Imaging Equipment

成像及流式細胞術設施

Confocal Microscope

LSM 800 with Airyscan1

LSM 800 is an inverted confocal setup equipped with 4 lasers (405 nm, 488 nm, 561 nm, 640nm) and Airyscan for sub-diffraction resolution to 140 nm. The microscope has 2 confocal detectors and a highly sensitive GaAsP detector, each of the detector’s wavelengths is tunable per nanometer. The motorized stage, advanced tiling module, photo-bleaching module enables the multi-dimensional (multi-channel, multi-position) imaging, photo-manipulation and big tissue tile scanning. Applications:

XY 2D / Z-stack 3D imaging
DIC/Phase contrast imaging
Tile scanning & stitch; multiple position imaging
Fluorescence recover after photobleaching (FRAP)

Detailed Configuration

Standard Operation Protocol – Carl Zeiss LSM800

Staff-in-charge: Ms WU, Vincci| 3917-9137 | imaging.cpos@hku.hk

LSM 880 with Airyscan1

The Zeiss LSM880 with Airyscan 1 is an inverted confocal microscope. The system is equipped with 7 laser lines 405nm, 458nm, 488nm, 514 nm, 561nm, 594nm and 633 nm. The newly designed excitation beam is elongated in y which acquires four lines of image information instead of only one with just one horizontal scanner movement. It offers high sensitivity, enhanced resolution in x, y and z, and high image-acquisition speed in one system and achieve a 1.7× higher resolution in all spatial dimensions, 120 nm laterally and 350 nm axially which allows fast biological processes with minimal phototoxicity. The stage-top live cell chamber, the motorized stage, advanced tiling module, photo-bleaching module enables the multi-dimensional (multi-channel, multi-position, time-lapse) live cell imaging, photo-manipulation and big tissue tile scanning.

Applications:

XY 2D / Z-stack 3D imaging, DIC/Phase contrast; Spot/line Scan;
Live cell imaging;
MPLX mode for fast biological processes tracking
Experiment Designer: Convenient for complex imaging routines set up
Tile scanning & stitch; multiple position imaging
Fluorescence recovery after photobleaching (FRAP), Fluorescence resonance energy transfer (FRET)

Detailed Configuration
Standard Operation Protocol – Carl Zeiss LSM880
Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

LSM 900 with Airyscan2

The LSM900 is an inverted confocal microscope with 4 solid state lasers (405nm, 488nm, 561nm, 640 nm) and Airyscan2 scanner for sub-diffraction resolution of 120 nm laterally and 350 nm axially. Airyscan2 in Multiplex mode can acquire up to four image lines with high SNR in a single sweep enabling unique combination of high speed, high resolution and high sensitivity. New ZEN software modules, including “Sample Navigator” and “Zen Connect” will simplify the imaging setup procedure. The stage-top live cell chamber, the motorized stage, advanced tiling module, photo-bleaching module enables the multi-dimensional (multi-channel, multi-position, time-lapse) live cell imaging, photo-manipulation and large tissue tile scanning.

Applications:

XY 2D / Z-stack 3D imaging, DIC/Phase contrast; Spot/line Scan;
Live cell imaging;
MPLX mode: Fast biological processes tracking
Tile scanning multiple position imaging.
Fluorescence recovery after photobleaching (FRAP), Fluorescence resonance energy transfer FRET
Sample Navigator: Good for whole specimen positioning;
ZEN Connect : Work with images from multiple sources;
Experiment Designer: Useful for complex imaging routines set up

Detailed Configuration
Standard Operation Protocol – Carl Zeiss LSM900
Staff-in-charge: Ms WU, Vincci| 3917-9137 | imaging.cpos@hku.hk

LSM 900 Inverted Confocal

The LSM900 inverted confocal microscope is equipped with four solid state lasers (405nm, 488nm, 561nm, 635 nm) and two PMT detectors. The motorized stage, advanced tiling module enables the multi-dimensional (multi-channel, multi-position) imaging and large tissue tile scanning.

Applications:

XY 2D / Z-stack 3D imaging.
Tile scanning & stitch.
Multiple position imaging.

Detailed Configuration
Standard Operation Protocol – LSM 900 Inverted Confocal
Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

LSM 900 Upright Confocal

LSM900 upright confocal is an upright confocal microscope equipped with 4 lasers (405 nm, 488 nm, 561 nm, 640nm), two PMT detectors and one transmitted light detector. The motorized stage, advanced tiling module enables the multi-dimensional (multi-channel, multi-position, time-lapse) imaging and big tissue tile scanning. ZEN software modules, including “Sample Navigator” and “Zen Connect” will simplify the imaging setup procedure.

Applications:

XY 2D / Z-stack 3D imaging.
DIC/Phase contrast imaging.
Tile scanning & stitch.
Multiple position imaging.
Sample Navigator: Good for whole specimen positioning.
ZEN Connect : Work with images from multiple sources.

Detailed Configuration
Standard Operation Protocol – LSM 900 Upright Confocal
Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

LSM 980 with Airyscan2

The Zeiss LSM980 system is an inverted laser scanning confocal microscopes with Airyscan 2 expands the excitation laser spot to allow imaging of eight lines in parallel simultaneously. It enables multiplex modes of SR-8Y, SR-4Y and CO-8Y for high speed imaging without sacrificing sensitivity or resolution. New ZEN software modules, including “Sample Navigator” and “Zen Connect” will simplify the imaging setup procedure. The stage-top live cell chamber, the motorized stage, advanced tiling module, photo-bleaching module enables the multi-dimensional (multi-channel, multi-position, time-lapse) live cell imaging, photo-manipulation and large tissue tile scanning.

Applications:

XY 2D/ Z-stack 3D imaging; DIC/Phase contrast imaging; Spot/line Scan;
Live cell imaging;
MPLX mode: Fast biological processes tracking
Tile scanning multiple position imaging.
Fluorescence recovery after photobleaching (FRAP), Fluorescence resonance energy transfer (FRET)
Experiment Designer: Convenient for complex imaging routines set up;
Sample Navigator: Good for whole specimen location;
ZEN Connect: Work with images from multiple sources

Detailed Configuration

Standard Operation Protocol – Carl Zeiss LSM980

Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

Perkin Elmer UltraView VOX Spinning Disc Confocal

PE UltraVIEW VoX spinning disk confocal is built for rapid and gentle imaging of live cell specimens. Highly sensitive EMCCD camera coupled with Yokogawa CSU-X1 spinning disk unit enables user to image with very low laser power to minimize effect of phototoxicity during long-term live cell experiments. Piezo z-stage is included in the setup for capturing volume up to 400 µm in z-axis with exceptional speed.

Applications:

XY 2D/ Z-stack 3D imaging; DIC/Phase contrast imaging
Live cell imaging (drosophila, C. elegans, zebrafish, organoid)
High Speed Imaging
Fluorescence recovery after photobleaching (FRAP), Fluorescence resonance energy transfer (FRET)

Detailed Configuration

Standard Operation Protocol – Perkin Elmer UltraView VOX Spinning Disc Confocal

Staff-in-charge: Ms WU, Vincci| 3917-9137 | imaging.cpos@hku.hk

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Super-resolution Microscope

Live-SR Super Resolution/TIRF Microscope

Live-SR Super-resolution/TIRF Microscope is an integrated imaging system equipped with a Live-SR Super-resolution Unit, a Yokogawa CSU-W1 Spinning Disk Unit, and an iLAS3 Ring-TIRF/FRAP/Ablation Unit. The Live-SR is based on an optically demodulated structured illumination technique with online processing. It enables super-resolution (105nm laterally) at high speed with low photo-toxicity. iLAS3 Ring-TIRF/FRAP with 4 laser lines (405nm, 488nm, 561nm, and 639nm) allows the observation of membrane-associated processes like cell adhesion, hormone binding, molecule transport, exocytotic and endocytotic dynamics. A high-power 355nm UV pulsed laser enables ablation for applications like DNA damage and axotomy.

Applications:

High Speed Super Resolution Imaging
Total Internal Reflection Fluorescence (TIRF)
Fluorescence Recovery After Photobleaching (FRAP)
Laser Ablation

Detailed Configuration
Standard Operation Protocol – Live-SR Super Resolution/TIRF Microscope
Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

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High Throughput Imaging System

Akoya Vectra Polaris

Vectra Polaris integrates both multispectral imaging and automated slide scanning to better visualize and quantify immunolabelled cells in tissue sections and tissue microarrays. With Tyramide signal amplification-based staining of multiple biomarkers (up to 8 overlapping fluorescent colors and DAPI), visualization is achieved by patented Liquid Crystal Tunable filter and unmixing algorithm. This fully automated system provides high speed whole-slide scanning at 10x to 40x in brightfield or fluorescence for up to 80 slides in a run. Supported by the tissue analysis software, quantification and spatial analysis of biomarkers can be accomplished in high throughput via machine learning and batch analysis.

Applications:

Brightfield microscopy of histological sections (e.g. DAB, H&E)
Whole-slide widefield fluorescent scanning (e.g. in-vitro GFP expression)
Multispectral imaging, quantification and analysis of immunolabelled sections (up to 8 fluorescent colors and DAPI)

Detailed Configuration

Standard Operation Protocol – Perkinelmer Vectra Polaris

Opal staining protocol
Akoya Opal Assay Development_Guide

Staff-in-charge: Ms. KWOK, Alice | 3917 9442 | imaging.cpos@hku.hk

Cytiva IN Cell Analyzer 6500HS

IN Cell Analyzer 6500HS is a laser-based line scanning high-content imaging system. High speed filter based widefield, EDGE confocal imaging modes with a sensitive PCO Edge 4.2 sCMOS camera. User could choose between fixed cell imaging, live cell incubation and hypoxic incubation ([Oxygen] adjustable 0.1% -20%). Combined with new IN Carta software, for object quantification streamlines workflow for high content analysis feature extraction.

Applications:

XY 2D/Z-stack 3D imaging
Slide scanning
High through-put imaging
Live cell imaging with hypoxia
3D culture imaging (< 300 µm organoid diameter)

Detailed Configuration
Standard Operation Protocol – Cytiva IN Cell Analyzer 6500 HS

Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

MACSima Imaging System

The MACSima™ Imaging System is an automated imaging platform for multiplexed RNA or protein visualization or RNA + Protein co-expression analysis in tissue samples. It is equipped with liquid handling system and sCMOS camera, combining cyclic fluorescent hybridization or cyclic immunofluorescence (DAPI/FITC/PE/APC) with high-resolution microscopy technoloy, enabling dozens of RNAs alongside hundreds of proteins detection in a single sample. Combined with MACSima IQ view analysis software, multidimensional multiomic datasets can be analyzed in high throughput.

Applications:

Fluorescence imaging of fixed tissue section or fixed cells
Multiplex imaging, quantification, and analysis of tissue sections
Same-section multiomics for diverse research fields, including Cancer research and oncology, Immunology, Neuroscience, Developmental biology

Detailed Configuration

Standard Operation Protocol – MACSima

Experiment Setup:

MICS_antibody_conjugates_v50.0.1.xlsx

Sample Prepration Protocols:

DAPI Pre-Staining Protocol

MACSima sample preparation protocol_adherent cells

MACSima sample preparation protocol_frozen tissue

MACSima sample preparation protocol_FFPE tissue

How to setup a MACSwell Imaging Frame

Antibody test protocol for the use in the MACSima Imaging System

RNAsky™ FFPE Sample Preparation Guide

RNAsky™ User Guide

Staff-in-charge: Dr. WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

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Light-sheet Microscopy

LiTone XL Light-sheet Microscope

The LiTone XL Light-sheet Microscope is a lightsheet setup that applies unique 4-side illumination to achieve sample penetration across the entire sample. The microscope is equipped with 4 solid lasers (405 nm, 488 nm, 561 nm, and 640 nm). With the sCMOS camera and high NA objective, the microscope provides lateral resolution to 500 nm and axial resolution to 1.5 um. The microscope has optics that are adaptable to a wide range of refractive indices, from water to solvent solutions, with an index of refraction ranging from 1.33 to 1.52. With its live cell imaging toolkit and the motorized stage, the microscope enables long-term imaging of live small transparent animal, such as zebrafish. The detachable sample bath and holder set allow for imaging of both soft and hard samples ranging in size from 0.5 mm to 25 mm.

Applications:

Cleared tissue/embryos imaging (e.g. cleared mouse brain/liver, cleared mouse embryo, etc.)
Fixed transparent tissue imaging (e.g. organoid, etc.)
Live small transparent animal imaging (e.g. zebrafish, etc.)

Detailed Configuration
Standard Operation Protocol – LiTone XL Light-sheet Microscope
Staff-in-charge: Ms. KWOK, Alice | 3917 9442 | imaging.cpos@hku.hk

ZEISS Lattice Lightsheet 7 Microscope

The Zeiss Lattice Light Sheet 7 Microscope is a light sheet setup that allows long term volumetric imaging of living cells. It features an illumination objective lens with 13.3X magnification and NA of 0.44, along with a detection lens with 44.83X magnification and NA of 1, enabling excitation and detection in perpendicular directions. The microscope is equipped with three lasers emitting at 488nm, 561nm, and 640nm, which can be modulated to create a thin light sheet for imaging samples. The deskewed resolution could reach 290 nm × 290 nm × 450 nm (XYZ) after deconvolution. It also includes an incubation system and autoimmersion system for long term stable live cell imaging. Standard sample carriers including slides, dish and mutiwell plate with No.1.5 coverslips are fully compatible with this microscope. This system allows for volumetric imaging of subcellular structures in attached or suspended cells, spheroids, or organoids with diameters up to 200 μm.

Applications:

Imaging of adherent cells and suspension cells (live cell imaging or fixed cells)
3D cell culture imaging (e.g. organoid, Spheroids, Cysts, Cells in Hydrogel)
Small evolving organisms imaging (e.g. Zebrafish embryos, C. elegans embryos, Drosophila embryos)

Detailed Configuration
Standard Operation Protocol – Zeiss Lattice Light Sheet 7 Microscope
Staff-in-charge: Dr.WANG Zhaoyue (Cindy) | 3917 9851 | imaging.cpos@hku.hk

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Other Imaging System

Olympus FVMPE-RS Hybrid Multiphoton System

Olympus FVMPE-RS Hybrid Multiphoton System is a multi-photon laser scanning microscope that allows fluorescence imaging deep into specimens at hundreds of micrometers in living cells and tissues. It is equipped with two Coherent Chameleon Vision II laser, tunable from 680nm to 1080nm, four NDD (non-descanned detector) detectors (PMT), upright microscope stand and dual scanners (Galva and Resonant Scanner). This enables simultaneous imaging of maximum four fluorescent channels on live samples. High NA water immersion objectives allow imaging directly into specimens in media. It is dedicated to intravital imaging of living, whole mount, thickly sliced specimens or small animals.

Applications:

Multiphoton imaging
Second harmonic generation (SHG)
Z-stack 3D imaging
Time-lapse live tissue/animal imaging
Fluorescence recovery after photobleaching (FRAP), Fluorescence resonance energy transfer (FRET) and photoactivation

Detailed configuration
Standard Operation Protocol – Olympus MP-ERS
Staff-in-charge: Mr. WU Kelly | 3917 9445 | imaging.cpos@hku.hk

Nikon Ti2-E Widefield Imaging

Nikon Ti2-E is an inverted fluorescence microscope equipped with motorized and intelligent components for advanced imaging applications at high speed and high sensitivity. The integration of an unprecedented 25mm field of view with industry-leading optics, a Perfect-auto-Focusing System and a stratum structure enable the Nikon Ti2-E to have extra expandability meeting the needs for various type of imaging.

Applications:

Widefield imaging (main application)
Phase contrast/DIC
XY 2D imaging
multispectrum fluorescent imaging
live cell imaging

Detailed Configuration
Standard Operation Protocol – Nikon Ti2-E Widefield Microscope
Staff-in-charge: Ms WU, Vincci| 3917-9137 | imaging.cpos@hku.hk

Carl Zeiss AxioZoom.V16

AxioZoom.V16 is a multi-zoom stereoscope with fluorescent illumination. High N.A. optics enable visualization of specimen ranging from large field-of-view to single cell observation. Flexible design allows illumination options of brightfield, dark field, oblique ring illumination, reflective fluorescence for DAPI / GFP / RFP.

Applications:

Color documentation of biopsy specimen
Screening small transgenic animal
stitching tissue section slides

Detailed Configuration

Standard Operation Protocol – Carl Zeiss AxioZoom.V16

Staff-in-charge: Dr. WANG, Cindy | 3917 9851 | imaging.cpos@hku.hk

Optical Tweezers/TIRF System

Optical Tweezers/TIRF system is an integrated platform enables users to control, manipulate and observe samples in real time with nanometer precision and femtoNewton resolution. The NanoTracker™ 2 optical tweezers module enables trapping and tracking samples from several µm down to 30nm by using a highly focused 1064nm laser beam for sensitive manipulation, force, and tracking experiments. The iLAS2 ring-TIRF Unit with 4 laser lines (405nm, 488nm, 561nm, and 640nm) is also attached to this system for TIRF imaging.

Applications:

Quantitative force measurements
Molecular motor mechanics
Binding/elasticity of DNA and proteins
Cell membrane dynamics and particle uptake into cells
TIRF Imaging

Detailed Configuration
Standard Operation Protocol – Optical Tweezers/TIRF System

Staff-in-charge: Dr. CHEN, Miao | 3917 9806 | imaging.cpos@hku.hk

Primo micropatterning/Color imaging system

The Primo micropatterning/color imaging system is an advanced microscopy system that combines the Nikon Ti2-E inverted microscope with the Primo 2 micropatterning unit. The Primo 2 micropatterning unit is specifically designed for micropatterning applications, allowing for precise control to create complex patterns and structures with high accuracy. The Ti2-E microscope offers exceptional optical performance, featuring a motorized scanning stage for precise and automated XY scanning, as well as a perfect focus system for sharp and clear imaging. With high-speed color camera, the system enables whole tissue color imaging.

Applications:

Micropatterning, microfabrication, hydrogel polymerization/photo-scission
Brightfield microscopy of histological sections (e.g. DAB, H&E)
XY 2D imaging
multispectrum fluorescent imaging

Detailed Configuration

Standard Operation Protocol – Color imaging system

Standard Operation Protocol – Primo micropatterning

Micropatterning Sample preparation protocols:

Staff-in-charge: Ms. KWOK, Alice | 3917 9442 | imaging.cpos@hku.hk

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Sample Preparation Equipment

PELCO easiGlow™ Glow Discharge Cleaning System

During the process of vitrification, the aqueous sample solution may form a droplet and fail to spread evenly on EM grids. Plasma cleaning is a key step in preparing EM grids for vitrification, especially those with hydrophobic carbon films. The PELCO easiGlow™ Glow Discharge Cleaning System is a highly effective method for cleaning EM grids and turning carbon film surfaces hydrophilic. With a glow discharge treatment, the PELCO easiGlow™ system can modify the surface properties of carbon films, allowing aqueous solutions to spread easily and ensuring even distribution of the sample on the grid. This process is essential for high-quality vitrification and subsequent cryo-EM imaging.

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