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.
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.
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.
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.
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.
Mouse cerebral cortex multi-color imaging
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).
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
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
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.
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
NEW Objective for Tissue Clearing Techniques
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
Hippocampus and Amygdala
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.
|NA 1.10 WD 2.0 Nano Crystal Coat|
|NA 1.10 WD 2.0 Nano Crystal Coat|
CFI Apochromat LWD|
|NA 0.95 WD 0.95 Nano Crystal Coat|
CFI Apochromat LWD|
|NA 1.15 WD 0.6 Nano Crystal Coat|
|NA 1.25 WD 0.18 Nano Crystal Coat|
CFI Plan Apochromat|
|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.
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
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.