SUPER RESOLUTION

Structured Illumination Microscopy (SIM)

SIM is an imaging technique that employs patterned excitation light to generate interference patterns (moiré patterns) that contain high frequency spatial information. By collecting a series of images with different interference patterns, specialized algorithms are able to extract super-resolution detail to produce a reconstructed final image that has approximately twice the resolution of traditional diffraction limited microscopes, ie. approx. 110 nm (XY), 300 nm (Z). This technique is ideally suited for 3D imaging of thin (<20 um), fixed samples that have excellent fluorescence labeling.

System name: Zeiss Elyra PS1

Objectives

  • 63x/1.4 PlanApo
  • 100x/1.46 alpha PlanApo

Camera

  • Andor iXon3 885 (SIM)

Lasers

  • 405 nm (150 mW)
  • 488 nm (200 mW)
  • 561 nm (200 mW)
  • 640 nm (150 mW)

Localization Microscopy (STORM/PALM)

Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) function by stochastically cycling fluorophores between ‘on’ and ‘off’ states in a controlled manner, followed by precise localization of the centre of each fluorescent molecule to within tens of nanometers. By processing thousands of images, the molecular coordinates of all labeled molecules are obtained with a resolution ten times better than traditional diffraction limited microscopes, i.e. approximately 20 nm (XY), 50 nm (Z). This technique is best suited for imaging the stoichiometric architecture of proteins within defined complexes, and is largely limited to fixed, cellular samples.

System Name: Zeiss Elyra PS1

Objectives

  • 63x/1.4 PlanApo
  • 100x/1.46 alpha PlanApo

Cameras

  • Andor iXon DU897 (PALM)

Lasers

  • 405 nm (150 mW)
  • 488 nm (200 mW)
  • 561 nm (200 mW)
  • 640 nm (150 mW)

Other

  • 3D: double-helix PSF

Stimulated Emission Depletion (STED)

STED microscopy overcomes the diffraction limit by overlapping the primary excitation laser with a secondary, donut-shaped, high-power laser beam. Irradiation by the secondary laser forces molecules in the outer region of the primary excitation area to return to the ground state, while leaving fluorophores in the centre of the excitation focus to form the high resolution image. This application can achieve resolutions up to 50 nm laterally, and up to 120 nm axially, and is best suited for 2D or 3D imaging of thin samples (<50 um) that have excellent fluorescence labeling.

System Name: Leica SP8 Lightning Confocal/STED

Objectives

  • 93x/1.3 STED (motCORR)
  • 100x/1.4 STED

Lasers

  • 405 nm
  • white light laser (470-670 nm)
  • argon (458/476/488/496/514 nm)
  • 592 nm (STED)
  • 660 nm (STED)
  • 775 nm (pulsed, STED)

Other

  • Resonance scanner

CONFOCAL

Confocal microscopy is a powerful technique in which a pinhole is used to block out-of-focus light in image formation, thereby generating improved contrast and resolution compared to widefield microscopes. Generally speaking, there are two types of confocal microscopes: point scanning and spinning disk. Although slower than a spinning disk confocal, point scanning confocal microscopes tend to achieve higher resolutions, and have more integrated solutions for specialized techniques such as fluorescence recovery after photobleaching (FRAP). On the other hand, spinning disk confocals tend to have significant advantages in both speed and phototoxicity, making them ideally suited for live cell imaging.

In recent years, the resolution of traditional point scanning confocal microscopes has been extended to the super-resolution realm by implementing three key components to a workflow:

  1. Closing the pinhole diameter below 1 Airy Unit,
  2. Using low-noise, high-sensitivity detectors,
  3. Combining pixel oversampling with modern deconvolution algorithms.

Taken together, these implementations can improve resolution by up to 40% compared to traditional confocal microscopes, i.e. 120 nm (XY), 400 nm (Z), and can be applied to most standard confocal applications.

A specialized application of confocal microscopy is multiphoton imaging. In this technique, a specialized infrared laser is used to selectively excite molecules located within a femtoliter volume of space, up to one millimetre deep within a sample. The primary advantage of multi photon imaging is reduced phytotoxicity and deep sample penetration, making it ideal for intravital microscopy.

Point scanning confocals

System Name: Leica SP8 Lightning Confocal/Light Sheet

Objectives

  • 5x/0.15
  • 20x/0.75
  • 40x/1.3 (motCORR)
  • 63x/1.3

Lasers

  • 405 nm
  • 448 nm
  • 488 nm
  • 552 nm
  • 638 nm

Detectors

  • 4x HyD, 1x PMT, resonance scanner

System name: Leica SP8 Lightning Confocal/STED

Objectives

  • 10x/1.4
  • 20x/0.75
  • 40x/1.3
  • 63x/1.4

Camera

  • Andor iXon3 885 (SIM)

Lasers

  • 405 nm
  • white light laser (470-670 nm)
  • argon (458/476/488/496/514 nm)

Detectors

  • 2x HyD, 3x PMT, resonance scanner

System Name: Zeiss LSM880 Airyscan

Objectives

  • 5x/0.16
  • 10x/0.45
  • 20x/0.8 PlanApo
  • 40x/1.1
  • 63x/1.4 PlanApo

Lasers

  • 405 nm
  • argon (458/476/488/496/514 nm)
  • 552 nm
  • 642 nm

Other

  •  32-channel spectral PMT, Airyscan Fast

System Name: Nikon A1R Confocal Microscope w/FCS + FLIM

Objectives

  • 10x/0.5
  • 20x/0.75
  • 40x/1.25
  • 60x/1.4
  • 100x/1.4

Lasers

  • 402 nm
  • 440 nm (pulsed)
  • 488 nm
  • 561 nm

Other

  • 2 x GaAsP PMT, HD resonance scanner, 32-channel spectral PMT

Spinning disk confocals

System Name: Quorum Spinning Disk 1

Objectives

  • 10x/0.25
  • 20x/0.75
  • 25x/0.8
  • 40x/1.2
  • 63x/1.4
  • 100x/1.4

Lasers

  • 405 nm
  • 491 nm
  • 561 nm
  • 640 nm

Camera

  • Hamamatsu C9100-13 EM-CCD

System Name: Quorum Spinning Disk 2

Objectives

  • 10x/0.4
  • 20x/0.75
  • 40x/0.9
  • 60x/1.27
  • 60x/1.35

Lasers

  • 405 nm
  • 491 nm
  • 561 nm
  • 642 nm

Camera

  • Hamamatsu C9100-13 EM-CCD

System Name: Quorum Spinning Disk 3

Objectives

  • 10x/0.4
  • 20x/0.8
  • 40x/1.1
  • 3
  • 63x/1.4

Lasers

  • 405 nm
  • 491 nm
  • 561 nm
  • 637 nm

Camera

  • Hamamatsu C9100-13 EM-CCD, Photometrics Prime 95B

System Name: Quorum Spinning Disk 4

Objectives

  • 4x/0.16
  • 10x/0.4
  • 20x/0.75
  • 40x/0.95
  • 60x/1.35
  • 100x/1.4

Lasers

  • 405 nm
  • 491 nm
  • 561 nm
  • 642 nm

Camera

  • Hamamatsu C9100-13 EM-CCD

Multiphoton imaging

System Name: Leica SP8 Confocal w/Two-Photon

Objective

  • 25x/0.95

Detectors

  • 1 x HyD, 2 x PMT, 2 x NDD-HyD

Lasers

  • Argon (458/476/488/496/515 nm)
  • 561 nm
  • 633 nm
  • Coherent Ultra II (680-1080 nm, >3.3 W)

WIDEFIELD

Widefield imaging is the method of choice for basic fluorescence imaging. It is most often applied to examining protein expression levels in cells and tissue, general patterns of localization, and population studies. It is especially powerful for high-speed imaging, as well as ratiometric measurements of intracellular pH, or other ion levels. Finally, it is the tool of choice for researchers looking to image cellular samples with white light (eg. DIC imaging of wound assays, beating cilia, etc.).

System Name: Zeiss Epifluorescence Microscope

Objectives

  • 5x/0.15
  • 10x/0.5
  • 20x/0.75
  • 40x/1.3
  • 60x/1.4
  • 100x/1.4

Camera

  • Hamamatsu Flash 4.0 v2 sCMOS

Excitation Filters

  • 340/25
  • 380/25
  • 377/50
  • 436/20
  • 472/30
  • 481/15
  • 500/20
  • 531/40
  • 640/20

Emission Filters

  • 430/25
  • 480/40
  • 500/20
  • 520/35
  • 535/30
  • 560/25
  • 610/60
  • 647/75

Dichroics

System Name: Nikon Epifluorescence Microscope

Objectives

  • 4x/0.13
  • 10x/0.3
  • 20x/0.5
  • 40x/0.6
  • 60x/1.4

Camera

  • Hamamatsu Orca R2
  • Micropublisher 5.0 RTV

OTHER

Light sheet fluorescence microscopy

In light sheet imaging, samples are illuminated perpendicularly to the direction of observation using a thin sheet of light that is projected through the sample. Although the lateral resolution of a light sheet microscope is comparable to a widefield microscope, its key advantages are greatly reduced phototoxicity, rapid imaging, and the ability to image large samples from multiple directions for increased clarity and resolution. This technique has been masterfully adapted for use with cleared tissues, such as brain, kidney, and other organs measuring up to several millimetres in thickness

System Name: Zeiss Light Sheet Z1

Illumination Objectives

  • 5x/0.1
  • 10x/0.2

Detection Objectives

  • 5x/0.16
  • 10x/0.5
  • 20x/1.0
  • 20x/1.0 (CLARITY, RI=1.45)

Lasers

  • 405 nm
  • 488 nm
  • 561 nm
  • 638 nm

Cameras

pCO Edge 5.5 x 2

System Name: Leica SP8 Lightning Confocal/Light Sheet

Illumination Objectives

  • 6x/0.05
  • 5x/0.07
  • 5x/0.15

Detection Objectives

  • 5x/0.15
  • 10x/0.3
  • 25x/0.95

TwinFlect Mirrors

  • 5 mm
  • 5 mm
  • 8 mm
  • 8 mm (glycerol)

Lasers

  • 405 nm
  • 448 nm
  • 488 nm
  • 552 nm
  • 638 nm

Detectors

  •  pCO Edge 5

Fluorescence Lifetime Imaging (FLIM)

FLIM is an excellent technique for mapping the local environment and molecular interactions of a fluorophore. A fluorophore’s lifetime (ie. the amount of time spent in the excited state) is a quantitative signature that can be used to extract information about its surrounding environment: ion concentrations, pH, viscosity, and molecular interactions. Furthermore, because fluorescence lifetime measurements are not affected by probe concentrations or photobleaching, and are independent of excitation, FLIM is an ideal tool for measuring FRET in living cells.

System Name: Nikon A1R Confocal Microscope w/FCS + FLIM

Objectives

  • 10x/0.5
  • 20x/0.75
  • 40x/1.25
  • 60x/1.4
  • 100x/1.4

Lasers

  • 440 nm (pulsed)

Other

  • PicoQuant TCSPC
  • PicoQuant hybrid PMT x 2 (520/35, 624/40)

System Name: Olympus cellTIRF/Lambert FLIM

Objectives

  • 4x/0.16
  • 20x/0.75
  • 40x/0.95
  • 60x/1.49
  • 150x/1.45

Camera

  • Li2CAM iCCD (w/GenIII GaAs intensifier)

LED

  • 402 nm
  • 446 nm
  • 483 nm
  • 540 nm

Fluorescence Correlation Spectroscopy (FCS)

FCS is a correlative analysis of fluorescence fluctuations over time within a defined volume. As fluorophores enter and exit the observed volume, the resulting fluctuations in intensity encode parameters such as diffusion rates, molecular volume, binding affinities, and colocalization. This tool is especially useful for measuring the mobility of rapidly diffusing cytosolic proteins.

System Name: Nikon A1R Confocal Microscope w/FCS + FLIM

Objectives

  • 10x/0.5
  • 20x/0.75
  • 40x/1.25
  • 60x/1.4
  • 100x/1.4

Lasers

  • 440 nm (pulsed)

Other

  • PicoQuant TCSPC
  • PicoQuant hybrid PMT x 2 (520/35, 624/40)

Total internal reflection fluorescence (TIRF)

TIRF microscopy is a specialized technique that allows for imaging of fluorescent molecules located within 200 nm of the coverslip. It accomplishes this by the generation of a fluorescent evanescent wave that decays exponentially as it projects upward from the surface of the coverslip. In doing so, only fluorophores located 100-200 nm from the coverslip are excited, resulting in very high signal-to-noise imaging of the basolateral surface of cells. This technique is especially useful for observing membrane associated processes such as ligand binding, cell adhesion, and vesicle trafficking from the cell surface.

System Name: Olympus cellTIRF/Lambert FLIM

Objectives

  • 4x/0.16
  • 20x/0.75
  • 40x/0.95
  • 60x/1.49
  • 150x/1.45

Camera

  • Hamamatsu C9100-13 EMCCD

Lasers

  • 491 nm
  • 561 nm

Automated slide scanning

Digital slide scanning is the ideal solution for imaging whole tissue sections. Up to 250 slides can be automatically scanned in a high-throughput fashion using high-resolution 20x and 40x objectives, in both brightfield and fluorescence modes. The resulting images can be analyzed to provide area quantification, complex colour analysis, cytonuclear quantitation, etc. This technology is featured as a service in the Facility, with the turnaround typically being 24 hours.

System Name: 3DHistech Pannoramic Flash II Slide Scanner

Objectives

  • 20x/0.8
  • 40x/0.95

Cameras

  • pCO Edge 4.2 sCMOS
  • CIS VCC-FC60FR19CL

Fluororescence Filter Cubes

  • DAPI
  • GFP
  • Cy3
  • Cy5
  • Quad cube

All Imaging Facility equipment can be booked through QReserve.