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 HD scan head supports both high speed and high definition imaging. The wide dynamic range and reduced noise level raises the bar for image quality in resonant scanners.

High definition

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 HD 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 HD resonant scanner.  Sample courtesy of Martha Marvin, Ph.D., Williams College

A1R HD has a hybrid scan 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.

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

What is a hybrid scan head?

This mechanism allows flexible switching or simultaneous use of two scanners (resonant and galvano) with the use of a hyper selector.

The A1R HD's galvano scanner enables high-resolution imaging of up to 4096 x 4096 pixels. In addition, with its scanner driving and sampling systems, plus image correction technology, high-speed acquisition of 10 fps (512 x 512 pixels) is also possible.

Image of a zebrafish labeled with four probes (captured with galvano scanner)
Nucleus (blue): Hoechst33342, Pupil (green): GFP, Nerve (yellow): Alexa Fluor^® 555, Muscle (red): Alexa Fluor® 647

Photographed with the cooperation of: Dr. Kazuki Horikawa and Prof. Takeharu Nagai, Research Institute for Electronic Science, Hokkaido University

Increased light detection efficiency realizes high image quality

The low-angle incidence method utilized on the dichroic mirrors increases fluorescence efficiency by 30%.

Conventional 45°  incidence angle method Reflection-transmission characteristics have high polarization dependence

Low-angle  incidence method Reflection-transmission characteristics have lower polarization dependence

By employing the hexagonal pinhole, higher brightness equivalent to that of a circular pinhole is achieved.

Square pinhole 64% of the area of the circle

30% more light

Hexagonal pinhole 83% of the area of the circle

Nikon's original dual integration signal processing technology (DISP) has been implemented in the image processing circuitry to improve electrical efficiency, resulting in an extremely high S/N ratio.

High-speed spectral imaging

Acquisition of a 32-channel spectral image (512 x 512 pixels) with a single scan in 0.6 second is possible. Moreover, 512 x 32-pixel images can be captured at 24 fps.

Accurate, high-speed unmixing

Accurate spectral unmixing provides maximum performance in the separation of closely overlapping fluorescence spectra and the elimination of autofluorescence. Superior algorithms and high-speed data processing enable real time unmixing during image acquisition.

Actin of HeLa cell expressing H2B-YFP was stained with Phalloidin-Alexa Fluor® 488. 
Spectral image in the 500-692 nm range captured with 488 nm laser excitation 
Left: Spectral image, Right: Unmixed image (green: Alexa Fluor® 488, red: YFP) 
Specimen courtesy of: Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Faculty of Medicine, Osaka University

Wide band spectral imaging

Simultaneous excitation with four lasers selected from a maximum of eight wavelengths is available, enabling spectral imaging across wider bands.

V-filtering function

Filter-less intensity adjustment is possible by selecting desired spectral ranges from 32 channels that match the spectrum of the fluorescence probe in use and combining them to perform the filtering function.

High-sensitivity spectral image acquisition

With a GaAsP PMT, the A1-DUVB tunable emission detector delivers flexible detection of fluorescent signals with higher sensitivity, in both the galvano and resonant imaging modalities.

Variable acquisition wavelength range

User-defined emission bands can collect images within a selected wavelength range, replacing the need for fixed bandwidth emission filters. 
Users can define the emission bandwidth range to as little as 10nm. Spectral images of multi-labeled specimens can be acquired by capturing a series of spectral images while changing detection wavelengths.

Based on the application, virtual bandpass mode and continuous bandpass modalities are selectable on the A1-DUVB.

VB (Variable Bandpass) mode

VB (Variable Bandpass) mode allows maximum 5ch color image

Unmixed Image CB (Continuous Bandpass) mode

CB (Continuous Bandpass) mode allows maximum 32 ch spectrum imaging

HeLa cells labeled with five-color fluorescence, Nucleus: DAPI, Vimentin: Alexa Fluor® 488, Lamin: Alexa Fluor® 568, Tubulin: Alexa Fluor® 594, Actin: Alexa Fluor® 633 
Specimen courtesy of: Dr. Tadashi Karashima, Department of Dermatology, Kurume University School of Medicine

Optional second channel detector

An optional second GaAsP PMT provides flexibility in detection. Users can divert selected wavelengths to the second fixed bandwidth emission channel by inserting a dichroic mirror, while simultaneously utilizing the user-definable emission band on the first channel. The second detector allows FRET, ratio imaging and other applications requiring simultaneous multi-channel imaging.

Accurate spectral unmixing

Multi-channel images acquired with the A1-DUVB can be spectrally unmixed by using the spectra of reference samples, or the spectra within the acquired images.

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.

Basic operation

Optical setting

Configuration of A1+ with N-SIM

System integration of Ti2-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.