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Acquired in 2024, funding for this instrument was provided by Canada Foundation for Innovation (Biosciences Research Infrastructure Fund) and ß÷ßäÉçÇø Government infrastructure awards to Dr. Tom Hobman.

STELLARIS 8 is a true confocal point scanning system, including a White Light Laser as excitation light source, an Acousto Optical Beam Splitter (AOBS) and a highly sensitive, prism-based spectral detection design with computer controlled adjustable bandwidth for all fluorescence channels. STELLARIS 8 offers enhanced detection efficiency. The extended detection range up to 850 nm plus the expanded excitation range in the visible from 440 nm up to 790 nm allow the application and separation of an extended range of spectrally overlapping fluorophores, up to five simultaneously. Including TauSense, a set of tools based on fluorescence lifetime information, it provides an additional contrast, improved image quality and separation of spectrally overlapping flurophores.

LIGHTNING allows for optimal extraction of image details and maximum resolution, thus expanding the imaging portfolio both in the classical range and beyond the diffraction limit.

Features a tandem scanning system to provide highest axial resolution with galvanometric scanner, switchable between resonant and non-resonant mode.

Equipped with Power HyD S detector, new standard for detection. All-round detector, high performance throughout the spectrum, two times increased photon detection efficiency in the blue-green range compared to standard multialkaline photomultiplier. Leica Si-based technology is compatible with TauSense in combination with the white light laser.

Includes Power Hybrid Detector HyD X: Specialized for Tau-STED in the visible up to far-red (depending on configuration). Leica GaAsp-based technology.
Compatible with TauSense technology in combination with the white light laser.

Inverted research microscope with touch screen based on the DMi8 series, with motorized 6x nosepiece, closed loop, cooling system and spill protection. Quick switching from coarse to fine drive. Prepared for confocal imaging with laser safety equipment.

Based around a Leica inverted DMI8 CS Premium inverted microscope base, our Stellaris 8 has the following configuration:

• Lenses: 10x/0.4 NA, 20x/0.75 NA, 40x/1.3 NA Oil, 40x/1.25 NA Glyc.(motorized), 63x 1.4 NA Oil, 63x/1.2 Water (motorized, 0.22 mm WD)
• Transmitted light modes: BF, Pol, DIC
• 6 detectors, 3 HyD S and 2 HyD X detectors, each equipped with spectral detection and tau sense detection, transmitted light PMT.
• 405 nm Diode laser
• White Light Laser (440-790nm in 1nm increments)
• Acousto-Optical Beam Splitter
• Tunable Filters
• Z Super Galvo stage
• TokaiHit stage top incubation system
• LED Fluorescence lamp
• AFC Hardware autofocus
• Resonant scanner
• LAS X Software with Dye Finder, Navigator and Lightning

Acquired in 2010, thanks to a CFI grant to the CEGIIR at the ß÷ßäÉçÇø. Based around a Leica inverted DMI 6000 B microscope base, our SP5 has the following configuration:

• Lenses: 10X/0.3, 20X/0.5, 40X/1.25 Oil, 60X/1.2 Water, 100X/1.44 Oil
• 5 detectors, 3 standard PMTs and 2 HyD detectors, each equipped with spectral detection
• 405 nm Diode laser
• Argon ion laser (458 nm, 476 nm, 488 nm, 496 nm, 514 nm)
• Green HeNe (543 nm)
• Red HeNe (633 nm)
• Acousto-Optical Beam Splitter
• Z Galvo stage

Purchased from , this spinning disk confocal has the following configuration:

• Olympus IX-81 motorised microscope base
• Yokagawa (#1) spinning disk confocal scan-head
  • Sedat Dichroic with reflections at 405/491/561/640 for standard 4-colour imaging
• Lenses: 20X/0.85 Oil, 40X/1.3 Oil, 60X/1.42 Oil
• Illumination:
  • X-Cite-Mini+ LED Fluorescent Light Source for eyepiece visualization
  • LMM5 from Spectral Applied Research for laser merging.
  • 44mW 405nm pumped diode laser (for blue dyes, e.g. DAPI)
  • 50mW 491nm pumped diode laser (for green dyes, e.g. GFP, YFP, Alexa 488, FITC)
  • 50mW 561nm pumped diode laser (for red dyes, e.g. mCherry, Alexa 546, Cy3, TRITC)
  • 45mW 642nm pumped diode laser (for far-red dyes, e.g. Cy5, Alexa 633, Alexa 647)
• Filter Cubes for use with eyepieces (BRIGHTLINE filter sets from ):
• Emission Filters for use during Confocal Imaging: The system is fitted with two interchangeable filter wheels, one for imaging standard 4 colour (Blue, Green, Red, Far-Red) "fixed" samples, and one optimised for imaging fluorophores commonly found in "live" cell experiments. Note that with these emission filters, there are no excitation filters necessary, as the lasers provide single wavelength excitation. Additionally, there is a choice of three dichroic mirrors housed in the confocal scan unit. Most commonly used, the Sedat dichroic is compatible with 4 colour fixed imaging. The second choice is a dichroic that maximises the emission from GFP (EGFP), but at the expense of separating its emission from other fluorophores. Last is a dichroic that is designed for separating CFP from YFP.
  • "Fixed" filter wheel (for use with "fixed" samples)
    • Polariser (for DIC imaging)
    • DAPI (460/50)
    • GFP (515/30)
    • Cy3 (595/50)
    • Texas Red (620/60)
    • Cy5 (690/50)
  • "Live" filter wheel (for use with "live" samples)
    • 436/24
    • CFP (470/24)
    • GFP (520/40)
    • YFP (540/30)
    • RFP (595/50)
    • Cy5 (700/75)
• Imaging using a Hamamatsu Orca-Fusion BT Digital sCMOS
• Acquisition using Perkin Elmer's Volocity
• Chamlide TC-A Live Cell Chamber (37°C incubator + 5% CO2 atmosphere). Can accommodate either 18mm or 25mm diameter coverslips.
• ASI MS-2000 motorised XY stage with a piezo Z insert that has 100µm travel

Purchased from , this spinning disk confocal has the following configuration:

• Olympus IX-81 motorised microscope base
• Yokagawa CSU-X1 spinning disk confocal scan-head
  • Sedat Dichroic with reflections at 405/491/561/640 for standard 4-colour imaging
  • CFP dichroic with reflections at 440/491/561/640 for 4-colour imaging using CFP.
• Lenses: 20X/0.85 Oil, 40X/1.3 Oil, 60X/1.42 Oil, 100X/1.4 Oil
• Illumination:
  • from Lumen Dynamics for visualization with eyepieces
  • LMM5 from Spectral Applied Research for laser merging.
  • 44mW 405nm pumped diode laser (for blue dyes, e.g. DAPI)
  • 40mW 440nm pumped diode laser (for CFP)
  • 50mW 491nm pumped diode laser (for green dyes, e.g. GFP, YFP, Alexa 488, FITC)
  • 50mW 561nm pumped diode laser (for red dyes, e.g. mCherry, Alexa 546, Cy3, TRITC)
  • 45mW 642nm pumped diode laser (for far-red dyes, e.g. Cy5, Alexa 633, Alexa 647)
• Filter Cubes for use with eyepieces (BRIGHTLINE filter sets from ):
• Emission Filters for use during Confocal Imaging: The system is fitted with two interchangeable filter wheels, one for imaging standard 4 colour (Blue, Green, Red, Far-Red) "fixed" samples, and one optimised for imaging fluorophores commonly found in "live" cell experiments. Note that with these emission filters, there are no excitation filters necessary, as the lasers provide single wavelength excitation. Additionally, there is a choice of three dichroic mirrors housed in the confocal scan unit. Most commonly used, the Sedat dichroic is compatible with 4 colour fixed imaging. The second choice is a dichroic that maximises the emission from GFP (EGFP), but at the expense of separating its emission from other fluorophores. Last is a dichroic that is designed for separating CFP from YFP.
  • "Fixed" filter wheel (for use with "fixed" samples)
    • DAPI (460/50)
    • GFP (525/50)
    • TRITC (593/40)
    • Texas Red (620/60)
    • RFP (650/100)
    • Cy5 (700/75)
  • "Live" filter wheel (for use with "live" samples)
    • CFP (470/40)
    • YFP (545/40)
    • DsRed (593/40)
    • mCherry (641/75)
    • Cy5 (700/75)
    • Empty position
• Imaging using a Hamamatsu EMCCD (C9100-13)
• Acquisition using Perkin Elmer's Volocity
• Chamlide TC-A Live Cell Chamber (37°C incubator + 5% CO2 atmosphere). Can accommodate either 18mm or 25mm diameter coverslips.
• ASI MS-2000 motorised XY stage with a piezo Z insert that has 100µm travel.

Funding for this instrument was provided by Canada Foundation for Innovation and ß÷ßäÉçÇø Government infrastructure awards to Dr. Michael Hendzel.

With Lattice SIM, the sample area is illuminated with a lattice spot pattern instead of grid lines as in conventional SIM. This leads to a dramatic increase in imaging speed. In addition, the lattice pattern provides higher contrast to allow a more robust image reconstruction. Since the sampling efficiency of lattice pattern illumination is 2× higher compared to classic SIM, you need less laser dosage for sample illumination. This lattice illumination makes SIM a preferred live cell imaging technique. The strongly improved photon efficiency of lattice illumination allows you to increase the imaging speed while achieving higher contrast and lower photo dosage.

• High-powered CW lasers: 405nm, 488nm, 561nm, 642nm
• Incubation chamber for adjustable temperature, humidity, and CO2 control.
• Lenses: 63x/1.4 Oil for structured illumination, 63x/1.46 Oil for TIRF and SMLM, 63x/1.2 Water for SIM on thick / highly scattering samples, 40x/1.3 Oil for SIM Apotome mode/thick samples, 25x/0.8 LD multi-immersion lens with correction collar, 5x/0.16 Dry.
• 2 Duolinked sCMOS cameras for fast, 2-channel simultaneous acquisition (light is separated by a set of dichroics = no moving parts). Can also acquire 4 channels sequentially.
• Burst mode processing allows for SIM imaging up to 255 fps for 2D short-duration but very fast time lapse acquisition.
• Acquisition in Leap mode accelerates the volume (z) imaging speed by three times and at the same time decreases the light dosage on your sample.

Applied Precision's OMX super resolution microscope that uses structured illumination to break the diffraction barrier.

• Lasers: 405, 488, 561, 642
• Solid state conventional illumination for: DAPI, CFP, Alexa488, YFP, mCherry, Alexa568, Alexa633, Alexa647, Cy5
• Lenses: 60X/1.42 Oil for structured illumination or deconvolution, 60X/1.49 for TIRF
• RING TIRF
• 3X sCMOS cameras to simultaneously acquire 3 channels (light is separated by a set of dichroics, no moving parts). Can also be used to acquire 4 channels sequentially.
• Two modes of microscopy possible — either structured illumination or fast live cell imaging. Fast live imaging means 10+ stacks per second — dependent on the thickness of your sample and the exposure time required to image your signal.
• 37°C incubation and 5% CO2

Zeiss AxioObserver.Z1

• z motor focus with 25nm step
• Brightfield, phase contrast, DIC transmitted techniques in motorized condenser
• 6x motorized fluorescence filter turret with DAPI/BFP, AF488/GFP, Rhod/Cy3, Cy5, DIC analyzer cube, and QUAD cube for fast DAPI/GFP/Cy3/Cy5
• Colibri LED light sources 365, 470, 555, and 625
• HXP120 metal halide source
• Motorized xy stage with holders for slides 3x1", dishes 35-60mm, and multiwell plates
• Axiocam HRm monochrome cooled CCD camera and Axiocam 105 color camera for imaging histological stains.

AxioVision imaging has MultiChannel overlay/merging, z-stack, timelapse, mark and find, Mosaix, deconvolution, 3d rendering, Apotome optical sectioning.

Objectives:
10x/0.3 Phase PN, 20x/0.4 Phase LD, 20x/0.8 PA, 40x/1.3 PA Oil, 63x/1.4 PA Oil, and 100x/1.3 PN Oil.

Based around a Leica inverted DMI 6000 microscope base, this microscope has the following features:

• 100X/1.49 Oil
• 40X/1.3 Oil
• 20X Dry
• 10X Dry
• 4X Dry

• for DAPI (360/40 excitation, 400 Dichroic, 470/40 emission)
• for AlexaFluor488 (470/40 excitation, 495 dichroic, 525/50 emission) (Also works for other common Green dyes, e.g. FITC, GFP)
• for AlexaFluor594 (560/40 excitation, 595 dichroic, 645/75 emission) (Also works for some common red dyes, e.g. AF568, DsRed, Cy3m TRITC, Texas Red)
• for AlexaFluor647 (620/60 excitation, 660 Dichroic, 700/75 emission) (Also works for other far-red dyes, e.g. Cy5, Atto647N)
• Triple B/G/R cube for fast acquisition ( 400/20; 495 /15; 570/20 excitation, 415; 510; 590 Dichroic, 465/20; 530 /30; 640/40 emission)

Funding for this instrument was provided by Canada Foundation for Innovation and ß÷ßäÉçÇø Government infrastructure awards to Dr. Meghan Riddell.

The Cell Discoverer 7 is a closed-box, inverted microscope that is ideal for live-cell imaging. The fully contained incubation system allows for stable environmental control of temperature, humidity, and CO2 over long time periods. This microscope has motorized correction collars, a sample finder, and thickness detector, so it can automatically compensate for different sample holders (multi-well plates, slides, chambers etc.), sample thicknesses, and immersion media.

Illumination modes: Fluorescence and brightfield

Lenses: 5x/0.25NA (Dry, 5.1mm working distance), 20x/0.7NA (Dry, 2.2mm working distance), 50x/1.2NA (water immersion, 0.84mm working distance)

Optovar: 0.5x, 1x, 2x

Excitation wavelengths: Colibri light sources with the following LEDs:

• 385 (DAPI / Hoechst)
• 470 (GFP / AF488)
• 567 (TdTomato / AF568)
• 625 (Cy5 / AF647)

Emission Filters:

• 425/30
• 514/30
• 592/24
• 681/45

Camera: Axiocam 712 monochrome sCMOS

Other features:

• Automatically applied/removed objective (water) immersion.
• Sample finder, providing image of sample and holder and automatic focusing.
• Motorized correction collars, enhancing imaging in plastic well-plates (5x, and 20x only), and thick samples.
• Plate adapters for holding multi-well plates, 35mm petri dishes, 60mm petri dishes, standard slides (76x26 mm), and Lab-Tek chambers (57x26mm).
• Live cell incubation (CO2, humidity, and temperature control).
• Pipette insert port for adding solutions without disturbing environmental conditions.
• Separate analysis workstation for image processing, and deconvolution.

Funding for this instrument was generously provided by the Striving for Pandemic Preparedness ß÷ßäÉçÇø Research Consortium (SPP-ARC).

Historically, widefield microscopy has not been best suited for the imaging of large samples/specimen volumes. The image background, mainly originating from out-of-focus regions of the observed sample, significantly reduces the contrast, the effective dynamic range, and the maximal possible signal-to-noise ratio (SNR) of the imaging system. Instant Computational Clearing (ICC) is the core technology in THUNDER Imagers. It detects and removes the out-of-focus background for each image, making the signal of interest directly accessible. At the same time, in the in-focus area, edges, and intensity of the specimen features remain. In addition to ICC, the THUNDER microscope can perform an immediate post-processing, decision-mask- based 3D deconvolution dedicated to either thin samples (small volume computational cleaning - SVCC) or thick samples (large volume computational clearing - LVCC).

Illumination modes: Fluorescence, Brightfield, DIC, Polarized Light

Lenses: 2.5x/NA (Dry), 10x/0.3NA (Dry), 20x/0.75NA (multi-immersion), 40x/0.95NA (Dry), 63x/1.4NA (Oil immersion).

Excitation wavelengths: 8 discrete light sources with the following LEDs:

• 390 (DAPI / Hoechst)
• 440 (CFP)
• 475 (GFP / AF488)
• 510 (YFP)
• 555 (TdTomato / AF568)
• 575 (RFP / AF594)
• 635 (Cy5 / AF647)
• 747 (Cy7 / AF750)

Emission Filters:

• Quad bandpass filter cubes:
  • DFT5 (435/30, 519/25, 594/32, 695/58)
  • CYR7 (473/22, 539/24, 641/78, 810/80)

Camera: Leica K8 Monochrome sCMOS

Other features:

• Navigator function for fast whole specimen overview and tilescanning.
• THUNDER Live allows computationally cleared images to be viewed instantly in the live viewer to allow you to optimize ICC parameters by using immediate image feedback.
• Live cell imaging capabilities with various sample holder stage inserts for multi-well plates, 35mm petri dishes, 60mm petri dishes, standard slides (76x26 mm), as well as CO2, humidity, and temperature control.
• External emission filter wheel for “clean-up” of emission detection.
• Leica’s image analysis software (Aivia) available for image processing, analysis and segmentation workflow, to extract data from your images.

Funding for this instrument was generously provided by the University Hospital Foundation.

Creates a virtual slide image by digitizing the entire sample on the slide. The system works with both brightfield and fluorescence contrast, and can accommodate 100 standard sized slides at a time. User designed scan profiles enable unattended, automated scanning. Our system can digitise up to 5 fluorescence channels (e.g. DAPI, AF488, AF568, AF647 and AF750). Fluorescence images are captured on a state of the art Hitachi sCMOS camera (Orca Flash 4.0), while full colour images from histological colorimetric stains are captured by a progressive scan Hitachi HV-F202SCL 3X CCD camera. Fluorescence excitation is provided by an integrated, multi wavelength LED light source with excitation wavelengths of 385nm, 469nm, 555nm, 631nm and 735nm. Our system has 3 scanning lenses, 10X, 20X and 40X that give images with pixel sizes of 440nm, 220nm and 110nm, respectively. Shading correction as well as other post-acquisition processing and analysis is provided by a nearline image processing/analysis computer, optimised to view, interrogate, analyze and process the exceptionally large sized files (typical pixel dimensions: 100,000 X 100,000) that the slide scanner generates.

Acquired in 2021, funding for this instrument was provided by Canada Foundation for Innovation and ß÷ßäÉçÇø Government infrastructure awards to Dr. Maria Ioannou.

The LSM 900 Airyscan2 is a confocal microscope that is equipped with the second generation of the Airyscan2 detector allows Super/resolution Imaging ~120 nm at High Sensitivity through an area detector with 32 circularly arranged detection elements.

Apart from the Airyscan2 detector It is equipped with 2 conventional photomultiplier detectors and 405, 488, 561 and 640 nm diode lasers and 10x ,20x (Air) and 40x and 63x oil immersion lenses on a Zeiss Axis-Observer inverted microscope.

Since this microscope is heavily used by the Ioannou Lab, usage is only permitted for live-cell experiments that have been planned and discussed in advance with the CIC team.

Funding for this instrument was provided through an ß÷ßäÉçÇø Cancer Foundation Game Changers Award to Dr. Kris Baker.

The Akoya PhenoCycler-Fusion 2.0 that combines the PhenoCycler in combination with the PhenoImager cutting-edge tool system for studying cells in tissues, offering powerful insights for medical research. The PhenoCycler-Fusion 2.0 maps millions of cells at once, using up to 100+ biomarkers to identify and characterize cell types and their interactions with high precision. It combines fast fluidics (PhenoCycler) and sub cellular imaging (Pheno Imager) to analyze entire tissue samples quickly highlighting specific cells and biomarkers to reveal how they are involved in the development of diseases like cancer or immune disorders.

For the Faculty of Medicine and Dentistry, these tools are game-changers. They provide a research platform to explore complex diseases at a cellular level using a high number of biomarkers simultaneously, uncovering potential treatment targets.

The LSCM uses a point light source (laser) and a pinhole to reject out of focus light. It enables optical sectioning of fluorescently stained samples to provide a 3-Dimensional view of the structure. For more information on confocal microscopy, please go to resource section.

Funding for this instrument was provided through an ß÷ßäÉçÇø Cancer Foundation award to Dr. Joan Turner.

Applications

1. To examine fluorescently labeled specimens for single or multiple colors localization of molecules within cell/tissue.
2. To carry out physiological studies such as ratio imaging, time-lapse recording of live cell/tissue.
3. Photo manipulation of the fluorescence signal (e.g. FRAP (photo bleaching after fluorescence recovering); FLIP (Fluorescence Loss Imaging after photobleaching), photoactivation/switching, etc.) to study the kinetics of molecules in living cells.

This system is mainly used for detection of fluorescence labeling in fixed samples. However, it is also equipped with necessary accessories (temperature, CO₂ controls, O₂) for live cell imaging. Additionally, the system is equipped with an FCS module which also allows imaging using the sensitive Avalance Photodiode Detector (APD, Zeiss Confocor 3, see FCS section below).

The instrument is equipped with a spectral detector which uses a PMT (Photon Multiplier Tube) array and optical grating elements to collect spectral information for fluorophores. Spectral information from 405nm to 720nm with step size of 10.7nm can be detected with the setup. Then, using the built-in, non-linear un-mixing tool, signals from different fluorophores can be separated mathematically. The system eliminates the need for emission filter and offers the advantage of being able to separate fluorophores with extensive emission spectra overlap. For example, GFP and YFP can be separated using this setup (which is impossible with conventional filter based imaging system). The system is also particularly useful for imaging highly auto-fluorescence sample with fluorescence imaging as auto-fluorescence has very different spectral signature than fluorophores.

Zeiss LSM 710 Specifications

Microscope: Zeiss AxioObserver (inverted)

Objectives:

• 10x 0.45NA EC Plan-Neofluar
• 20x 0.8NA Plan-Apochromat
• 40x 1.3 oil plan-Apochromat
• 40x 1.2NA Water C-Apochromat
• 63x 1.4NA Oil DIC Plan-Apochromat

Laser Lines:

• Diode 405nm
• Diode 440nm
• Argon: 458, 488, 514 nm
• Solid state: 561 nm
• HeNe: 633 nm

Detectors:

• 3 fluorescence PMTs
• 1 transmission PMT
• 2 APDs

This is a complex system with multiple capabilities:

1. Super resolution microscope with STED (STimulated Emission depletion) which is capable of obtaining fluorescence images with resolution down to <50nm.
2. Multiphoton microscope with tunable laser from 680-1064nm.
3. Fluorescence Lifetime Imaging system based on time domain for FLIM measurements.
4. Fluorescence correlative Spectroscopy system for detecting molecular associations.
5. Combination of optics and software module capable of obtaining fluorescence images down to ~120nm.

STED uses a donut shaped depletion laser to modify the point spread function of the beam down to sub-diffraction limit level to achieve super resolution microscopy at resolution below 50nm . The system is equipped with 594nm, 660nm and 775nm depletion lasers for variety of dyes. For detailed system information, please visit the .

Multi-photon microscope uses an ultra-fast pulsing laser to excite fluorescent molecules. Briefly, 2 (multi)-photon microscopy is based on the principle that a given fluorescent molecules which normally would only be excited by absorbing the energy of a single photon of certain wavelength can also be excited by combination of the energies produced by simultaneous absorption of 2 (or multi-) lower-energy photons of double (multiple) wavelength. The emission of the fluorescence is quadratically dependent on the excitation intensity. This steep dependence of absorption rate on photon concentration gives multi-photon Laser Scanning microscope the intrinsic three-dimensional resolution with the depth of field defined by:

1. intensity of the excitation light
2. numerical aperture of the lens used
3. the wavelengths of the lights.

This z-resolution is comparable to conventional Laser Scanning confocal microscope.

Funding for this instrument was provided through an Canada Foundation for Innovation award to Dr. Michael Hendzel.

Advantages over confocal microscope

1. Longer wavelength used for excitation allows imaging deeper into thick specimens due to less scattering of longer wavelength (IR) light.
2. Photo-bleaching and damaging are confined to the focus points only.
3. It is possible to image UV dyes with infra-red light. This is particularly useful for live cell imaging where UV light is highly damaging to the cells

Therefore, multi-photon microscopy is highly suitable for imaging of live, thick specimens.
The system is also capable of measuring fluorescence life time with the equipped pulse lasers. The system is equipped with a white laser which is pulsed and could be tuned continuously from 470-690nm in addition to the IR laser with tuning range of 680-1024nm. In addition, the system is able to perform Fluorescence correlative spectroscopy, deconvolution and high speed imaging.

In the below image, COS cells were stained with an anti-nuclear pore protein conjugated to Star635P dye and imaged with Confocal (top) and STED (bottom) at the same optical plane to demonstrate the improvement in resolution with STED (scale bar 2um).

Leica Falcon SP8 STED system with Spectra-physics femto-second laser Specifications

Microscope: Leica SP8 (inverted)

Objectives:

• 5x 0.15NA HC Plan-Fluotar
• 10x 0.4 NA HC Plan Apo
• 25x 0.95NA Water HC Fluotar
• 86x 1.2NA Water HC Plan-Apo
• 100x 1.4NA Oil Plan-Apo

Laser Lines:

• Ti:Saphire: Dual beams with fixed at 1064nm and tunable 680-1064nm
• White Laser II: continuously adjustable wavelength from 470 to 690nm
• Argon: 458, 488, 514nm
• Diod: 405nm

Detectors:

• 2 PMTs
• 3 Hybrid detectors
• 1 transmission PMT

The PerkinElmer UltraVIEW system (PerkinElmer Life Sciences Inc., MA, USA) is a Yokogawa (Yokogawa Corp. Japan) Nipkow spinning disk confocal system. It uses a spinning disk with multiple pinholes to achieve confocality (e.g. the rejection of out-of-focus lights). Nipkow disk refers to scanning disk with symmetrically placed spirals of pinholes through which illumination light is passed. Such pinholes split illumination lights into multiple 'minibeams'. When the disk spins, the light scans the sample in a raster pattern. Emission lights from the sample are detected through the pinhole to generate a confocal image of the sample that can be detected (with an EMCCD (Electron Multiplification Charge-Coupled Device) camera). Because the pinholes on a Nipkow disk must be placed up to 10 diameters apart in order to avoid cross-talking problem, the light throughput of traditional Nipkow disks is only ~1% of the light shining onto the disk. The Yokogawa scanhead has overcome this problem by using an innovative, collector disk containing microlenses placed in front of the Nipkow disk. The microlenses ensure that most of the light illuminating the disk is focused onto the pinholes. Transmission efficiency is thus increased from ~1% to 70% of the light falling on the disks allowing the sample to be illuminated with a sufficient quantity of light.

Funding for this instrument was provided through Canada Foundation for Innovation awards to Dr. Gordon Chan.

Advantages of the UltraVIEW system

The system has mainly 2 advantages over a conventional Laser Scanning confocal microscope:

1. Higher image speed
2. Lower photo-toxicity

The Laser Scanning Confocal Microscopy (LSCM) is a sequential scanning system where a single point of the specimen is illuminated at a time and LSCM uses a point detector (Photon Multiplier Tube (PMT)). Therefore, for LSCM, an expensive scanner is needed and electronic processing is necessary for image formation. The LSCM system is consequently relatively slow (typically 0.5-10sec/frame, slower scan can take up to a minute/frame). Whereas Nipkow disk system uses multi-point scanning disk and a cooled CCD camera which is a parallel array detector. This enables the Nipkow system to acquire images at higher speed (up to 360frames/sec) than with LSCM (typically 0.5-1 frame/sec). This will allow many rapid cellular processes (e.g. calcium imaging, vesicular trafficking) to be monitored in real time. More importantly, it has been demonstrated that the spinning disk confocal system reduces phototoxicity and photobleaching by as much as 5 folds comparing to a LSCM. It is speculated that the reduction in phototoxicity and photobleaching is probably due to the fact that the system splits the laser light into thousands of minibeams.

While the mechanism of such reduction in phototoxicity and photobleaching is still a subject of studying, spinning disk confocal is becoming increasingly the instrument of choice for live cell imaging. The system is particularly powerful for applications such as real-time (4-D) confocal microscopy, calcium signaling, vesicular trafficking; green fluorescence protein studies.

System Description

The system is based on a Zeiss Axiovert 200M inverted microscope with the following laser lines for excitation of fluorophores: 404nm, 440nm, 488, 514nm, 561nm and 633nm. It is equipped with a piezo focusing motor and all necessary accessories (Temperature, CO₂) for high speed live cell imaging. The scope is mounted with a high precision motorized stage for monitoring of multiple positions in a single sample holder. This system was upgraded with a FRAP (Fluorescence Recovery After Photobleaching) module in 2009.

Applications

The system is well suited for high-speed, live cell imaging with reduced phototoxicity. The system is particularly useful for 3-D timelapse to monitor fast cellular events because of the high focusing/acquisition speed than other systems in the Facility. With its FRAP module, FLIP, FRAP and photoactivation could be performed on the system as well.

Perkin Elmer Ultraview ERS FRAP Specifications

Microscope: Zeiss Axiovert 200M (inverted)

Objectives:

• 10x 0.3NA EC Plan-Neofluar
• 20x 0.8NA Plan Fluar
• 40x 1.3NA oil Plan-Neofluar
• 63x 1.2NA Water C-Apochromat
• 63x 1.4NA Oil DIC Plan-Apochromat
• 100X 1.4NA, oil DIC plan-Apochromat

Laser Lines:

• Diode: 405nm
• Diode: 440nm
• Argon: 458, 488, 514nm
• Solid state: 561nm
• Solid state 633nm

Detectors: Hamamatsu EMCCD 1Kx1K 8um pixel

Funding for this instrument was provided through a Canada Foundation for Innovation award to Dr. Sue-Ann Mok. The Cytation C10 confocal imaging reader combines automated confocal and widefield microscopy with conventional multimode microplate reading in a unique, proprietary design. The automated water-immersion objectives capture more light, driving lower exposure times and reducing phototoxic impacts on live cells.

The spinning-disk confocal module and accompanying disk pinhole options allow a researcher to look deeper into thick sample biology with improved clarity and detail. High-quality components used in the system, including a Hamamatsu scientific CMOS (sCMOS) camera, Olympus objectives and laser-based illumination enable excellent image quality.