Nikon A1Si Laser Scanning Confocal Microscope - Biology ...

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.
Figure 1. Principals of LSCM confocal
microscopy. From Nikon Microscopy U
For more information about confocal microscopy, visit Nikon MicroscopyU at:
http://www.microscopyu.com/articles/confocal/index.html
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).
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
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. (Nikon A1 website).
Figure 4: Virtual
Filtering (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:
Objective Magnification Working Distance Numerical Aperture
CFI Plan Apo Dry 10X 4 mm 0.45
CFI Plan Apo VC Dry 20X 1 mm 0.75
CFI Plan FLUOR MI 20X 0.35 mm 0.75
CFI Plan FLUOR Oil 40X 0.2 mm 1.30
CFI Plan Apo VC Oil 60X 0.13 mm 1.4
CFI Plan Apo VC Oil 100X 0.13 mm 1.4
• Lasers: AOTF-modulated LU4A laser unit:
Laser Wavelength Power
Diode laser Melles/Griot 640 nm 10 mW
DPSS laser Melles/Griot 561 nm 10 mW
Argon-Ion laser Melles/Griot 457/488/514 nm 40 mW
Diode laser Melles/Griot 408 nm 17 mW
• 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, 3 filter wheels allowing up to 6 filter cubes each:
Filter Wheel Filter Cubes Installed
Filter Wheel 1 450/50 480DCXR, 485/40 520DCXR
Filter Wheel 2 525/50 560DCXR
Filter Wheel 3 545/40, 600/50 640DCXR + 650LP
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/