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Multiphoton Confocal Microscope System A1R MP+

Faster, deeper, sharper multiphoton confocal imaging

New multiphoton and confocal microscope system for high speed, high resolution and high sensitivity multiphoton excitation and confocal fluorescence imaging.
Nikon’s A1R MP+ multiphoton confocal microscope is a unique multiphoton imaging system featuring a fast, high resolution galvanometer scanner and an ultra-high speed resonant scanner that is capable of frame rates from 30 fps at 512 x 512 pixels to as fast as 420 fps in band scan mode. 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.

Resonant Scanner Enables Imaging Up To 420fps

Nikon’s exclusive resonant scanner is capable of imaging a wide area at a much higher speed than a non-resonant scanner, making it possible for 420-fps imaging using point scanning technology. The NDD for multiphoton microscopy makes it possible to image fast and deep through the thickest specimens. Nikon’s optical pixel clock system allows more stable and more evenly illuminated imaging— even at high speeds.

High Speed Imaging
Visualization of intravital microcirculation
Blood cells in blood vessels within a living organism were excited by a femtosecond pulsed IR laser with the A1R MP+‘ ultrahigh-speed resonant scanner, and their movements were simultaneously captured in three successive fluorescence images at 30 fps (30 msec), with three separate color channels.
Three fluorescent probes are simultaneously excited and imaged–nucleus (blue), endothelium (green), and plasma (red). The long-wavelength ultrafast laser in combination with the ultrahigh-speed resonant scanner effectively reduces photodamage and makes time resolved multiphoton imaging of biomolecules possible.
Image resolution: 512 x 512 pixels, Image acquisition speed: 30 fps, Objective: water immersion objective 60x
Imaged with the cooperation of: Dr. Satoshi Nishimura, Department of Cardiovascular Medicine, the University of Tokyo, TSBMI, the University of Tokyo, PRESTO, Japan Science and Technology Agency

 

Revolutionary Hybrid Confocal Scan Head

The A1R+ has a hybrid scanner head that incorporates both an ultrahigh-speed resonant scanner and a high-resolution galvano scanner. Simultaneous photoactivation and ultrafast imaging using these two scanners allow acquisition of rapid changes after photoactivation and enables observation of intermolecular interaction.

A1R-Plus-Hybrid-Scanner
HeLa cells expressing Yellow Cameleon 3.60 were excited with 457 nm laser light. After stimulation with histamine, calcium ion concentration dynamics were observed. The (blue) emission of CFP and the (yellow) emission of YFP are shown as green and red channels respectively. The graph displays fluorescence intensity (vertical) versus time (horizontal). The green and red lines in the graph indicate the intensity change of CFP emission (green) and YFP emission (red) from the region of interest (ROI). Along with the increase of calcium ion concentration in the cell, the intermolecular FRET efficiency between CFP and YFP within Yellow Cameleon 3.60 increases, the CFP fluorescence intensity decreases, and the YFP fluorescence intensity increases. Imaging laser wavelength: 457 nm, Image size: 512 x 512 pixels, 30 fps (with resonant scanner) Photos courtesy of: Dr. Kenta Saito and Prof. Takeharu Nagai, Research Institute for Electronic Science, Hokkaido University

 

in vivo High-Speed imaging

The Nikon resonant scanner is capable of high-speed 420-fps imaging. Unique to this design is a resonant scan mirror capable of imaging full fields of view at much higher speeds than traditional galvano scanners. Nikon’s optical pixel clock system, which monitors the position of the resonant mirror in real time, adjusts the pixel clock to ensure more stable, geometrically correct and more evenly illuminated imaging even at high speeds. This enables the successful visualization of in vivo rapid changes, such as reactions in living organisms, dynamics and cell interactions.

A1R-MPplus_invivo_hi_speed

 

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 A1R 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 UniversityDr. Shigenori Nonaka,
National Institute for Basic BiologyDr. 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: 1024×1024 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

NEW Dual IR Beam Option Enables Simultaneous Two-Color Multiphoton Imaging

With the dual IR beam option, users can now simultaneously excite two different fluorophores such as GFP and mCherry.  This capability enables ultra-fast two-color multiphoton imaging, ideal for dynamic specimens.  The dual beams can also be used for stimulation at a specific wavelength and imaging with a different wavelength, reducing time delays normally present when using a single beam which requires mode locking to change wavelengths.

live-brain-slice

Live brain slice expressing YFP and Rhod-2, simultaneously excited with 900nm and 1040nm wavelengths (CFI75 LWD Apo 25x 1.1 NA W 1300nm objective).  A live slice culture from a YFP-VGAT rat stained with rhod-2 AM was imaged to measure mitochondrial calcium transients in hippocampal interneurons.

Image courtesy of Richard Kovacs, Ph.D., Institute for Neurophysiology, Charité-Medical University, Berlin, Germany

 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

Apo_LWD_20x

CFI Apochromat LWD 20xWI λS

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
20xWI λS
NA 0.95 WD 0.95 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

Objectives for multiphoton microscopy

Auto Laser Alignment when Changing Multiphoton Excitation Wavelength

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.

Two Types of Scanning Heads Enable High-speed, High-quality Imaging

Scanning Heads

A1R MP+ has a hybrid scanning head that incorporates both a high-resolution galvano scanner and an ultrahigh-speed resonant scanner. The galvano scanner enables imaging up to 4096 x 4096 pixels and high-speed acquisition of 10 fps (512 x 512 pixels). A new A1R MP+ system is now available which is compatible with a wavelength of 1300 nm.

Scan head input/output port 3 laser input ports
3 signal output ports for standard, spectral and optional detector*1
Laser for multiphoton microscopy Compatible laser Mai Tai HP/eHP DeepSee,
InSight DS+*2,
InSight DS+ Dual Option*3 (Spectra-Physics)
Chameleon Vision Ⅱ, Chameleon Discovery*3 (Coherent)
Modulation Method: AOM (Acousto-Optic Modulator) device
Control: power control, return mask, ROI exposure control
Incident optics 700 – 1080 nm*4 *5, 700 – 1300 nm*2 *3, auto alignment
Laser for confocal microscopy (option) LU-N3 3 laser unit 405 nm, 488 nm, 561 nm laser are installed; built-in AOTF

*Cannot used with spectral detector.

LU-N4/LU-N4S 4laser unit 405 nm, 488 nm, 561 nm, 640 nm laser are installed; built-in AOTF

*Use LU-N4S when using the spectral detector.

LU-NV Laser unit Compatible laser: 405 nm, 445 nm, 458 nm, 488 nm, 514 nm, 532 nm, 561 nm, 594 nm, 640 nm, 647 nm; built-in AOTF
NDD for multiphoton microscopy Type Compatible with 1080 nm
Episcopic GaAsP NDD (for Ni-E/FN1/Ti-E), Diascopic GaAsP NDD (for Ni-E, FN1), Diascopic NDD (for Ni-E only): Detectable wavelength range 380 – 650 nm*6
Episcopic GaAsP NDD compatible with 1300 nm (for Ni-E/FN1) : Detectable wavelength range 380 – 750 nm
Detector 4 PMTs
(3 GaAsP PMTs and 1 normal PMT for GaAsP NDD)
Filter cube 450/50, 450/70, 492, 525/50, 550/88, 575/25, 610/75, 629/53, 641/75, 732/68
Standard fluorescence detector (option) Detectable wavelength range 400 – 750 nm (400 – 650 nm when using IR laser)
Detector A1-DU4 4 Detector Unit: 4 normal PMTs
A1-DUG GaAsP Multi Detector Unit: 2 GaAsP PMTs + 2 normal PMTs
Filter cube 6 filter cubes commonly used for a microscope mountable on each of three filter wheels
Recommended wavelengths for multiphoton/confocal observation: 450/50, 482/35, 515/30, 525/50, 540/30, 550/49, 585/65, 594LP, 595/50, 700/75
Diascopic detector (option) Detectable wavelength range 440 – 645 nm
Detector PMT
FOV Square inscribed in a Φ18 mm circle
Image bit depth 4096 gray intensity levels (12 bit)
Scan head Type A1-SHRM (compatible with 1080 nm)/A1-SHRMV (compatible with 1080 nm and visible light photoactivation/IR imaging)/A1-SHRM3 (compatible with 1300 nm)/
A1-SHRMD (compatible with 1300 nm and Dual IR)
Standard image acquisition Scanner: galvano scanner x2
Pixel size: max. 4096 x 4096 pixels
Scanning speed:
Standard mode: 2 fps (512 x 512 pixels, bi-direction), 24 fps (512 x 32 pixels, bi-direction)
Fast mode: 10 fps (512 x 512 pixels, bi-direction), 130 fps (512 x 32 pixels, bi-direction) *7
Zoom: 1-1000x continuously variable
Scanning mode: X-Y, X-T, X-Z, XY rotation, Free line
High-speed image acquisition Scanner: resonant scanner (X-axis, resonance frequency 7.8 kHz), galvano scanner (Y-axis)
Pixel size: max. 512 x 512 pixels
Scanning speed: 30 fps (512 x 512 pixels) to 420 fps (512 x 32 pixels), 15,600 lines/sec (line speed)
Zoom: 7 steps (1x, 1.5x, 2x, 3x, 4x, 6x, 8x)
Scanning mode: X-Y, X-T, X-Z
Acquisition method: Standard image acquisition, High-speed image acquisition, Simultaneous photoactivation and image acquisition
IR laser wavelength range 700 – 1080 nm*4 *5, 700 – 1300 nm*2 *3
Dichroic mirror Low-angle incidence method
Position: 8
Standard filter: 405/488, 405/488/561, 405/488/561/638, 400 – 457/514/IR, 405/488/543/638, IR total reflection*2 *3, BS20/80, IR, 405/488/561/IR
Pinhole 12 – 256 μm variable (1st image plane)
Spectral detector*8(option) Detectable wavelength range 400 – 750 nm (400 – 650 nm when using IR laser)
Number of channels 32 channels
Spectral image acquisition speed 4 fps (256 x 256 pixels), 1000 lps
Wavelength resolution 80 nm (2.5 nm), 192 nm (6 nm), 320 nm (10 nm)
Wavelength range variable in 0.25 nm steps
Unmixing High-speed unmixing, Precision unmixing
Compatible microscopes ECLIPSE Ti-E inverted microscope, ECLIPSE FN1 fixed stage microscope, ECLIPSE Ni-E upright microscope (focusing nosepiece type)
Z step Ti-E: 0.025 μm, FN1 stepping motor: 0.05 μm
Ni-E: 0.025 μm
Option Motorized XY stage (for Ti-E/Ni-E), High-speed Z stage (for Ti-E), High-speed piezo objective-positioning system (for FN1/Ni-E)
Software Display/image generation 2D analysis, 3D volume rendering/orthogonal, 4D analysis, spectral unmixing
Image format JP2, JPG, TIFF, BMP, GIF, PNG, ND2, JFF, JTF, AVI, ICS/IDS
Application FRAP, FLIP, FRET (option), photoactivation, three-dimensional time-lapse imaging, multipoint time-lapse imaging, colocalization
Control computer OS Microsoft Windows®7 Professional 64 bit SP1
CPU Intel Xeon E5-2667 (2.90G.Hz/15MB/1600MHz)
RAM 16 GB (4GB x4)
Chipset Intel® C602 chipset
HDD 300 GB SAS 3GB/s (15,000 rpm) x2, RAID-0 configuration
Graphic controller NVIDIA Quadro 600 (PCI Express/dual monitor), or
NVIDIA Quadro 4000 (PCI Express/dual monitor)
Optical drive Super multi-drive, max. 16x or faster
Extension slot PCI slot x1
PCI Express 3.0 (x16) x3 (for graphics)
PCI Express 3.0 (x16 mechanical, x8 electrical) x1
PCI Express 3.0 (x8 mechanical, x4 electrical) x1
PCI Express 2.0 (x8 mechanical, x4 electrical) x1
Network interface Serial x1, RJ45
LAN port 10/100Base-T 1 port (for controller)
10/100/1000Base 1 port (for I/F board for external LAN)
Monitor 1600 x 1200 or higher resolution, dual monitor configuration recommended
Vibration isolated table 1800 (W) x 1500 (D) mm required (for FN1/Ni-E), or 1500 (W) x 1500 (D) mm required (for Ti-E)

*1 FCS/FCCS/FLIM is possible in combination with third-party systems.

*2 When using a scan head compatible with 1300 nm.

*3 When using a scan head compatible with 1300 nm and Dual IR.

*4 When using a scan head compatible with 1080 nm.

*5 When using a scan head compatible with 1080 nm and visible light photoactivation/IR imaging.

*6 400 – 650 nm when using diascopic NDD.

*7 Fast mode is compatible with zoom 8-1000x and scanning modes X-Y and X-T. It is not compatible with Rotation, Free line, CROP, ROI, Spectral imaging, Stimulation and FLIM.

*8 Compatible with galvano scanner only