Nikon A1Si Laser Scanning Confocal Microscope

Confocal microscopy uses optical methods to remove the out-of-focus blur of fluorescence microscopy images. In widefield microscopy, not only is the plane of focus illuminated but also much of the specimen above and below this point, resulting in out-of-focus blur from these areas. This out-of-focus light leads to a reduction in image contrast and a decrease in resolution by obscuring important structures of interest, particularly in thick specimens.

light pathway

Figure 1

Principals of LSCM confocal microsopy.
From Nikon MicroscopyU

In the confocal microscope most of the out-of-focus structures are suppressed at image formation. This is obtained by an arrangement of diaphragms, which conjugate points of the path of rays, act as a point source and as a point detector respectively. The detection pinhole does not permit rays of light from out-of-focus points to pass through it (Figure 1).

To obtain a full image, the point of light is moved across the specimen by scanning mirrors. The emitted/reflected light passing through the detector pinhole is transformed into electrical signals by a photomultiplier and displayed on a computer monitor. The confocal scanning microscope allows optical sectioning of samples up to 150 microns thick. If you are resolving thick samples or doing 3-D reconstruction, confocal is your first choice.

In the Laser Scanning Confocal Microscope (LSCM) a single point of light is moved across the specimen by scanning mirrors and the image is built up pixel by pixel. The emitted/reflected light passing through the detector pinhole is transformed into electrical signals by a photomultiplier and displayed on a computer monitor. The confocal scanning microscope allows optical sectioning of samples up to 150 microns thick. If you are resolving thick samples or doing 3-D reconstruction, confocal is your first choice.

For more information about confocal microscopy, visit Nikon MicroscopyU at:

http://www.microscopyu.com/articles/confocal/index.html

The Biology Department Nikon A1Si LSCM was installed in 2009. It offers many features for acquisition and analysis of high quality fluorescent and Differential Interference Contrast images. The A1Si features 4 Melles Griot lasers ranging from 408 nm (near blue) to 640 nm (far red) that can either be imaged by separate dedicated photomultipliers to allow up to 5 channel imaging (4 fluorescent + 1 transmitted), or the lasers can instead be directed to a 32 channel spectral detector for analysis at 2.5, 6, or 10 nm intervals allowing spectral analysis of a sample over a wavelength range of up to 320 nm. In addition, the spectral detector output can be binned into up to 4 channels to allow fine-tuning of emission output for unusual fluorophores (Virtual Filtering). Regions of Interest (ROI's) can be chosen from the full 32-channel output and unmixed into separate channels in real time so that even objects with very close emission maxima can be cleanly separated from one another (Spectral Unmixing). The 408 nm laser allows direct visualization of DAPI- stained samples for quick and accurate analysis of cell nuclear material, and can be used to bleach or activate ROI's for FRAP or photoactivation analysis. The rapid and accurate Z motor allows for rapid 3D acquisition in the Z plane and can easily be coupled to a time-lapse macro for 4D imaging. In addition, FRET, FLIP and colocalization studies can be performed. Finally, Nikon's unique low-incident angle dichroics and hexagonal pinhole combine to allow much lower laser powers to be used for sensitive live imaging.


Figure 2

Spectral acquisition and spectral unmixing (from Nikon A1 website)

Figure 3

30% fluorescence intensity leads to lower laser powers. (from Nikon A1 website)

Figure 4

Virtual Filtering (from Nikon A1 website)

System Specifications

  • Microscope and illumination sources: Nikon Ti-E inverted microscope with DIC optics and PFS perfect focus stage controller. Broadband illumination withTl-PS100W halogen lamp illuminator and Intensilight C-HGFI precentered fiber optic mercury lamp fluorescent illuminator.
  • Objectives
  • Lasers: AOTF-modulated LU4A laser unit:
  • Scanner: A1-SHS galvanometer scan head, Max 4096 X 4096 px resolution, Max scan speed of 4 fps at 512 X 512 px resolution, 1-1000X continuously variable zoom, 8 position low-angle incidence dichroic mirrors, 12-256μm variable hexagonal pinhole.
  • Detectors:
    2) Standard Detector: 4 PMT, 2 filter wheels allowing up to 6 filer cubes each:

    2) Spectral Detector: 32 channel lambda detector. Max speed of 4 fps at 256 X 256 px resolution. Resolution: 80 nm at 2.5 nm step, 192 nm at 6 nm step, 320 nm at 10 nm step size. Real-time spectral unmixing; binning of up to 8 consecutive channels for virtual filtering.

  • Software: Nikon Elements software: 2D, 3D and 4D acquisition and object analysis, 3D volume rendering, Spectral Unmixing.
  • Applications: FRAP, FLIP, FRET, photoactivation, 3D timelapse, colocalization.
  • OS: Microsoft Windows 7 64-bit
  • Output: ND2, JPG200, JPG, TIFF, BMP, GIF, PNG, JFF, JTF, AVI, ICS/IDS
  • Freeware: Nikon Elements NIS viewer software can be downloaded from the following URL:
    http://www.nis-elements.com/
  • For more information about the Nikon A1 Confocal, please see:

 
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