Multifunctional confocal laser scanning microscope Booking

The Optical Microcharacterisation Facility at Macquarie University manages 2 laser scanning microscopy systems. Other accessories include an incubation system without gas flow but with a Bachofer Perfusion Chamber.

1. Technical specifications of Leica TCS SP / SP2 system
   • Fluorescence microscope details
   • List of available objectives
   • Light Detectors
   • Filters
   • Scan parameters
   • Software
   • Laser sources and accessories
   • Combined 405 nm FLIM and multiphoton FLIM
   • Multiphoton accessories
2. Technical specifications of TCS SL system
   • Objectives
   • Laser Sources
   • Other features
   • Detailed Description
3. Management and Operation
4. Access

Technical specifications of Leica TCS SP / SP2 system

It can do measurements of transmitted light, fluorescence and laser scanning fluorescence imaging. It has a z-stage with rotation capabilities, with z-resolution of 40 nm.

Fluorescence microscope details

• 50 W Hg lamp
• Filter block A: UV excitation filter 340-380 nm, long pass filter LP425 nm typically used for AMCA or DAPI.
• Filter block I3- blue excitation BP 450-490 long pass emission LP515 nm typically used for FITC or CY2.
• Filter block N2.1 - green excitation BP515-560, long pass emission LP590 typically used for TRITC or Cy3.

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List of available objectives

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Light Detectors

Four photomultipliers are available, three for confocal fluorescence and one for transmitted light. Range: 400 nm-850 nm.

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Filters

AOBS (Acousto-Optical Beam Splitter). It has continuously adjustable bandwidth and center wavelength (NO NEED FOR DICHROIC FILTERS ANY MORE).

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Scan parameters

• Line frequency up to 2800 lines/sec.
• Frame rates 4.2 fps at 512x512; 28 fps at 512x32 pixels
• Scan resolution up to 4096x4096 pixels
• Zoom 1...32x
• Scan rotation -5...+95 degrees, optically uncompromised solution
• Scan field diagonal 22mm (intermediate image plane).

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Software

1. LCS Basic Software to operate the microscope:

   • Easy to use, user configurable interface
   • Drop down menus for software utilities (user profiles and templates
   • Large icons for main functions and large arrow shaped buttons for main processes.
   • Context sensitive online help system
   • User access by individual login
   • Image Viewing & Processing
   • Display of individual images
   • Movie function
   • Image play mode for visualization of series with play speed control
   • Gallery display
   • LUT display, selection of LUTs
   • Selection of channel for display
   • Display of overlay images
   • Image storage and retrieval
   • Maximum Projection
   • Average Projection
   • Transparency Projection
   • Topological Projection
   • Stereo Pair Projection
   • Orthogonal slicing view (xy, xz, yz)
   • 3D View with move, zoom and pan function
   • Quantification
   • Interactive length, area, angle
   • Pixel, line (user defined) and area (user defined) intensity with statistical analysis
   • Height step measurement from height profiles
   • ntensity profile along z-series with height step interactive measurement
   • Intensity histogram of whole image or ROI
   • Charts and statistic data with export function
   • Rescaling of charts.
   • Analysis charts for time related experiments.
   • Image Annotation
   • Copying images and charts into annotation sheet
   • Variable size and position of transferred objects
   • Addition of text, arrows and lines.
   • Changing colour and size of annotated objects.
   • Printing of annotation sheets
   • Storage and retrieval of annotation sheets
   • Image Utilities
   • Software zoom
   • Image merging of series or single images
   • Image separation of series or single images
   • Arithmetic and logical operations between images
   • Arithmetic and logical operations between images and constants
   • Bit conversion from12 bit to 8 bit & vice versa.
   • Brightness and contrast operators.
   • Gamma correction operator
   • Segmentation function with thresholding
   • Leveling function with linear, quadratic and cubic models
2. LCS Physiology Software
• Live cell imaging, time lapse recordings
• Trigger handling, spot-bleach feature
• Ion-concentration measurement.
3. LCS Multicolor Software
• Colocalisation of 2channel and 3channel recordings.
• Cytofluorograms in 2D and 3D display.
• Histogram-segmentation and masking.
4. LCS Microlab
Software packages for
• FRAP,
• Fly-back FRAP,
• FRET (acceptor bleaching, sensitized emision)
5. LCS 3D Software
   • Volume rendering,
   • 3D animations,
   • 3D filter,
   • Stereo images and animations,
   • Average projection,
   • Maximum point projection,
   • SFP
6. LCS Macro Developer Software
• Task automation based on MS VBA technology,
• includes VBA IDE.
7. LCS 2nd Workstation package complete for additional workstations
• includes: 3D,
• Physiology,
• Multicolor,
• Materials packages

Can be copied anywhere we like but can only be used with the single dongle that we have. To use it on your machine you will need to borrow the dongle.

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Laser sources and accessories

• Red (He-Ne) 633nm 10 mW
• Orange (He-Ne) 594 nm 2.5 mW
• Green (DPSS laser) 561 nm, 10 mW
• Blue (Ar laser) 458nm/5mW;
• 476nm/5mW;
• 488nm/20mW,
• 496nm/5mW;
• 514nm/20mW
• Pulsed diode laser 405 nm for FLIM and confocal imaging
• Tsunami tunable 700 nm-1000 nm with nitrogen purge

Tsunami is a pulsed fs laser passing through a ps pulse stretcher. It is essentially used for multiphoton excitation but will, of course also do single photon excitation if required. The stretcher is useful as fs laser has a huge peak power and may damage samples easily.

We also have a 8 channel Acousto-Optical Tunable Filter for laser line selection and attenuation.

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Combined 405 nm FLIM and multiphoton FLIM

• Uses time correlated photon counting
• FLIM PMT < 200 ps response time
• Pixel resolution 256x256
• 256 time channels per pixel
• SPC Image software for data analysis
• Repetition rate either 40 MHZ or 80 MHz (so measured lifetimes can be within ns range)

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Multiphoton accessories

• Tsunami laser continuously tunable 700-1000 nm with nitrogen purge.
• Pumped by a new Verdi 5 W.
• 200 fs pulses BUT a ps Pulse stretcher in the path
• EOM - allows continuous computer control of laser intensity
• Dual channel non-descanned PMT for reflected light and fluorescence
• Dual channel PMT for bright field and transmitted fluorescence

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Technical specifications of TCS SL system

It is a spectral confocal system, that requires dichroic filters (no AOBS). In the future it will be dedicated also to TIRF.

• Inverted stand
• 50 W Hg lamp
• Fluorescence Filter blocks: I3 (blue excitation BP 450 -490; long pass emission LP 515) typical for FITC or Cy2 etc.
• N2.1 (green excitation BP 515 - 560; long pass emission LP 590) typical for TRITC or Cy3 etc.

Objectives

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Laser Sources

• Ar 458nm/5mW;
• 476nm/5mW;
• 488nm/20mW;
• 514nm/20mW
• HeNe 543nm/1.2mW
• HeNe 633nm/10mW )

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Other features

• Variable, computer controlled pinhole diameter
• Line frequency up to 2000 lines/sec.
• Frame rates 3 fps at 512x512; 20 fps at 512x32 pixels
• Scan resolution up to 2048x2048 pixels
• Zoom 1...32x
• Scan field diagonal 22mm (intermediate image plane)
• Multi-dimensional series acquisition
• Sequential scan in stack, frame and line mode
• Three channels for confocal fluorescence or reflected light.
• Continuously adjustable bandwidth and center wavelength.
• Spectral steepness factor <= 1%
• Transmitted light detector for recording bright field images.
• Four main beamsplitters mounted on motorized slider
• RSP 500, DD 458/514, TD 488/543/633 & RT 30/70 for fluorescence and reflection.
• Acousto-optical tunable filter for simultaneous control of 6 laser lines. Allows fast switching between lines and continuous attenuation of each line individually.
• LCS Physiology Software License

A TIRF objective will be delivered in 2005.

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Detailed Description

Description from other website (manufacturer and local):

1. http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf

Description of microscopy and usage:

Multifunctional confocal laser scanning microscope with time resolved and two-photon imaging capabilities

The microscopy system is built on a foundation of a modern laser scanning confocal fluorescence microscope. This microscope is equipped with the Fluorescence Lifetime Imaging accessory (FLIM), both described below.

  Laser scanning fluorescence microscopy

A fluorescence microscope works by exciting fluorochromes in the studied specimens, which deexcite by releasing light at other wavelengths. The fluorophores can be inherent or native in the specimen, or deliberately introduced for example in a process of fluorescent labelling of biological samples or impurities in materials. The laser scanning fluorescence microscope system comes equipped with several lasers emitting at a number of lines in the UV and visible spectral ranges, while short pulse lasers are used for multiphoton and time-resolved applications. Light dispersing elements such as prism or grating disperses the fluorescence spectrum onto the detector. Thus the spectral signature is acquired for each pixel of the scanned image. This information can, subsequently, be used for the digital separation of images into component dyes. Software driven artificial colouring can be used to emphasise minute colour variations in the microscope image. The software enables to separate even widely overlapping emission spectra. Thanks to a high performance graphics card such images can be used for fast presentation of 2D and 3D graphics and animations.

 Laser scanning microscopy forms a foundation for a variety of techniques including fluorescence resonance energy transfer (FRET). FRET is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. In conjunction with the recent developments in fluorescent marker technologies FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves.

Since their broad introduction in the early 90s laser scanning microscopes have led to a breakthrough in imaging science. The feasibility of multi-photon excitation, (see below) and the superior contrast of these instruments make them an ideal choice for fluorescence imaging.

Confocal imaging

In confocal microscopy the collected light is reflected or emitted by a single plane of the specimen. This leads to high contrast due to effective suppression of light scattered from outside the focal plane. Moreover, as number of such images generated with the focal length shifted in small steps can be combined in a three dimensional stack, which is accessible to digital processing. Thus such microscopes have the optical sectioning capability – as slices of the specimen can be examined without mechanical cutting and direct specimen preparation. These two important advantages have led to a widespread use of confocal imaging in many areas of science and technology.

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Fluorescence Lifetime Imaging (FLIM)

The powerful technique of time-resolved fluorescence significantly enhances the capabilities of conventional laser scanning microscopy, by offering deeper insights into chemical composition of the examined specimens. This very important capability makes it possible to produce fluorescence signatures that fully characterise the sample in a matter of minutes. Importantly, time-resolved fluorescence imaging is complementary to conventional fluorescence imaging. This facility is of importance in many biological applications, where the objective is frequently to make a fine distinction between two parts of a specimen with broad and very similar fluorescence curves.Applications of time-resolved fluorescence microscopy are as diverse as those of fluorescence and specimens can be examined rapidly and non-destructively.

The fluorescence of molecules is not only characterised by the emission spectrum, it also has a characteristic lifetime. Any energy transfer between an excited molecule and its environment changes this fluorescence lifetime in a predictable way. Since the lifetime does not depend on the concentration of the chromophore, fluorescence lifetime imaging is a direct approach to all effects that involve energy transfer. Typical examples are the mapping of cell parameters such as pH, ion concentrations or oxygen saturation by fluorescence quenching, or FRET between different chromophores in the cell. Furthermore, combined intensity- lifetime imaging is a powerful tool to distinguish between different fluorescence markers in multi-stained samples and between different natural fluorophores themselves. These components often have ill-defined fluorescence spectra but are clearly distinguished by their fluorescence lifetime.

Thanks to these characteristics, the Fluorescence Lifetime Imaging (FLIM) has become a new powerful tool to investigate molecular interactions, reactions and energy transfer including in cells and subcellular structures. These effects cause changes in the fluorescence quantum efficiency and thus in the fluorescence lifetime. Since the fluorescence lifetime does not depend on the unknown dye concentration it is a direct measure for the quantum efficiency. It therefore gives a more direct access to the investigated effects than the fluorescence intensity. Furthermore, the fluorescence lifetime can be used to separate the fluorescence of different luminophores in the cells if the components cannot be distinguished by their fluorescence spectra. Recording time-resolved fluorescence images is achieved by combining the Laser Scanning Microscope with pulsed laser excitation and a new Time-Correlated Single Photon Counting (TCSPC) Imaging technique introduced by Becker and Hickl.

The central part of the system is a modern laser scanning confocal microscope. Its key components include:

• an upright microscope stand with a z-stage that enables fine focusing ,
• a set of microscope objectives (Items 2 and 3), and other standard microscope accessories,
• a set of four detection channels used either separately or together in a single experiment
• a confocal scan head to ensure laser scanning operation,
• a control workstation driving the system,
• a suite of computer packages for data capture and analysis
• several steady-state excitation sources that are selected to suit a particular investigation, and accompanying microscope accessories.

In addition to the core confocal laser scanning microscope have an additional module that supplies the time-resolved imaging capability (FLIM) together with essential microscope accessories for femtosecond operation needed to integrate the FLIM module with the microscope.

The microscope is supplied with several excitation sources (lasers) and accompanying accessories, such as computer-controlled tunable acoustooptic filters, pinholes and optics. All excitation sources are of significant utility due to the selectiveness of the fluorophores used for fluorescent labelling or staining. However amongst the available excitation wavelengths, shorter wavelengths are more useful as they allow to excite a greater range of fluorophores.

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Management and operation:

The facility is supervised by, A/Prof E.M. Goldys. Time-sharing arrangements for access to instrumentation stipulate that no distinction will be made between the users from the collaborating institutions, Access rates and policies for external users (non-partner institutions and industry) are in line with similar facilities in other academic institutions.

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Access :

The instrument is operated and managed as a service facility. Macquarie University can provide formal and informal training and follow up advice to users, but once trained, the work is essentially done by users themselves through ‘’licensing’’. Researchers who make occasional use will be assisted by Macquarie staff.

The details of access rules are accessible from this website (at http://www.physics.mq.edu.au/~goldys/MicroscopeBooking ).

Only licensed users can make the bookings, other prospective users have to contact Prof E.M. Goldys, goldys @ ics.mq.edu.au.

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