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Multiphoton Confocal Microscope

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Faster, deeper, sharper multiphoton confocal 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.

Key Features

New High Resolution 1K Resonant Scanner

A1R-1K-HD-Scanner

Comparison of a large FOV image and detailed image of fine structures in a cleared* 2 mm brain slice of H-line mouse.

Photographed with the cooperation of: Drs. Ryosuke Kawakami, Kohei Otomo, and Tomoni Nemoto, Research Institute for Electronic Science, Hokkaido University

*RapiClear1.52, SunJin Lab

Nikon’s new resonant scanner mounted in the A1R+ scan head supports both high speed and high resolution imaging. The wide dynamic range and reduced noise level raises the bar for image quality in resonant scanners.

High resolution

A new resonant scanner achieves finely detailed images with a maximum resolution of 1024 x 1024 pixels (15 fps). A newly developed sampling method produces sharper images with any configuration: even at lower resolution settings. When combined with Nikon’s high NA objective lenses, the A1R+ can achieve absolute optical precision.

Large field of view

With both 1024 x 1024 pixel resolution and a large field of view (FOV18), the new resonant scanner delivers higher throughput in various imaging applications.

High speed

The fast acquisition speed of the resonant scanner (up to 420 fps depending on scan area) is able to capture images with a very short dwell time, minimizing excitation time and light energy exposure of the samples.

Multicolor

Up to 5 channel (four-channel episcopic detector plus diascopic detector) simultaneous imaging is possible.

Zebrafish heart and blood cells imaged with 1K resonant scanner.  Sample courtesy of Martha Marvin, Ph.D., Williams College


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 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

deep-imaging_6
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


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

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

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.

Three dimensional multiphoton image of a 1 dpf transgenic zebrafish (Tg(h2afv:GFP; EF1α: mCherry-zGem)).  Specimen was treated with Phenyltiourea (PTU), to inhibit melanin synthesis and clarified with optical clearing solution LUCID. This transgenic line expresses mCherry-tagged geminin and GFP-tagged histones.  Specimen was imaged with a CFI75 Apo 25xW MP1300 (1.10 N.A., 2.0 W.D.) objective and simultaneously excited with 900nm and 1040nm wavelengths.

Image courtesy of: Drs Toshiaki Mochizuki and Ichiro Masai, Developmental Neurobiology Unit, OKINAWA Institute of Science and Technology Graduate University

zebra-CAAX-34D-tail-z-stack---Deconvolved-8-iterations_2_crop_color-int_UNMIX-float4web4

Lateral view of the trunk of a transgenic zebrafish at 34 hpf (Tg(h2afv:GFP; EF1α: mCherry-CAAX)).  Specimen was treated with Phenyltiourea (PTU), to inhibit melanin synthesis and clarified with optical clearing solution LUCID.  This transgenic line expresses mCherry-tagged membrane proteins (purple) and GFP-histones (green).  Muscle fibers was also visualized using SHG (blue).  Specimen was imaged with a CFI75 Apo 25xW MP1300 (1.10 N.A., 2.0 W.D.) objective and simultaneously excited with 900nm (for SHG and GFP) and 1040nm (for mCherry) wavelengths.

Image courtesy of: Drs Toshiaki Mochizuki and Ichiro Masai, Developmental Neurobiology Unit, OKINAWA Institute of Science and Technology Graduate University


NEW Objective for Tissue Clearing Techniques

CFIPlan10xCGlyc

In the field of neuroscience research, there is an ever-increasing need to image deeper into intact brain tissues while maintaining high resolution and clarity.

CFI Plan Apochromat 10xC Glyc was developed to provide deep imaging capabilities for use with a wide variety of clearing techniques. 

The support of multiple refractive indices allows observation with not only water, oil and glycerin, but also with various optical clearing liquids. 

This new objective provides broad chromatic aberration correction and high transmission rates by incorporating Nikon’s exclusive Nano-Crystal Coat technology. 

This long-working-distance objective provides clear, high-contrast images deep inside the tissue with its high numerical aperture.

Whole mouse brain imaging with optical clearing method

CFIPlan10xCGlyc-diagram

PFC

Striatum

Hippocampus and Amygdala

Optical slices of H-line mouse whole brain after clearing with LUCID-A 
Objective: CFI Plan Apochromat 10xC Glyc (NA 0.5, WD 5.5) 
Photos courtesy of: Drs. Ryosuke Kawakami 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

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

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.


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.



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