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Multiphoton Confocal Microscope System

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Streamlined multiphoton confocal for deeper, faster multiphoton imaging.

Nikon’s A1 MP+ multiphoton confocal microscope is a unique multiphoton imaging system featuring a high resolution galvanometer scanner. New 4 channel non-descanned detectors with higher sensitivity, reduced dark current and broad spectral range allow for real time unmixing of closely spaced probes for accurate and high-contrast spectral imaging. This is especially important in multiphoton microscopy because of the overlap of emission spectra of probes and autofluorescence, which is often unavoidable when using a single laser line.

Key Features

Deep Specimen Imaging with High-sensitivity Non-Descanned Detectors (NDD)

The fluorescence emissions from deep within a specimen are highly scattered in multiphoton excitation, and therefore the conventional detector using a pinhole cannot provide bright fluorescent images. The episcopic NDD in the A1 MP+ is located close to the back aperture of the objective to detect the maximum amount of scattered emission signals from deep within living specimens.

In vivo image of deep areas of cerebral cortex of a mouse

The cerebral cortex of an H-line 5-week-old mouse was studied with the open skull method. The entire shape of dendrites of pyramidal cells in layer V expressing EYFP were visualized from the bottom layer into a superficial layer. In addition, the fluorescence signal of white matter in deeper areas was also studied.

A1R-MPplus_NDD
Left 3D reconstruction image  

Photographed with the cooperation of:

Dr. Tomomi Nemoto,
Research Institute for Electronic Science,
Hokkaido University

Dr. Shigenori Nonaka,
National Institute for Basic Biology

Dr. Takeshi Imamura,
Graduate School of Medicine,
Ehime University

Right    Z-stack images      
  Top:
dendrites located in superficial layers in the layer V pyramidal cells
25 µm from the surface
 
  Middle:
basal dendrites in the layer V pyramidal cells
625 µm from the surface
 
  Bottom:
fluorescence from white matter
Excitation wavelength: 950 nm
Objective: CFI75 Apo 25xW MP (NA 1.10 WD 2.0)
 

Mouse cerebral cortex multi-color imaging

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deep-imaging_7
deep-imaging_8

Simultaneous acquisition of three channels in anesthetized YFP-H mouse using IR excitation of 950 nm and imaging Second Harmonic Generation (SHG) and two fluorescence emissions.
Cyan: SHG signal of dura mater
Yellow: EYFP pyramidal neurons in layer V of the cortex
Red: SRB-labeled blood vessels

Photographed with the cooperation of:
Drs. Ryosuke Kawakami, Terumasa Hibi and Tomomi Nemoto, Research Institute for Electronic Science, Hokkaido University

3D volume rendering images

Three-dimensional volume renderings of a kidney labeled with Hoxb7/myrVenus marker (Chi et al, 2009 Genesis), using depth-code pseudocolor volume rendering to reference Z depths (pseudocolored by depth - 1 μm step for 550 μm).

A1R-MPplus_NDD_2

Objective: CFI Apochromat 25xW MP, Scan zoom: 1x, Z step size: 1 μm, IR excitation wavelength: 930 nm
Image resolution: 1024x1024 pixels, Image volume: 460 μm (length) x 460 μm (width) x 600 μm (height)
Photographed with the cooperation of Dr. Frank Costantini and Dr. Liza Pon, Columbia University Medical Center, New York


1300nm Imaging

In addition to the GaAsP NDD compatible with a wavelength of 1080nm, there is a new model for upright microscopes that is compatible with a wavelength of 1300 nm. This new NDD enables deep imaging up to 1.4 mm in combination with a newly developed scanning head A1R MP+ that is compatible with a wavelength of 1300 nm.

Deep brain imaging in in vivo mouse with the GaAsP NDD compatible with the 1300 nm wavelength

gaasp1

Photographed with the cooperation of: Drs. Ryosuke Kawakami, Terumasa Hibi and Tomomi Nemoto, Research Institute for Electronic Science, Hokkaido University

In vivo imaging of an anesthetized YFP-H mouse (4-week-old) via open skull method. Visualization of the entire layer V pyramidal neurons and the deeper hippocampal neurons. Deep imaging achieved for 3-dimensional imaging of hippocampal dendrites up to 1.4 mm into the brain.

Captured with episcopic GaAsP NDD for 1300 nm and CFI75 Apochromat 25xW MP1300 objective lens (NA 1.10, WD 2.0 mm)
Excitation wavelength: 1040 nm

Mouse brain in vivo dual color imaging with the GaAsP NDD compatible with the 1300 nm wavelength

gaasp2

The cerebral cortex of an anesthetized YFP-H mouse (4-week-old) was studied with the open skull method.
Alexa594 was injected into the tail vein to visualize the blood vessel.

Photographed with the cooperation of: Drs. Ryosuke Kawakami, Terumasa Hibi and Tomomi Nemoto, Research Institute for Electronic Science, Hokkaido University


Nikon High-NA Objectives are Ideal for Multiphoton Imaging

High-NA objectives have been developed that highly correct chromatic aberrations over a wide wavelength range, from ultraviolet to infrared. Transmission is increased through the use of Nikon’s exclusive Nano Crystal Coat technology.

In particular, the CFI75 Apochromat 25xW MP/MP1300 objective lenses provide an industry leading highest numerical aperture of 1.10 while still maintaining a 2.0 mm working distance. They also have a collar that corrects spherical aberrations depending on the depth of the specimen and a 33° manipulator pipette access angle, making it ideal for deep multiphoton imaging and physiology research applications.
Nano Crystal Coat is a Nikon exclusive lens coating technology using an ultralow refractive index nanoparticle thin film originally developed for the semiconductor fabrications industry. The Nano Crystal Coat particle structure dramatically reduces stray reflections and boosts transmission over a wide wavelength range, producing images with higher signal-to-noise (S/N) ratios.

high-NA1

CFI75 Apochromat 25xW MP CFI Apochromat LWD 40xWI λS

high-NA2

CFI Apochromat 40xWI λS CFI Plan Apochromat IR 60xWI

Objectives for multiphoton microscopy

CFI75 Apochromat
25xW MP1300
NA 1.10 WD 2.0 Nano Crystal Coat
CFI75 Apochromat
25xW MP
NA 1.10 WD 2.0 Nano Crystal Coat
CFI Apochromat LWD
40xWI λS
NA 1.15 WD 0.6 Nano Crystal Coat
CFI Apochromat
40xWI λS
NA 1.25 WD 0.18 Nano Crystal Coat
CFI Plan Apochromat
IR 60xWI
NA 1.27 WD 0.17 Nano Crystal Coat

Auto Laser Alignment when Changing Multiphoton Excitation Wavelength

Auto Laser Alignment

When the multiphoton laser wavelength or group velocity dispersion pre-compensation is changed, the multiphoton laser beam positional pointing at the objective back aperture may also change, resulting in uneven intensity across the image, or a slight misalignment between the IR and visible laser light paths.

Verifying the IR laser beam pointing and setting the alignment has traditionally been difficult. The A1R MP+ auto laser alignment function, housed in the Incident Optical Unit for the multiphoton excitation light path, automatically maximizes IR laser alignments with a single click in NIS-Elements C.


Increased flexibility and ease of use

NIS-Elements C control software enables integrated control of the confocal imaging system, microscope and peripheral devices with a simple and intuitive interface. Diverse reliable analysis functions are also available.

System integration of Ti-E inverted microscope for multi-mode imaging is possible by equipping the confocal microscope system with N-SIM/N-STORM super resolution microscope system, TIRF system, spectral detector and Perfect Focus System. All systems can be controlled by a single NIS-Elements platform.



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