Confocal Microscopy

SAHMRI Light Microscopy Core Facility

Facility Manager: Dr. Makoto Kamei makoto.kamei@sahmri.com

The Light Microscopy Core Facility at SAHMRI was established in 2014, and is operated by the Hopwood Centre for Neurobiology.

Three light microscopes are available in the Facility, two of which are live-imaging capable. The Facility is available for use by SAHMRI, its partners, and by external academic and commercial users.

For further information about the Facility or your project-specific microscopy requirements please contact the Facility Manager.

Instruments

Leica TCS SP8X-MP Confocal Microscope

The confocal microscope has both confocal and multi-photon imaging capabilities. It has four laser excitation light sources:

  • 405 nm laser to excite DAPI/Hoechst dyes;
  • Argon laser for GFP/YFP excitation;
  • NKT Photonics Supercontinuum White Light Laser to provide excitation throughout the visible light spectra; and a
  • Spectra Physics Mai Tai DeepSee laser to provide multiphoton excitation

The instrument has nine light detectors (four internal, four non-descanned, and one bright field). The White Light Laser is fully tuneable within the 470 and 670 nm range, and is capable of emitting eight lines of excitation simultaneously. The multiphoton laser is also tuneable within the 740 to 1040 nm range. Four internal detectors are fully tuneable to detect only the range of wavelengths of interest, and the four external non-descanned detectors provide better emission light detection for multiphoton imaging by bypassing the confocal pinhole. The combination of the tunable laser light sources and the tunable light detectors provides unparalleled flexibility in confocal and multiphoton imaging. It has been used in characterising unpublished novel fluorophores utilising the tunable excitation light source and the detectors.

1. Leica TCS SP8X-MP Confocal Microscope

The Confocal Microscope is also equipped with a Ludin chamber, providing an optimal environment for live cell imaging, such as time-lapse imaging, fluorescence recovery after photobleaching (FRAP), and Förster Resonance Energy Transfer (FRET) experiments. It has appropriate holders for histology slides, chambered coverslips, 30 mm culture dishes, and tissue culture plates. Acquisition of images over time can be automated with the fully-motorised stage and the control software. For live cell imaging, the resonance scanner operates at 12 kHz, equating to acquisition of ~40 frames per second at 512 x 512 pixel resolution), and the focus compensation mechanism tracks the bottom of the cell imaging chamber to compensate for any focus drift during imaging.

2: Human skin fibroblast cells were stained for Golgi, actin, lysosomes and nuclei. Z-stacks of images were captured on the Leica TCS SP8X Confocal Microscope. Individual z-stacks were processed by maximum projecting them then all channels combined to produce the merged image. Images were processed using FIJI software.

3:Tg(Thy1-GFP)Mmouse hippocampus was fixed and the refractive index matched using CUBIC. Z-stack images were captured on the Leica TCS SP8X Confocal Microscope. Neurons are represented in green. Refractive index matching allowed the user to capture 250 um thick dataset on a single-photon microscope. Images were processed using FIJI software.

4:Tg(Thy1-GFP)Mmouse brain was imaged forneurons (green) and collagen (red) using the Leica TCS SP8MP multiphoton microscope. Collagen was visualized using Second Harmonic Generation, a visualization technique without the need for staining. Images were processed using FIJI software.

Nikon Live Imaging Microscope

The Nikon Ti-E inverted microscope provides live ratiometric imaging, and is equipped with a high-speed cooled EMCCD camera (capable of imaging up to 60 frames per second at 512 x 512 pixel resolution) coupled with a fast-triggered filter wheel, and a fast-switching LED fluorescence light source. This combination permits fast fluorescence image acquisition for ratiometric analysis. Filter sets available for use are: CFP/YFP, GFP/RFP, and BCECF. The microscope is also equipped with a stage-top incubator to maintain cell viability during time-lapse imaging experiments. The Nikon Perfect Focus system automatically maintains the focus on the cells of interest by sensing the bottom of the imaging chambers, and corrects for focus drifting over time.

5: Nikon Ti-E ratiometric imaging microscope

Zeiss Axio Fluorescence Microscope

The Zeiss Axio upright fluorescence microscope is used to image fluorescently-labelled slides. It is equipped with an LED fluorescence light source, four fluorescence filters (DAPI, FITC, Cy3 and Cy5), and a camera.

6: Zeiss Axio upright fluorescence microscope 

7: HeLa cells were stained for nuclei (blue), microtubules (green) and lysosomes (red). Images for each channel were acquired on the Zeiss Axio upright fluorescence microscope. Images were processed using FIJI software.

8: HeLa cells were stained for nuclei (blue), golgi (green), endoplasmic reticulum (red) and lysosomes (magenta). Images for each channel were acquired on the Zeiss Axio upright fluorescence microscope. Images were processed using FIJI software.

Training

All users require training before operating these instruments autonomously. Only trained users are permitted to book the microscopes on PPMS.

SAHMRI users should register their training requests on PPMS:

Leica confocal microscope https://au.ppms.info/sahmri/req/?pf=7&training=tr...

Nikon ratiometric imaging microscope https://au.ppms.info/sahmri/req/?pf=7&training=tr...

Zeiss fluorescence microscope https://au.ppms.info/sahmri/req/?pf=7&training=tr...

PPMS user accounts can be requested at https://au.ppms.info/sahmri/areq/?pf=7.

Non-SAHMRI users should contact the Facility Manager to discuss training and booking arrangements.

Standard Fee Structure

Confocal microscope

$58.00/hour peak hours (8.00 am to 6.00 pm Monday to Friday)

$40.00/hour off-peak hours (6.00 pm to 8.00 am Monday to Friday, weekends and public holidays)*

Nikon Live Imaging Microscope

This microscope is available for use on Mondays and Tuesdays only unless prior arrangements are made. Please contact Dr. Pirjo Apaja (pirjo.apaja@sahmri.com) if use outside these days is required.

$40.00/hour peak hours (8.00 am to 6.00 pm Monday and Tuesday)

$30.00/hour off-peak hours (6.00 pm to 8.00 am Monday, Tuesday, weekends and public holidays)*

Zeiss Axio Upright Fluorescence Microscope

$10.00 per hour (peak/off-peak/weekends/public holidays)*

Discounts are possible for long-term time block bookings. Non-standard fees apply to commercial use. Please contact the Facility Manager for further details.

*: off-peak/weekends/public holiday use available to SAHMRI users only

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