Confocal Microscopy Laboratory

The Confocal Microscopy Laboratory is located at Blk S1A, #03-07 and #01-01 (CBIS lab), Department of Biological Sciences, NUS. The lab is equipped with several imaging systems which have different designs and configurations to meet variable research application requirements.

Access to the lab is contingent on adherence to procedures governing usage and usage policy. All users must undertake system training conducted by facility staff as well as OSHE online laser safety training before they are allowed to access the facility.

Find out more about online booking, facility charges and how to apply for an NEA N3 laser operation license.

To request training for microscopy, please submit the following documents to https://forms.gle/F14iZqUg9D4B3Du6A

To request access to Leica Cryostat1850, please submit soft copy of completed RA and user registration form to core facility staff.

The Confocal Microscopy Lab currently houses the following equipment:

3I diSPIM (Marianas LightSheet™)

quick reference

manual

booking

Live imaging directly involves inevitable tradeoffs of spatial resolution, temporal resolution and phototoxicity. This is especially true when interrogating imaging in three dimensions and/or three-dimensional samples over time (3D and/or 4D imaging) to obtain a complete picture of many dynamic subcellular processes.

3I diSPIM (click for full-size image).

Selective Plane Illumination Microscopy (or Light Sheet Microscopy) uses a separate excitation lens perpendicular to the widefield detection lens to illuminate only the plane of interest within sheet of light. Its low photo toxicity, photo bleaching, high signal to noise ration and high imaging speed make it the most favorite tool for live imaging.

Marianas LightSheet™ from Intelligent Imaging Innovations, Inc. (3I, USA) merges dual inverted selective plane illumination (diSPIM) with the power and flexibility of a live-cell microscopy system. It employs two orthogonal objectives positioned at 45° above the specimen plane. By alternating between the objectives for imaging and excitation, diSPIM captures two volumes that are fused and deconvolved to achieve isotropic resolution. Unlike capillary-based light sheet methods, diSPIM allows for standard specimen preparation in standard dishes and standard media.

The 3I diSPIM at CBIS/DBS confocal core, offers a single- (iSPIM) or dual- (diSPIM) sided light-sheet arrangement on an inverted microscope with custom built OKO environment chamber for live cell imaging and photon manipulation unit for FRAP. 3D isotropic resolution of 360 nm in x-y-z is achievable (by beads). Samples are prepared on conventional glass coverslips and agarose gel for monolayer cell culture and small organisms imaging respectively.

Features

  • Modalities: Single and dual inverted SPIM (iSPIM and diSPIM)
  • iSPIM x-y-z resolution: 360 x 360 x 760 nm
  • diSPIM x-y-z resolution: 360 x 360 x 360 nm
  • SPIM objectives: Nikon water dipping objectives: CFI NIR Apo 40X/0.8 NA WD 3.5 mm, x2; CFI Plan Fluor 10x/0.3NA WD 3.5mm, x2.
  • Finder objectives: Zeiss 40x and 10x
  • Sample platform: Rectangular coverslips (1.5#, 24 x 50 mm) or agarose on petri dishes
  • Excitation sources: 405nm (100mW), 488nm (50mW), 561nm (50mW) and 640 nm (100mW)
  • Dichroic mirror: Shemrock410/504/582/669 nm BrightLine® quad-edge dichroic beamsplitter
  • Emission filters Shemrock440/521/607/700 nm BrightLine® quad-band bandpass filter
  • Light-sheet camera: Twin Hamamatsu ORCA-Flash4.0 V3 sCMOS
  • Environmental control: diSPIM custom designed OKO incubator with temperature, CO2 and humidity control
  • Software: Slidebook 6
  • Operating system: Windows 10
  • Data transfer: 10 GB network connection – no external hard drives allowed
  • Usage: Staff-assisted and self-use allowed (following training)

Applications

  • 3D-5D Live cell imaging: multi-channel, 3D and time lapse imaging for isotropic resolution
  • 3D-5D Live small organism imaging
  • Advanced F-techniques: FRAP

Software

FV3000

quick reference (basic)

quick reference (advanced)

manual

booking

One new Olympus FLUOVIEW FV3000 confocal has been setup in confocal core since March 2017. FV3000 is designed not only for high sensitivity and speed imaging for live cell on 2D-6D (x,y,λ,z,t,p), but also microplate imaging and complex screening.

Our FV3000 is equipped with both galvanometer and high-speed resonance scanners. The resonance scanner acquires full field of view at 30 frames per second with high sensitivity and low phototoxicity, which is critical for velocity measurements and capturing dynamic events, such as for beating heart, blood flow, calcium signaling, etc. With Olympus’ exclusive silicone objectives providing clear and bright images at depth, and Olympus IX2-ZDC2, Z drift compensator, our FV3000 is optimized for time-lapse 3D imaging for culture cells.

For small organisms or large pieces tissue, FV3000 facilitates macro to micro imaging at very low to high magnification (1.25X up to 100X) so that our end user is able to see the entire organism in context and focus on the interested region with high resolution information.

With Olympus’ patented Super Resolution (FV-OSR) imaging, our FV3000 provides an easy-to-use way for boosting resolution beyond the diffraction limit in fixed tissues.

Overall, the FV3000 has a range of standard and optional advanced application features including FV-OSR, spectral unmixing, photo-conversion, photo-stimulation and complex cell cycle imaging, to meet our users’ imaging requirement to answer different research questions.

Olympus FLUOVIEW FV3000 confocal

Features

  • Olympus IX 83 inverted microscope, with motorized XY stage IX3-SSU.
  • Environmental chamber with laser safety interlock, temperature (32C to 42C).
  • Oko lab stage insert chamber with adaptors, with CO2, humidity and temperature control
  • IX3-HO35D, 35mm dish holder and IXs-HOS, slide glass/slide chamber holder
  • Wide field light Source: coolLED pE 300 excitation system (365nm, 470nm, 565nm)
  • Filter cube on microscope  :
    • U-FUNA: Ex BP360-370, DM410, Em LP420
    • U-FBNA: Ex BP470-495, DM505, Em BP510-550
    • U-FGWA: Ex BP 530-550, DM570, Em BP575-625
  • Confocal DM:
    • DM405/488/561/640
    • DM405/488
    • DM405-445/514
    • BS10/90
    • DM 405/445/514/561/640

Objectives

  • PLANON 1.25x: Plan Apochromat Objective 1.25x/0.04, WD 5.0, Air
  • Olympus UPLSAPO10x2: U Plan Super Apochromat objective 10x/0.4, WD3.11, Air
  • Olympus UPLSAPO20x: U Plan Super Apochromat objective, 20x/0.75, WD 0.6 (spring) Air
  • UPLSAPO30xS: U Plan Super Apochromat Silicone immersion objective, 30x/1.05, WD 0.8, Silicone oil
  • Olympus UPLSAPO60xW: U Plan Apochromat Water Immersion Objective, 60x/1.2 (CC0.15-0.2), WD 0.28 Water
  • Olympus UPLSAPO60xO: U Plan Super Apochromat Objective 60x/1.35, WD 0.15 Oil
  • Olympus PLAPON60xOSC2: Super chromatic aberration correction, N.A. 1.4, WD 0.12 (chromatic aberration compensation range 405-650nm) (stand-by) Oil

Laser

  • Laser Diode : 405nm (50mW); 488nm (20mW); 561nm (20mW); 640nm (40mW)
  • Laser Diode: 445nm (75mW); 514nm (40mW)

Scanner

  • FV31-HSU-P: Hybrid scanning unit including xy galvanometer scanner set and X resonant Y galvanometer scanner. Highest scanning speed: 30fps@512×512 and 438 fps@512×32

Detector

  • FV31-SD: 2 CH spectral detector unit by Multi-Alkali PMT
  • FV31-HSD: 2CH High Sensitivity-Spectral detector unit by Cooled GaAsP PMT
  • FV31-LETD: Transmitted detector for BF/DIC

Software

Applications

  • 2D-6D Live cell imaging: multi-channel, 3D, multiple location and time lapse imaging
  • Fixed slide imaging, tile scan
  • Advanced F-techniques: FRAP, FRET
  • Spectral Unmixing

Zeiss LSM 800 Confocal with Airyscan

Zeiss LSM 800 (https://www.youtube.com/watch?v=–9F1Xa2I_A) with Airyscan super-resolution module is a compact confocal laser scanning microscope based on Zeiss invert microscope and equipped with four lasers. It is stationed in Confocal Lab, S1A #03-07 as the replacement of LSM 510 Meta.

quick reference

Zenblue manual

laser information

registration form | booking

Airyscan detector is a hexogen array of 32 GaAsP detectors in hexagonal lattice, each detector approx. 0.2AU in diameter thus total detection area approx. 1.25AU in diameter. When it scans across sample, both resolution and SNR is improved. After detector-wise deconvolution and pixel reassignment, LSM800 Airyscan is able to achieve 1.7x improvement over the set by the Abbe limit. Zeiss patented linear scanning regime gives a constant signal-to-noise level and uniform exposure to the illuminating laser throughout the scanned area. The GaAsP detectors, fast linear scan and short light path, grants LSM800 one of the most sensitive microscopes in our confocal core. However, GaAsP detectors are more sensitive than the Photo Multiplier Tubes (PMTs) typically in the green and red region but not in the far red region.

Airyscan detector is a hexogen array of 32 GaAsP detectors in hexagonal lattice, each detector approx. 0.2AU in diameter thus total detection area approx. 1.25AU in diameter. When it scans across sample, both resolution and SNR is improved. After detector-wise deconvolution and pixel reassignment, LSM800 Airyscan is able to achieve 1.7x improvement over the set by the Abbe limit. The two GaAsP detectors, Zeiss fast linear scan and its short light path, LSM800 is one of the most sensitive microscopes in the core. While GaAsP detectors are more sensitive than the Photo Multiplier Tubes (PMTs) typically in the green and red region, but not in the far red region.

Its software, Zen Blue, has tiling function for large field of view imaging and includes the Shuttle & Find module for Correlative Microscopy.

On our system, the super-resolution Airyscan mode is only available for 63x objective.

Features

  • Microscope: Zeiss Axio Observer 7, with motorized XY stage
  • Wide field fluorescent microscopy:
    ·       Light Source: Solid-State Light Source Colibri 7, Type RGB-UV. Equipped with 4 solid state LED lamps. 630nm, 555nm, 475nm and 385nm
    ·       Filter cube: Filter set 90 HE LED (E) for fluorescent dyes like DAPI, FITC, TRITC and Cy5 with excitation wavelengths 385, 475, 555 and 630 nm. Beam splitter QBS 405 + 493 + 575 + 653 and emission filter QBP 425/30 + 514/30 + 592/30 + 709/100.
  • Confocal scan:
    ·       Scanner: Two galvanometric scanning mirrors with ultrashort line and frame flyback
    ·       Scanning speed: up to 8 images/sec (Airyscan up to 1.6 images/sec) with 512 × 512 pixels at bi-direction scan
    ·       Scanning Field: 12.7 mm × 12.7 mm in the intermediate image plane, with full pupil illumination.
    ·       Pinhole: Master pinhole with preset size and position; can be adjusted as desired for multitracking.
    ·       Beampath: one Main beam splitter (MBS) for 4 laser lines (405, 488, 561 and 640nm). Two patented Variable Secondary Dichroics (VSDs) at 10 degree angle to incident beam for most effective excitation light suppression.
  • Sample holder: universal sample holder and multiple well plate holder
  • XY stage: Motorized XY scanning stage, for Mark & Find function (xy) as well as Tile Scan (Mosaic Scan)

Objectives

  • Objective Plan-Apochromat 10x/0.45 M27
  • Objective Plan-Apochromat 20x/0.8 M27
  • Objective Plan-Apochromat 63x/1.4 Oil DIC M27 (airyscan)

Lasers

  • Laser Diode : 405nm (5mW); 488nm (10mW); 561nm (10mW); 640nm 5mW)

Detectors

  • LSM 800 Confocal GaAsP-detection module consists of two highly sensitive GaAsP-PMT detectors Two GaAsP (typical QE 45%) and one Airyscan detector for 63x
  • Transmitted light detector: Nil

Software

  • Zenblue 2.6

Applications

  • LSM 800 Confocal GaAsP-detection module, consists of two highly sensitive GaAsP-PMT detectors Two GaAsP (typical QE 45%) and one Airyscan detector for 63x
  • Transmitted light detector: Nil
  • Wide field detection: Zeiss AxioCam ERc 5S color camera

Leica TCS SP5 X with two photon excitation laser

Leica TCS SP5X Two-photon
The Leica TCS SP5X Two-photon (click for full-size image).

The Leica TCS SP5X Confocal Micro-scope System with multi-photon laser (MP) consists of an inverted microscope DMI 6000 CS with a XY scanning stage, Z motorized focus as well as a Super-z galvo stage, for higher speed and repeatability. It uses an Acousto Optical Tunable Filter (AOTF) for laser line selection and intensity control with micrscosecond precision. An Acousto Optical Beam Splitter (AOBS®) replaces fluorescent filters and beam splitters in the light path. It is a spectrally tunable element which is fast, switchable and without any moving parts. In combination with AOTF, the AOBS offers a more light efficient system. This optical element allows the excitation light to excite the sample and the emission passes straight through the crystal.

quick reference

manual

booking

The Spectral Detector SP ® is free from barrier filters and secondary dichroic filters and therefore highly transparent and extremely sensitive. Light emitted from the sample is dispersed by a high efficient prism. The resultant spectrum is directed towards two tunable slits. Two motorized sliders acts as barriers and transmit a step-lessly tunable band to the sensor. The full spectrum can be split into several channels without any loss of energy. The Spectral Sliders offer a flexible system for wavelength discrimination which can be used to minimize cross talk. The SP5 has also a Tandem Scanner which comprises of a conventional scanner for high resolution morphology imaging (8kx8k) and a resonant scanner for fast imaging. Conventional speed ranges from 10-1400Hz whereby resonant speed is 8000Hz.

For laser excitation, the user has a choice of different lasers: a diode 405, a multiline Argon, a White Light Laser (WLL) and a Ti-sapphire IR laser. With white laser light, users can freely choose any excitation line within the continuous range of 470nm to 670nm in 1nm increments to perfectly match fluorophores with minimum cross-excitation, and turn down laser power to prevent specimen damage. There are up to eight excitation lines can be used simultaneously. The multiphoton excitation allows imaging of fluorophores deep into intact tissue (beyond the range of confocal microscopy). This penetration is due to the greatly reduced scattering of infrared light and the fact that excitation is restricted to the focal point of the pulsed laser.

Features

  •  Leica DMI 6000 CS Bino Inverted Research Microscope with motorized xy stage and SuperZ-Galvo Stage
  •  Fluorescent illumination: Leica EL6000
  • Filter system: BGR filter system: UV, BP420/30, BP465/20; Blau, BP495/15, BP530/30,Grun, BP570/20, BP640/40

Objectives

  • Leica HC PL APO 10x/0.40 CS
  • Leica HC PL APO 20x/0.70 lmm Corr CS
  • Leica HCX PL APO 40x/0.85 Corr
  • Leica HCX PL APO 63x/1.20 W CORR CS
  • Leica HC PL APO 100x / 1.40 Oil CS2

Lasers

  • Diode laser, 405nm 50mW
  • Argon Ion Laser: 458nm 5mW, 476nm 5mW, 488nm 20mW, 496nm 20mW, 514nm 20mW
  • White laser: 470nm to 670nm, with 1 nm increments, with 8 channels AOTF for simultaneous control of 8 laser lines, each excitation wavelength provides 1.5mV
  • Multi Photon: Chameleon Ultra II, Ti-Sapphire, tuning range 680-1080 nm, ~4W at 800nm

Detectors

  • One Channel TLD Bright field Detector
  • Three Multi-Alkali PMT detectors
  • Two non-descanned detectors (NDD) for the multi-photon with filter block for FITC / TRITC

Software

  •   Leica Application Suite Advanced Fluorescence (LAS AF)

Applications

  • High Resolution imaging
  • High Speed imaging
  • Spectral imaging
  • Advanced F-techniques with wizards: FRAP, FRET AB/SE
  • Excitation lambda scans
  • Photo activation / ablation experiments

PerkinElmer UltraView Vox Spinning Disk

The UltraView VoX is an optimum system for live cell imaging. It consists of Yokogawa® CSU-X1 spinning disk head, EM-CCD camera, ProSync control box, photoKinesis unit etc.

quick reference

Volocity user guide

booking

A pair of rotating disks, pinhole disk, with thousands of pinholes and collector disk, with 20,000 microlenses, is housed in the scan unit. Light from the UltreView’s lasers, delivered by fiber optics into the scanning unit, illuminates a window of -1,000 of the 20,000 microlenses on the collector disk. Microlenese focus the laser light through the pinholes, and the objective lens focused laser light onto the sample. The spinning of the disk caused the sample to be scanned by 1,000 points of light at once, i.e. multi-point illumination. Fluorescence emission from the sample passed back through the pinholes and the emission light is separated from excitation light by the dichroic mirror. The multi-point fluorescence emission is focused onto a CCD detector and read out as a single image.

Perkin Elmer Spinning Disc (click for full-size image).
Perkin Elmer Spinning Disc (click for full-size image).

Spinning disk confocal microscope collects multiple points simultaneously rather than scanning a single point at a time, which produces not only the high speed but also protect biological sample with a lower dose of laser light. With additional benefits from the EMCCD camera, the Ultraview spinning disk confocal is highly suitable for live cell imaging of weakly expressing samples which are prone to photobleaching.

Features

  • Olympus IX 81 inverted microscope, with 400um range Piezo Z stage
  • Environmental chamber VoX Env. Chamb, with CO2, temperature (32C to 42C) and humidity control
  • Wide field light Source: coolLED pE excitation system (365nm, 470nm, 565nm, 635nm)
  • Ultraview Dichroic mirror:
    • Dichroic 1: 405/488/568/640
    • Dichroic 2: 405/440/514/640 (used for 640nm in FS)
    • Dichroic 3: 405/440/488
  • Ultraview filter wheel:
    • Position 2: for 488nm (single band)
    • Position 3 for 405nm and 561nm (dual band)
    • Position 4 for 440nm and 640nm (dual band)
    • Position 5 for 514nm
    • Position 9 for 405nm (single band)
    • Position 7: (FS) : 405/440/488/561
    • Position 8: (FS) : 405/440/515/640

Objectives

  • Olympus UPLFLN 4x/0.13 Air
  • Olympus UPLNSAPO 10x/0.40 Air
  • Olympus UAPO N 20x/0.70 Water (stand-by)
  • Olympus UPlanSApo 20x/0.60 Air
  • Olympus UAPO N 340 40x/1.15 Water
  • Olympus UPLSAPO 60x/1.20 Water
  • Olympus UPlan SApO 100x/1.4 Oil

Laser

  • Solid state diode laser with Diode module: 405nm (50mW); 445nm (40mW); 640nm (40mW)
  • Solid state diode laser with DPSS module: 488nm (50mW); 514nm (25mW); 561nm (50mW)

Scanner

  • Yokogawa high speed spinning disk CSU-X1-A3 scanner, scanning speed 1,500 ~ 5,000 rpm

Detector

  • EM CCD camera C9100-50 (Camera 1): 14-bit, 1K x 1K, cooled frame transfer electron multiplication CCD, 30 fps at full frame, 8µm X 8µm cell size
  • EM CCD camera C9100-13 (Camera 2): 16-bit, 512 x 512 pixel, dual read-out mode (EM-CCD read-out and normal CCD read-out mode), 32fps at full resolution, 16µm X 16µm cell size

Software

Volocity®

Applications

  • Live cell imaging and high Speed imaging
  • Advanced F-techniques: FRAP, FRET

Nikon Biostation IM Q (Plastic Model)

manual

booking

The Nikon BioStation IM-Q is a compact cell incubator and monitoring system that allows users with minimal microscopy experience to conduct live cell imaging without a steep learning curve. The BioStation IM-Q time-lapse imaging system facilitates a broad array of short-term and long-term time-lapse experiments, including studies of cell growth, morphology, and protein expression, by providing consistent environmental control of temperature, humidity and gas concentration in combination with phase and fluorescence imaging of exceptional quality. Additionally, researchers can select from two types of microscope cameras. Perfusion capabilities further expand experimental possibilities.

Nikon Biostation (click for full-size image).
Nikon Biostation (click for full-size image).

Features

  • Biostation-IM-C Chamber, for 3.5cm plastic petridish, comes with No focus drift design and a vibration damping chassis. Fully motorized XYZ
  • Illumination light source for phase contrast: Red LED Light
  • Epi-fluorescence observation: HG Precentered Fiber illuminator Intensilight C-HGFIE
  • Objective: 20X/0.5, 0.6 -2.0mm, with magnification of 10X, 20X and 40X
  • CO2 Gas mixer TK-0003MIGM-SN01, with built in humidifier (270ml) and temperature heater
  • Humidity control at 95% or higher, Temperature control at 37°C (+/- 0.1°C)
  • Epi-fluorescence observation: HG Precentered Fiber illuminator Intensilight C-HGFIE
  • Fluorescent filter: detailed as followings. However, the machine can accommodate only two filter cubes at one time
DAPI ET GFP ET mCherry ET YFP Blue GFP
Excitation Filters 340-380 450-490 540-580 490-510 379-401
Dichroic Mirrors 400 495 585 515 420
Barrier Filter 435-485 500-550 592-667 520LP 435-485
  • Magnification from 10X to 40X (air), NA 0.5
  • Built in cooled 1.3 Mega pixels Monochrome CCD Camera
  • BS-IM Perfusion Induction Unit
  • BS-IM Perfusion Medium Exchange Unit
  • Operation Software: Biostation IM. Multichannel, multipoint (stitching up to 6mm x 6mm), time lapse can be set up easily by following the instructions on the GUI.
  • Image format: .nex file created once the experiment starts. After capturing, a folder is created which stores the images as .ics and .ids files. Data are able to be exported to: JPG, TIFF, BMP, PNG and AVI
  • PC with Intel QuadCore Q8400 (2.66GHz /4M / 1333MHz), 4GB Ram, 22 inch LCD monitor, 1T hard disk, 1GB graphic card, DVD-RW and license window Vista.
  • Application: live cell fluorescent imaging

Leica Cryostat 1850

quick reference

manual

booking

The Leica CM1850 cryostat design incorporates emphasis on power savings,increased efficiency and operator safety. The result is a versatile cryostat with an optimized cooling system, rapid specimen freezing and smooth specimen orientation for the high-quality sectioning demanded in routine histology and clinical pathology. Up-to-date technology with an ergonomic design – for faster and safer sectioning.

confocal lab: Leica Cryostat 1850
Leica Cryostat 1850 (click for full-size image).

Features

  •  The Leica CM1850 is a powerful cryostat for rapid freezing and sectioning of tissue samples.
  • Preservation of enzyme activity for enzyme histochemistry and antigenicity for immunohistochemistry and in situ hybridization.
  • Retention of substances that is soluble in routine processing solutions, such as lipids.
  • Preservation of cell morphology without exposure to chemicals and/or heat.
  • Can be performed on fixed and unfixed tissue specimens.
  • Section thickness setting 1 – 60 μm; Specimen feed 25 mm; Vertical stroke 59 mm; Maximum specimen size 55 x 55 mm