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Resonant Scanning Confocal System

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Damos vida a las imágenes: captura de imágenes confocal de alta velocidad y resolución.

Captura imágenes confocales de alta calidad a velocidad ultrarrápida y sensibilidad mejorada con un explorador resonante y otro galvánico, el sistema confocal A1R+ de Nikon es una poderosa herramienta para captar y visualizar la dinámica e interacción intracelular.

  • Dinámica e interacción 
    • Explorador híbrido
    • Explorador resonante de alta velocidad
  • Brillo 
    • 30% de aumento en fluorescencia
    • Detector PMT GaAsP
  • Espectro 
    • Captura de imágenes en 32 canales a 24fps
    • Desmezclado espectral preciso

Key Features

Hybrid scanner for ultrafast photoactivation imaging

A1R+ 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.

A1R-Plus-Hybrid-Scanner

Células HeLa expresando amarillo camaleón 3.60, calibradas con luz láser de 457 nm. Después de la estimulación con histamina se observó la dinámica de concentración de iones de calcio. La emisión (azul) de CFP y la emisión (amarilla) de YFP se muestran como canales verde y rojo, respectivamente. La gráfica muestra la intensidad de fluorescencia (vertical) en comparación con el tiempo (horizontal). Las líneas verde y roja del gráfico indican el cambio de intensidad de la emisión de CFP (verde) e YFP (rojo) desde la región de interés (ROI). Junto con el aumento de la concentración de iones de calcio en la célula, la eficiencia de FRET entre CFP y YFP dentro del amarillo camaleón de 3.60 aumentos, la intensidad de fluorescencia de CFP disminuye y la intensidad de fluorescencia de YFP aumenta. Longitud de onda del láser de la captura de imágenes: 457 nm, tamaño de la imagen: 512 x 512 píxeles, 30 fps (con explorador resonante)

Fotos cortesía de: Dr. Kenta Saito y prof. Takeharu Nagai, Instituto de Investigación para la Ciencia Electrónica, Universidad de Hokkaido

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.

hybrid-scanner-1

Ultrafast imaging with a resonant scanner

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A1R+'s resonant scanner has an ultrahigh resonance frequency of 7.8 kHz. It allows imaging of intercellular dynamics at 30 fps (512 x 512 pixels) and 420 fps (512 x 32 pixels). The field of view of the scanned area is larger than that of the galvano scanner. The Nikon original optical clock generation method realizes high image quality even at the highest speed. The fiber-optic communication data transfer system can transfer data at a maximum of 4Gbps.

Mouse-Blood-Vessel

Vaso sanguíneo de ratón administrado con tetrametilrodamina y naranja de acridina y observado a 120 fps (8 ms/fotograma, con explorador resonante); rojo: vaso sanguíneo, verde: imágenes en mosaico del núcleo visualizadas cada 8 ms; las flechas indican el flujo de glóbulos blancos en el vaso.

Fotos cortesía de: Dr. Satoshi Nishimura, Departamento de la Medicina Cardiovascular, Universidad de Tokio, Programa de Educación de Nano-Bioingeniería, Universidad de Tokio, PRESTO, Agencia Japonesa de Ciencia y Tecnología


High-resolution imaging with a galvano scanner

galvano-2

The A1R+'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.

galvano-1

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
conventional-45
Reflection-transmission characteristics have high polarization dependence
  Low-angle
 incidence method
low-angle
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

Orificio cuadrado

64% of the area of the circle
30% more light
pinhole-arrow
 
hexagonal-pinhole

Orificio hexagonal

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.


Fast and accurate spectral imaging: A1-DUS Spectral Detector Unit

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

spectral-imaging-1

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.

spectral-imaging-2

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.

spectral-imaging-3

Bright spectral imaging: A1-DUVB GaAsP Detector Unit

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.

gaasp-1

VB (Variable Bandpass) mode

gaasp-2

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

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Unmixed Image CB (Continuous Bandpass) mode

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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.


Simple operation of complex applications

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.

Software-1

Basic operation

Software-2

Optical setting

Software-3

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.



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