Olympus FV3000 User guide

 Last updated on Mar 2022 
BEGINNERS SAFETY MANUAL FOR 
OLYMPUS FV3000 CONFOCAL (#02-20) 
Produced by Shaalini, Graham Wright, Fiona Chia, Cris Barzaghi, Melvin 
Safety Guidelines ...................................................................................................................2 
1 Lasers ...............................................................................................................................2 
2 Immersion Oil...................................................................................................................2 
3 Metal Halide Lamps (MHL) ..............................................................................................2 
Training ..................................................................................................................................3 
1 To arrange a training session:..........................................................................................3 
Online Booking and System Access........................................................................................4 
1 Acknowledgements..........................................................................................................5 
Operation Procedures............................................................................................................5 
1 Switching ON Protocol .....................................................................................................5 
2 Objective lens...................................................................................................................6 
2 Software Initiation ...........................................................................................................7 
3 Setting up directory to save images ................................................................................8 
4 Laser Configuration Settings............................................................................................9 
5 Using the LSM Imaging Tab............................................................................................10 
6 Image Adjustments –Setting the Gain and Offset properly..........................................11 
7 How to do a Z stack for a 3D Image (xyz).......................................................................11 
8 For Time-lapse (xyzt)......................................................................................................12 
9 Reuse previous settings and export images ..................................................................12 
10 Switching OFF Protocol................................................................................................13 
Troubleshooting...................................................................................................................14 
 
 

Last updated on Mar 2022

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 3 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 kim wipes

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.

1.3 Metal Halide Lamps (MHL)

Metal halide lamps are fluorescence lamps that emit 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

Last updated on Mar 2022

of 30 minutes to allow sufficient time for it to warm up and run properly. The lamp has

an enforced cooldown of 300 seconds (5 minutes) after shutdown where the lamp

cannot be switched on until the 300 seconds is up.

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

hours. The timer indicator on this particular lamp power box blinks when the timer

crosses 1,900 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.

•Important! If you hear an explosion and see the burner indicator turn red from blue,

save your work and evacuate immediately.

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 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.

Last updated on Mar 2022

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]rg.sg)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.

•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.

Last updated on Mar 2022

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.

4.1 Switching ON Protocol

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

(b) Switch on the Master switch on the wall (1) & PC (2). Wait until windows icon pops

up.

(d) (Optional) Turn on the Metal Halide lamp (5), if needed.

(e) Turn on the Touch screen panel power button at the back (6) and wait for 5s to light

up.

(f) Switching ON Lasers,

•To use 405/488/561/640nm, switch on the power button on the main

combiner (7), then turn the key to “ON” (7A) and finally turn on the

individual lasers that you need.

•To use 445/514nm, switch on the power button on the sub combiner (8),

then turn the key to “ON” (8A) and finally turn on the individual lasers that

you need.

(g) (Optional) Turn on the incubator (9) if required. It takes 10-15mins to stabilize.

(h) Mount your slide onto the sample holder. If you need Live cell Imaging, turn on the

OkaLab incubator and the facility CO2(10). It takes 10mins to stabilize. If you require

help mounting the stage incubator holder onto the stage, please request help from

the TLL bioimaging department.

(i) Log in with your TLL user account and double click on “FV31S-SW” to load the

software.

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 Mar 2022

(j) Load the FV31S-SW software and check if the model is “OLYMPUS IX3-SSU”. Then

click OK. Wait for complete initialization and DO NOT move any components on the

microscope during the initialization process.

a. If you are starting it at the morning of the first day of the week, check that

the turret is set to 10x objective or lower, then click “YES”. Cleaning the stage

will take some time.

b. Otherwise, click “NO” to skip cleaning.

4.2 Objective lens

Objective lens

Immersion media

1.25x/0.04 Plan Apochromat

Air

10x/0.4 U Plan Super Apochromat

Air

20x/0.75 U Plan Super Apochromat

Air

40x/1.35 U APO N 340 II

Oil (Blue Bottle)

60x/1.42 U Plan Super Apochromat

Oil (Blue Bottle)

100x/1.4 U Plan Super Apochromat

Oil (Blue Bottle)

30x/1.05 U Plan Super Apochromat

Silicon (Green Bottle)

60x/1.3 U Plan Super Apochromat II

Silicon (Green Bottle)

Last updated on Mar 2022

4.2 Software Initiation

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

username and password.

Double click on:

(A) The HW configuration screen will appear:

Select Enable XY stage control

Click on OK and wait for complete initialization. Do

not move anything on the microscope during the

initialization process.

(B) Execution of IX3-SSU clean-up will pop up:

If you are the first user of the week: Rotate the

turret so that either the 1.25× or 10× objective is in

place. Click on YES and wait for complete

initialization. The cleaning process uses ultrasonic vibrations to dislodge dust that has

accumulated on the stage mechanism. This adds time to the initialization process. Do not

place anything on the stage mechanism until the initialization process is complete.

If you are not the first user of the week: Click on NO and wait for complete initialization. Do

not move anything on the microscope during the initialization process.

On the FV31S-SW software, press on the ocular tab to

observe the specimen using either bright field/DIC or

fluorescence mode.

•For Brightfield imaging, select Ocular>Ocular>DIA.

Select Cube turret #1: Empty

Under DIA, ON both TD shutter and Trans lamp.

Increase intensity of Trans lamp for specimen

viewing.

•For Fluorescence imaging, select Ocular > Ocular >

EPI. Select Cube turret #1: DAPI/FITC/TRITC

Select the appropriate EPI ND Filter to control the

fluorescence intensity.

Then EPI shutter: Open

※Keep it Closed until you are actively viewing your sample.

Last updated on Mar 2022

4.3 Setting up directory to save images

If this is your first time using the FV3000, you need to change your default folder away from

the Fluoview software default, which is in the C: drive.

•Go to Configuration →Preference tab →File/Folder

•Uncheck Use default folder

•Press the Browse button to change the folder to either inside the D: or E: drives.

To save an image:

•Under Acquire tab, select the folder icon to select your directory to save all your images.

•Please save in your folder. You have several different options on where you can save

your images.

➢Option 1: D: or E: →Users →shaalini (create your own folder)→BPAE

cells.oir. Do keep in mind that we will delete any images which are older than

3months. Please make sure to do a backup.

➢Option 2: In TLL //research_cmn/drive folder in your respective lab folders.

➢Option 3: In your own lab drive.

•Key in the title of your image. Subsequent images will be saved in a running sequence

no.

•Your images will be saved in *.oir proprietary format.

Important: please do not save any data on the C: drive. Images saved in C: drive will be

deleted immediately!

Last updated on Mar 2022

4.4 Laser Configuration Settings

Under PMT Setting tab, click on Dye and Detector. A list of

commercially available dyes along with its excitation and emission

values are available for selection.

1. Choose the appropriate dye.

2. Select Add.

3. It will immediately be reflected under phase 1 where each dye

will be allocated to its respective channel. A transmitted light

channel will also be shown by default.

4. Click OK.

•Under Laser ND filter: Select 10%.

※If you need more than 10% laser power you can switch

off the ND filter, but most imaging will not need that high

power of a laser.

•Set your laser power for all your channels at 1%.

•Emission range could be altered to include a broader range of

signal otherwise you could use the default settings.

•For detectors:

➢HSD: keep HV within 350-500. More than 500 will

produce intense background noise.

➢SD:keep HV within 600-800. More than 800 will

contribute to intense background noise.

Under PMT settings tab:

•Mode: Select VBF

•Average: average a given number of scans to get a

higher signal to noise ratio.

➢In Line mode the averaging will be done on a line-

by-line basis.

➢In Frame mode the averaging will be done frame-

by-frame. Drawback: slower scanning time, more

bleaching, double exposure effect if your sample

moves. Averaging of 2 times will help you to get

better quality images if you can afford to do so.

No averaging or Line averaging is recommended if you are doing live-cell imaging.

•Sequential scan: Select Line to prevent crosstalk among the fluorophores. If you select

None, simultaneously imaging is done where cross talk is prominent.

•Confocal Aperture: press Auto button to make sure pinhole size is set to optimal

confocal imaging at Airy Disk ×1. This gives the most optimum size for the thinnest

optical slice while not sacrificing imaging quality. Increasing the size of the Airy Disk from

Last updated on Mar 2022

1× gives a brighter image while making the optical slice thicker, while decreasing the size

of the Airy Disk from 1× gives a darker image while not making the optical slice thinner.

4.5 Using the LSM Imaging Tab

Under Scan Settings tab, in which you can set the parameters for your imaging:

•XY

➢Type: normally, Galvano is used. For live imaging,

resonant imaging is preferred for its fast imaging

but resolution will be lower.

➢Mode: Normally, mono-directional mode is used.

For fast imaging, roundtrip can be selected where

phase correction has to be adjusted.

➢Interlace: Normally OFF is preferred. If 2x is

selected, it would capture the image by skipping

every 2 lines of the image.

➢Speed: the slower, the better your image will be

(a higher signal: noise ratio; but it takes longer

and its more prone to photobleaching of your

sample).

➢Scan Size: the number of pixels in your image

(the more pixels the higher the resolution, to an

extent, but slower the acquisition)

➢Zoom: allows you to magnify your region of

interest, the arrows allow you to zoom images

only in the center. Ideal zoom which satisfy the Nyquist limit can be achieved by

zooming till the red arrow, any zoom beyond it will result in poor resolution and

result in more photobleaching/damage.

Click on Live (images according to your scan setting configuration)/Live 2x (2x faster

imaging)/Live 4x (4x faster imaging) to view a live image. You can now adjust parameters

while checking the result directly on the monitor. Be careful though, if you spend too long

doing this you will photo bleach and damage your sample. Start focusing your sample and

then center it moving the stage.

Now, based on the image, you can adjust the brightness using:

•Excitation laser power: The higher the laser power, the higher the risk of

photobleaching. Also, this will alter both the brightness of the fluorescent image and

that of the transmitted light detection.

•Gain: This adds computer processing to the signal from the detectors into the image to

increase/decrease its brightness. For optimum signal to noise ratio, keep at 1.00×.

•HV: Keep below 500/800 for SD/HSD detectors for a low-noise image

•Offset: adjusts the background/black level

•Pinhole: the wider the pinhole aperture, the thicker is the optical slice –which means a

blurrier, but brighter image.

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