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 facility 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, usage fees and how to apply for an NEA N3 laser operation license.

The Confocal Microscopy Lab currently houses the following equipment:

 

Zeiss LSM 510 Meta

The Zeiss LSM 510 META system is a classic laser scanning microscopy adaptable to a wide variety of applications.

quick reference

manual

laser information

registration form | booking

It offers configuration for multifluorescence analysis, because it combines the spectral META detector (a 32-anode photomultiplier, generating a lambda series of images in one pass for the entire fluorescence spectrum) with two other single, conventional detectors. The presence of three pinholes (whereas most often, there is only one pinhole): one for each fluorescence detector differentiates this confocal microscope from many other models. The diameter of these pinholes is adjusted automatically, depending on the wavelength of the fluorescence to be captured, which amends the specificity and optimizes the signal acquisition.

Zeiss LSM 510 Meta (click for full size image).
Zeiss LSM 510 Meta (click for full size image).

This system shows an exceptional quality of system stability, excellent optics, real-time data management, friendly acquisition software with image resolution up to 8192 x 8192 pixels.

Features

  • Carl Zeiss Axiovert 200M
  • Fluorescent illumination: HBO Lamp
  • FL filters set 01(Ex BP 365/12, Bs FT 395, Em LP 397), set 09 (Ex BP 450-490,Bs FT 510, Em LP 515), set 20 (Ex BP 546/12, Bs FT 560, Em BP 575-640)

Objectives

  • Zeiss EC Plan-Neofluar 10x/0.3 with DIC sliders
  • Zeiss LD Plan-Neofluar 20x/0.4 Corr with DIC sliders
  • Zeiss EC Plan-Neofluar 40x/0.75 with DIC sliders
  • Zeiss Plan-Apochromat 63x/1.40 oil with DIC sliders
  • Zeiss C-Apochromat 63x/1.20 Water Korr (0.14-0.19mm), with DIC slide
  • Zeiss EC Plan-Neofluar 100x/1.3 oil with DIC sliders

Laser module (class 3b)

  • Argon multi-line gas laser (458, 477, 488, 514nm, 30.0mW)
  • Helium Neon (HeNe) gas laser (543nm, 1.2mW)
  • HeNe gas laser (633nm, 5.0mW)
  • Diode laser (405nm, 30.0mW)

Detector

  • Two Photomultiplier tubes (PMT), one META detector and one PMT for DIC

Software

  • Zen 2009

Applications

  • Z-stack: generation of a Z-series that dissect through the specimen to get 3D imaging
  • Co-localization of proteins: multi-color imaging to detect the distribution of various fluorescently-tagged proteins
  • Time lapse: observation and record sample response under certain treatment
  • Dynamic study in live cells, i.e. Fluorescent Resonant Energy Transfer (FRET), Fluorescent Recovery after Photo-bleaching (FRAP) and Fluorescent Loss in Photo-bleaching (FLIP), etc
  • Meta function: an advanced function to detect the full spectra of multiple fluorophores and generate references of distinct spectral signature to solve problem of emission cross-talk and tissue background auto fluorescence

 

Leica TCS SP5 X

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

laser information

registration form | 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.

The system is equipped with three highly efficient photomultiplier, SP, for simultaneous detection of different fluorescent dyes, one transmitted light detector for acquisition of DIC or bright field images.

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 channels SP detector: two normal PMT, one HyD detetor
  • 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

laser information

registration form | 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 500um 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

  • Hamamatsu Electron Multiplying Charge-Coupled Devices (EMCCDs)
  • 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

 

Olympus BX61

quick reference

CellSens manual

registration form | booking

The BX61 WI is an upright conventional fluorescence microscope from Olympus. It has been customized to cater variety research demanding on bright field and/or fluorescence imaging, from cell biology to animal working and material sciences.

The Olympus BX61WI (click to see full-size image).
The Olympus BX61WI (click to see full-size image).

Features

  • Olympus BX61 W1F motorized microscope (upright), with dry (by default) and/or oil condenser top lens (stand by) for high resolution bright field imaging
  • Incubation system: Tokai Hit INU G2E-UW (working with fixed microscope stage), with CO2, temperature and humidity control
  • Arm height gaining kit (stand by), rigid/fix stage and special objectives provide maximum workspace and ideal platform for animal working
  • Camera options: Monochrome camera for fluorescence labeled specimen and color camera for stained slides
  • Manual stage (by default) or motorized stage (stand by)
  • Nosepiece: single or dual swing nosepiece

Objectives

  • Olympus Mplan N 5x/0.10 Air
  • Olympus UPL SAPO 10x/0.40 Air
  • Olympus UMPlanFL N 10x/0.30 Water Dipping
  • Olympus UMPlanFL N 20x/0.50 Water Dipping
  • Olympus LUMPlanFL N 40x/0.80 Water Dipping
  • Olympus LUMFL N 60x/1.10 Corr Water Dipping
  • Olympus UPlan SApO 100x/1.4 Oil

Fluorescence Illumination

  • CoolLED pE-2 system with four LED wavelengths: (365nm, 470nm, 565nm, 635nm)
  • Fluorescence filter: DAPI350/50XEm460/50nm; ET470/40XEm525/50nm; Ex560/40XEm630/75nm; Ex620/60XEm620700/75nm; 20/80 beamspliter, allows 20% reflection/80% transmission

Camera

  • Olympus XM10 monochrome camera, 1,376 x 1,032 pixels, 6.45 μm x 6.45 μm, 15 fps at 1,376 x 1,032; 12 Bits
  • Olympus DP72, single chip color CCD camera, pixels 1360 x 1024 (maximum 4140 x 3096 with pixel shifting). Color/ standard gray scale/ custom gray scale

Software

  • cellSens Dimension V1.6

Applications

  • Fluorescence imaging for small animals
  • High resolution bright field imaging for live cell morphology study, and/or HE and immunochemical stained slides
  • Multi dimension fluorescence imaging: multi-location, time lapse, multi-channel
  • Reflected-light technique for the information on texture of smooth surfaces

 

Nikon Biostation IM Q (Plastic Model)

manual

registration form | 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

 

Cyntellect LEAP

quick reference

manual

registration form | booking

LEAP™ is a microplate-based automated live cell analysis and processing system. It is capable to do in situ imaging of cells using brightfield and, multi-wavelength fluorescence characteristics laser-based manipulation to perform cell purification and/or optoinjection.

Cyntellect LEAP (click for full-size image).
Cyntellect LEAP (click for full-size image).

This noninvasive in situ process allows various adherent and non-adherent cell types including primary cells, cancer cells and stem cells to be purified in a closed sterile microplate environment. LEAP applications include in situ purification of various cell types, rapid cell line development and stem cell processing and generation.  Basically, LEAP uses a powerful F-theta lens, galvanometer mirrors and pulsed laser to rapidly scan a large field-of-view without moving the sample, and eliminates of unwanted cells while keeping neighboring cells are virtually unaffected.

Features

  • Illumination light source for bright field: LED Light
  • Epi-fluorescence imaging: Hybrid-halide lamp (350-680nm)
  • Adjust magnification: 3x, 5x, 10x, 20x
  • Pulsed laser for ablation: 355nm and 532nm
  • 8 Excitation/dichroic/emission filter sets as follows:

cyntellectLeap-excitation-chart

 

Leica Cryostat 1850

quick reference

manual

registration form | 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