to

allow sufficient time for it to warm up and run properly.

•Check the timer on the power box to ensure the mercury lamp does not run past 2,000

hours.

•Do not look directly at the MHL when it is switched on as it can damage your eyes.

•Do not attempt to remove the light guide from the MHL, especially when it is on. Direct

emission of the MHL may set off fires.

•If there are no users in the next 30 mins, MHL must be turned off.

•Never attempt to change a bulb yourself. Please contact facility staff when you notice

that a bulb has reached between 2,000 –2,500 hours or is not functioning properly.

2. Training

Use of confocal microscopy and the online booking system is authorized by facility staff only. All

users must attend the confocal training session conducted by the Bioimaging facility staff

before they are allowed to access the confocal system.

2.1 To arrange a training session:

•Please email Bioimaging Facility ([email protected]) for a training session.

•Fill in the necessary particulars in the form here:

http://microscopy.tll.org.sg/pages/conf_training_form.html

(E.g. Lab, PI, sample, and a brief description of your project)

•We will help you identify the most appropriate microscope for your imaging needs.

•We will then arrange training session according to your availability within a week.

•Training sessions usually last 1 - 3 hours depending on users’ previous experience.

•Users receiving training are highly encouraged to bring their own samples so that staff

can adjust trainings to your imaging needs, but if not possible, staff have their own

samples that they can work with.

Once you have completed the training session, we will grant you access to both the confocal PC

and the online booking system. Please keep in mind that only bioimaging facility members are

Last Updated on Sep 2021

allowed to conduct the training. None of your lab members are allowed to conduct the training

for you.

If you need a refresher or some specific advice on anything microscopy related, please

approach any member of the Bioimaging Facility for assistance/help.

We also offer training on the various types of image analysis and image processing software

available here at TLL, including ImageJ/Fiji, Huygens and Imaris.

3. Online Booking and System Access

•Booking of all Light microscopes prior to use is COMPULSORY through microscopy

resource booking via TLL intranet (https://intranet.tll.org.sg/App/tll_intranet/booking_searches)

•Users are only allowed to book the confocal system that they have received training on. If

they wish to book other confocal system, they have to receive a separate training.

•Users are entitled to advance bookings of up to 2 weeks. They are advised to plan their

experiments accordingly to avoid any disappointments.

•During Office Hours (Weekdays from 8.30am to 6pm)

➢Each user is entitled to a MAXIMUM of 2 bookings per system per week.

➢Users whom are trained on multiple systems are entitled to a maximum of 3 bookings

per week but it has to be shared across the systems that they have received training

on.

➢Each booking must not exceed 3 hours.

➢If users have utilized all their entitled bookings for the week, “24Hr Rule” can be

applied where they can book the system in less than 24 hours in advance according to

its availability. If extra slots are booked within the 24-hour period, a note of “24Hr

Rule” should be made in the booking description.

•During Non - Office Hours (Weekdays after 6pm, Sat, Sun & Public holidays)

➢If users require more slots in a particular week, they can book on weekdays during

non-peak hours (after 6pm), on weekends (Sat and Sun) and on public holidays.

➢If extra time is required, bookings can be extended out of peak hours (E.g. 3 –7pm).

•Bookings exceeding these limits are subjected to cancellation without prior warning.

•Simultaneous bookings of two or more different systems are not allowed. Multiple

bookings for the same system on the same day during office hours is strictly prohibited.

•If users cannot attend a booked session for any reason, it is COMPULSORY that they

cancel their booking through TLL intranet and email ([email protected])to announce the

availability. If they are the last users for the day, they need to check if the system has

been switched off completely.

•If users are swapping a session with another user, they must change the booking details

accordingly.

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•Under any circumstances, users are not allowed to make bookings on behalf of other

people. Users who have received training but are yet to gain access to microscopy

resource booking may approach TLL Bioimaging department for booking assistance if they

need to use it urgently.

•If a user fails to show up within the first 30 minutes of their booking, the slot is forfeited

and is free for any user to use it.

•If any users violate any of these Booking rules, users will be subjected to the 3 strikes

policy.

3.1 Acknowledgements

If you use the TLL Microscopy and Imaging facility and/or have been trained or assisted any of

the bioimaging facility members in your research, then this should be acknowledged

appropriately in your publications and presentations.

4. Operation Procedures

Every confocal system in TLL Bioimaging Facility has its specific instructional manual which are

found in every confocal rooms. Strictly adhere to the correct order of operation for all system.

Failure to do so will result in disciplinary action from the facility. Any issues encountered during

the operation of the system are advised to seek help from the Bioimaging facility.

Modification, exchange or removal of components beyond this operational manual is strictly

prohibited and is only carried out by the manufacturer, Bioimaging facility or by experienced

users approved by bioimaging facility. During operation of laser microscopy system, do not look

into the laser beam directly as they are all Class 3b and Class 4 lasers.

1st offence

A warning will be issued along with the reminder of the rules.

2nd offence

A second warning will be issued and your respective PI will be notified.

3rd offence

Banned from using any of the facility’s microscope for 2 weeks.

Last Updated on Sep 2021

4.1 Switching ON Protocol

(a) SIGN IN the Logbook and record your START-TIMING.

(b) Switch on Laser Key (1), Laser Power (2), Scanner power (3) and PC Microscope (4).

(d) Switch on STED laser module (6), if you need it.

(e) Wait for PC to start up and log in with your TLL user name.

(f) Load LASx software and switch on the lasers that you are planning to use:

•Diode Laser: “ON” 405nm laser

•White Light laser: “ON” up to 70% power

•Argon laser: “ON” up to 70% power. (Make sure the argon lasers are ON for a

minimum of 2hrs before switching OFF as it requires sufficient time for warming up)

•STED laser: You may switch “ON” up to 100%. (Make sure the STED lasers are ON for

at least 1hr before switching OFF.)

(g) Do not overload the HyD detectors. Keep gain at max 100% as overloading will damage

the performance of HyD detectors over time. For best performance of the HyD detectors,

set gain at 100% and only use the laser power slider to moderate irradiation intensity.

(h) Finally, fill in the logbook.

4.2 Objective Lens

Objective lens

Immersion media

10x/0.4 HC Plan Apochromat

Air

20x/0.75 HC Plan Apochromat

Air

40x/1.3 HCX Plan Apochromat

Air

63x/1.2 HC Plan Apochromat

Water

63x/1.4 HC Plan Apochromat

Oil

100x/1.4 HCX Plan Apochromat

Oil

Last Updated on Sep 2021

4.3 Software initiation

Turn on the machine following the instructions pasted on the wall and inserted in the log book.

Wait for complete initialization of microscope and Windows after logging in with your TLL

username and password.

Double click on:

(A) The LAS X configuration screen will appear:

Configuration: Machine.xlhw must be selected.

Microscope: Check that DMI6000 is selected.

Click on OK and wait for complete initialization. Do not move anything on the microscope

during the initialization process.

4.4 Turning on the lasers

1. Under the Visible panel, click on the button to open the laser panel.

2. Click on the UV (405nm), Visible (Argon) and SuperContVisible2(WLL) bar to observe

405nm, Argon and WLL respectively in the dropdown menu.

3. White light laser (WLL) has excitation wavelengths from 470nm-670nm allowing you to

select specific excitation light source. Argon lasers have much higher power than the WLL

and has excitation wavelengths –458nm, 476nm, 488nm, 496nm and 514nm.

4. Turn on the laser that you need by clicking on the sliders. Make sure that WLL and

SuperContVisible2 are at 70% before carrying on with the next steps.

4.5 Using Dye Assistant tab

•Select the objective lens that you would like to image with your sample.

Last Updated on Sep 2021

•Then select, Dye assistant to set up your laser pathway.

1. Click to add the number of channels

you would like to image.

2. Click to select the dyes you are

using for each channel.

3. Then select the type of detector (HyD or

PMT) you would like to image with your

dyes. HyD is more sensitive than PMT.

4. The Dye assistant will give you a list of

laser settings options to select from.

Select the settings which has the least

amount of crosstalk. Sequential Scan is

always preferred as the amount of

crosstalk is lesser. Line sequencing is

much faster than frame/stack imaging.

5. Select your preferred laser setting option

and click apply.

Last Updated on Sep 2021

4.6 Setting Laser & Detector configuration

•Based on the no. of dyes, you have selected, it will give you

the total no. of seq. (E.g., if you have selected 3 dyes in

sequential mode, you will have 3 seqs corresponding to each dye.)

•Select each seq, and increase the laser power to 1% initially. Make sure the laser line is

activated by checking both the red button and the laser line shown at 2 and 3.

•Make sure that the HyD/PMT is activated. (Turn on the slider)

•Then, Select the fluorophore you are using. It will give you the emission spectrum of the

dye you are using.

•Choose an appropriate emission band to detect your fluorophore. (Important: Make sure

that the emission band is at least 8-10nm away from your excitation line.)

•Choose an appropriate detector pseudocolour (also known as a Look Up Table, or LUT).

•For PMT, set the Gain to approx. 700 - 800 and the Offset to 0.

•When using the HyD (2× more senstitive than PMT), default gain is set at 100.(Important:

Do not overload HyD, max is 100.)

•Gating: Removes the first few nanoseconds of the signal as a result of autofluorescence

from the sample or mounting medium.

oOnly usable on pulsed lasers such as the WLL. Constant mode lasers like the Argon lasers

will not work with the Gating function.

oChoose appropriate reference laser line for gating.

oNOTE: start with low laser power to avoid damaging the HyD detectors. HyD detectors

will shut down if an overload condition is detected.

Gating

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