Nikon A1 Si Single Photon

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 separately 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 low-incident angle dichroics and hexagonal pinhole combine to allow much lower laser powers to be used for sensitive live imaging.

Microscope and Illumination Sources

Nikon Ti-E inverted microscope with DIC optics and PFS perfect focus stage controller. Broadband illumination with Tl-PS100W halogen lamp illuminator and Intensilight C-HGFI precentered fiber optic mercury lamp fluorescent illuminator.

Objectives
Objective Magnification Working Distance NA

CFI Plan Apo

10X dry

4 mm

0.45

CFI Plan Apo VC

20X dry

1 mm

0.75

CFI Plan FLUOR

20X multi immersion

0.35 mm

0.75

CFI Plan FLUOR

40X oil immersion

0.2 mm

1.30

CFI Plan Apo VC

60X oil immersion

0.13 mm

1.40

CFI Plan Apo VC

100X oil immersion

0.13 mm

1.40

Lasers
Laser Wavelength Power

Melles/Griot Diode

408 nm

17 mW

Melles/Griot Argon-Ion

457/488/514 nm

40 mW

Melles/Griot DPSS

561 nm

10 mW

Melles/Griot Diode

640 nm

10 mW

Scanhead

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

Standard Detector: 4 PMT confocal detectors with the following Filter wheels:

Filter Wheel Filters

Filter Wheel 1

450/50 480DCXR, 485/40 520DCXR

Filter Wheel 2

525/50 560DCXR

Filter Wheel 3

545/40, 600/50 640DCXR + 650LP

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 version — : 2D, 3D and 4D acquisition and object analysis, 3D volume rendering, Spectral Unmixing.

Applications and Output

Applications: 

FRAP, FLIP, FRET, photoactivation, 3D timelapse, colocalization, point scanning, Tilescanning.

Output: 

.nd2, .jpeg,  .tiff,  .avi, .ics/.ids