Light microscopy

Access

BCO light microscopy core facility is part of the Biocenter Finland biological imaging platform services and a member of Euro-BioImaging ERIC through Finnish advanced light microscopy node.

Light microcopy core facility is open for all users. Open access to microscopy instruments and services outside University of Oulu is applied through Euro-BioImaging . Facility services include bright field, fluorescence, confocal, multiphoton and light sheet microscopy, optical projection tomography, live-cell imaging, cell and tissue manipulations and measurements, in vivo imaging, computer-based presentations and analysis of the image data, sequences and morphology. Assistance in sample preparation, in trouble-shooting, choosing the appropriate instruments, and in data analysis is also provided. Imaging instruments and computer rooms for data analysis are located on the fourth floor in the main building of the medical campus (Kieppi building, Aapistie 5A). Cell culture space to culture and manipulate cells before and between microscopic imaging is available. LM core microscopes and image analysis software are open to all researchers after the training. Training in the LM core is available by appointment. If there is a need for instruments outside the office hours (16.00-7.30) or on weekends when the building and the corridor doors are locked, the user should contact staff to get an electronic guest key.

Reservations

After the training a user account is created and the new user will have access to the reservation system. Imaging instruments are reserved through the iLab reservation system. By using the same online system, users can revise and cancel their reservations. Cancellations should be done as early as possible but at least 1 h before the reservation starts. Note that it is not allowed to use any microscope without a reservation, since the usage will be charged according to iLab logs. The reservations are made in one-hour blocks. Booking time of Zeiss LSM 780 confocal microscope is limited to four hours per day per user (during prime time: Mon-Fri, 8.00-16.00). Longer time lapse usage should be scheduled outside prime time hours.

Usage

It is not allowed to use microscopes without an instrument-specific training session given by the core facility personnel. Specific instructions and printed user guides are alongside the microscopes. It is not allowed to change the microscope assemblies without negotiating with the staff. Users should inform the staff in the case of malfunction. It is on the last users’ responsibility to turn off the microscopes at the end of the day. Microscope computers have temporary data storage only during the imaging and the user data should be transferred after the experiments. User data can be transferred from the instrument computers using a network drive. Further instructions will be provided by the core facility staff. The network drive is meant for data transfer only, not for personal storage.

Microscope usage time is charged according to the actual hours marked to iLab after imaging. There is a maximum expense limit of 4400€ / charging period (two six month periods: 1.1. – 30.6. and 1.7. – 31.12.) / research group, and hours exceeding that limit during that period will not be charged. Usage during the nightly hours between 24.00 and 8.00 has reduced price (see detailed prices from iLab). User training and support, usage of offline image analysis workstations (room 419A) and cell culture space are free of charge. Customized service packages are formed for industrial users.

Prices for academic users (€/hour)

Here is a visual guide that will help you track your microscopy method reporting. We also provide a general template followed by two specific examples of widefield and confocal imaging. You will find instrument-specific information on our webpages but we also suggest contacting our staff to assist with your method writing.

You can view the visual guide here as a PDF.

General template

Cells were [SAMPLE INFORMATION]. Imaging was performed using a [1. Microscope MAKE & MODEL]. The objective used was a [2. OBJECTIVE]. Live imaging was performed at [LIVE IMAGING]. Samples were imaged with [3. LIGHT SOURCE] with [4. FLUORESCENCE FILTERS]. Images were acquired [5. STAGE and 3D Capture] using a [6. CAMERA].

Acknowledgements template

Imaging was performed at the Biocenter Oulu Light Microscopy Core Facility, University of Oulu, Finland and supported by Biocenter Finland.

Working example: Widefield

Cells were grown (fluorophores?) on No. 1.5 coverslips, fixed with 4% PFA and mounted on slides using Prolong Gold. Imaging was performed using a Nikon Ti2-E widefield inverted microscope using Nikon NIS Elements 4.11 acquisition software. The objective used was a Nikon 63x Plan Apochromat 1.4 NA with Nikon oil immersion 1.518. Live imaging was performed with Okolab bold line heating system at 37°C, 20% O2 and 5% CO2 with images captured every 10 minutes for 24 hours. Samples were imaged with 475/28nm and 575/25nm excitation filters and 525/50nm and 600/30nm emission filters (Chroma). Images were acquired by taking a z stack of 30 slices with 300nm spacing using a Hamamatsu ORCA-Flash 4.0 v3 sCMOS camera with a pixel size of 6.5μm. Images were 16bit with pixel dimensions 2048x2048.

Working example: Confocal

Cells expressing GFP and RFP were grown on 35 mm Mattek dishes. Imaging was performed using a Leica TCS SP8 confocal with a DMI8 microscope using LAS X 3.5.2 acquisition software. The objective used was an HC PL APO 63x/1.20 W motCORR CS2. Samples were imaged with 488 nm and 561 nm solid-state lasers with emission windows at 500-550 nm and 580-630nm, the pinhole was set to Airy 1 and scan speed to 400 Hz. Live imaging was performed with Okolab bold line heating system at 37°C, 20% O2 and 5% CO2 with images captured every 10 minutes for 2 hours. Images were acquired by taking a z stack of 30 slices with 300nm spacing with a pixel size of 100nm.

BCO-TIC would greatly appreciate if the light microscopy core facility is acknowledged in the publications when services are provided. We also kindly ask you to send a notification about your BCO-TIC -supported publication(s) to Lauri Eklund (lauri.eklund (at) oulu.fi).

To register for an account:

To get started, you must register for an account:

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3636
2. In the upper-right-hand corner of the screen select the ‘Register using University of Oulu credentials’ option.
3. You will be directed to an authentication page where you will need to select your University and enter your HAKA credentials.
4. Once you have entered your credentials, click the ‘Login’ button
5. You will be directed to an iLab Registration page where you will need to select your PI/Lab, and verify your contact information.
6. Once your registration has been submitted, your PI will receive a notification that you have requested membership to their lab in iLab. They will need to approve your membership and assign any Cost centers for your use.

To Create an Equipment Reservation:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can schedule equipment time.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3636
2. At the upper right hand of the page click ‘Sign in’ and then select the ‘Sign in using HAKA credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Schedule Equipment tab and click on the ‘View Schedule’ button next to the instrument of interest. Click and drag on the time frame you would like to schedule your reservation for.
5. A window will pop up that will allow you to verify your reservations details and provide payment information before saving the reservation.

To Create a Service Request:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can create service requests.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3636
2. At the upper right hand of the page ‘Register using University of Oulu credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Request Services tab and click on the ‘Request Service’ button next to the service of interest.
5. You will be asked to complete a form before submitting the request to the core.
6. Your request will be pending review by the core. The core will review your request and either Agree to the work or they will ask for more information if needed.

Instructions:

1. Click here to log-in: https://oulu.ilab.agilent.com/service_center/show_external/3636
2. You will use your HAKA credentials to log into iLab
3. Once logged in, look for the link in the left hand menu that says 'my labs'. Hover-over and select your lab.
4. Set the auto-approval amount if you do not wish to approve service requests below a certain dollar amount. To do this, select the 'Members' panel and enter a dollar amount in the 'Auto Pre-Approval' amount and click 'save settings.’
5. To approve lab membership requests, select the ‘Membership Requests & Cost centers’ tab. New membership requests will show at the top of this page. Click “Approve” to accept a member into your lab. Click “Reject” if they are not a member of your lab.
6. To assign a Cost center to a member of your lab, find the member in the above list where it says, ‘Manage Cost centers.’ Select the checkbox(es) to the right of their name for the Cost center(s) you wish to assign them.

For high resolution and speed imaging of living and fixed samples. A white light laser and spectral detectors for most available fluorophores. Fast lifetime contrast (FLIM) imaging and real time adaptive deconvolution.

Sensitive multi-position 3D time-lapse imaging of tissues, organotypic cultures and cells. Freely selectable excitation and emission wavelengths. Equipped with a resonant scanner - suitable for imaging rapidly occurring cellular events and for fast large area imaging. Real time adaptive deconvolution (LIGHTNING) to maximize resolution and contrast in captured images. Integrated Fluorescence Lifetime Imaging (FLIM).

A fully motorized inverted microscope and a stage for multi-position and tile-scan imaging. Suitable sample formats: slides, 35-60mm dishes, multi-well plates (6 to 96-well).

A combination of a laser scanning confocal and a multiphoton microscope. For sensitive high resolution imaging of tissues, organotypic cultures and cells. Also for optically cleared thick light scattering samples. Label free imaging of e.g. collagen fibers (SHG) and structure interfaces (THG).

Spectral detectors for both confocal and multiphoton imaging. Real time adaptive deconvolution (LIGHTNING) to maximize the resolution and contrast in captured images. Integrated fast Fluorescence Lifetime Imaging (FLIM).

A fully motorized inverted microscope and a stage for multi-position and tile-scan imaging. Suitable sample formats: slides, 35-60mm dishes, multi-well plates (6 to 96-well).

Deep and fast imaging of thick samples and small animals in vivo. Dual output infrared laser with tunable (820nm-1300nm) and fixed (1040nm) beams.

For strongly light scattering samples; sensitive 3D time-lapse imaging of tissues, organotypic cultures and intravital (mouse, rat) imaging. Label free imaging of e.g. collagen fibers (SHG) and structure interfaces (THG). Equipped with a resonant scanner for scanning at high speed (suitable for rapidly occurring cellular events).

Fully motorized upright microscope and stage for multi-position imaging

Suitable sample formats: slides, 35mm and larger petri dishes, 6-well plates

Confocal laser scanning microscope for fixed samples.

For live-cell imaging, molecular dynamics (FRAP) and cell-surface events detection (TIRF).

Total internal reflection fluorescence (TIRF) microscopy with 405nm, 488nm and 561nm excitation

Fluorescence recovery after photobleaching (FRAP), DirectFRAP-module

Fully motorized inverted microscope and stage for multi-position imaging

Suitable sample formats: slides, slide chambers, 35mm and 50mm dishes, 6-well and 24-well plates

For live cell and tissue imaging, sensitive high resolution and spectral imaging of wide range of fluorophores in fixed and living specimens.

Fully motorized inverted microscope and stage for multi-position imaging

Suitable sample formats: slides, 35mm dishes, multi-well plates (6 to 96-well)

Motorized inverted epifluorescence microscope and a stage for multi-position imaging.

For microscopic to mesoscopic scale fixed and living samples. High speed sensitive 4D imaging in 3D culture environment.

Imaging from single cells to whole embryos over extended periods of time

Subcellular resolution and multi-dimensional real-time imaging of intact thick samples

Samples can be embedded to low melting temperature agar, Matrigel, collagen etc. and are imaged in an aqueous medium/water/PBS etc. filled chamber

A basic non-motorized fluorescence and bright field microscope with a monochrome camera. Possibility for hyperspectral imaging (under research and development).

ApoTome.3 for optical sectioning. Also for brightfield imaging with various transmission light contrast methods (brightfield, oblique illumination, dark field) and reflected light ringlight and gooseneck illuminations. Equipped with high sensitivity monochrome camera for fluorescence imaging and RGB-camera for color imaging.

Optical projection tomography (OPT) service is a part of the Biocenter Finland activities and is integrated to Oulu BioImaging (OBI) and Tissue Imaging Center (TIC) and the Transgenic and Tissue Phenotyping Core Facility.

OPT is a 3D imaging technology for biological specimens up to 1x1,5cm in size.

It is a convenient method for viewing the 3D anatomy, gene and protein expression patterns during embryo development, or separate organs and biopsies.

We are using Bioptonics 3001 device with the resolution 1024x1024 (~3,1micron) which allows analyzing specimens in 4 channels:

1. Bright field: in situ hybridization specimens (BMP purple, NBT/BCIP), beta-galactosidase and other stains;
2. GFP1 425/40nm exciter, 475nm LP emitter: auto-fluorescence and general morphology;
3. GFP 470/40nm exciter, 515nm LP emitter;
4. Texas red 560/40nm exciter, 610nm LP emitter.

The raw data obtained from scanner is further processed and analyzed by the 3D analysis software. TIC has license for the Bitplane Imaris software that allows data visualization and detailed morphometric studies of the OPT specimen.

We are offering:

• Tissue staining for the OPT (immuno-, beta-gal ect.)
• Sample preparation for OPT: embedding and clearing
• Scanning and reconstruction
• Data analysis (Quantification, 3D shape measurements, 3D and 2D linear measurements)
• Recovery of the specimens for the following use (histology analysis, IHC & ect.)

Please contact us to discuss about your specimen preparation and requests.

IVIS Spectrum is an in vivo imaging system for visualization, quantification and 3D reconstruction of both bioluminescent and fluorescent reporters in small animals. IVIS can be used to non-invasively follow and image disease progression and therapy, cancer metastasis, cell trafficking, microbial infection and gene expression patterns in living animals. The instrument is equipped with filters covering the blue to near infrared wavelength region, enabling the scanning of reporters over the range from 480-850 nm. In addition, the software provides tools that allow separation of signals from multiple reporters within the same animal. For fluorescence imaging both trans-illumination and epi-illumination modes are available.

IVIS Spectrum is located in Laboratory Animal Centre.

• Bitplane Imaris 3D modelling and analysis software.
• Huygens Professional for deconvolution and analysis.
• Offline-versions of the microscope control software for data analysis.

A fully motorized bright field microscope with a color camera. For all sized histological specimens with a maximum precision and reproducibility. Possibility for automated high resolution tile/mosaic scanning of large samples and extended focus depth imaging.