Instrument Name:

Nikon Two-photon Upright Microscope (AX R MP)  Room: X216

Introduction

Combining laser scanning confocal microscopy and two-photon excitation, AX R MP uses an infrared femtosecond laser as the light source to penetrate deep into tissues and nonlinearly excite fluorescent molecules. The long wavelength reduces laser-induced damage and phototoxicity while ensuring high spatial resolution (imaging depth > 1 mm) and supporting long-term live-sample imaging. The device can also be used in single-photon imaging mode.

Application

The system features high imaging depth and low phototoxicity, suitable for long-term real-time imaging of live animals. The principle that two-photon excitation only stimulates the focal-plane signal enables more precise photoactivation and photobleaching experiments. The tunable-wavelength lasers support multi-label sample imaging. Two sets of single-photon lasers allow synchronous optical stimulation and image acquisition. The complete objective set, including high-NA water objectives, is suitable for in vivo animal imaging.

Key Technical Specifications

Laser:

Continuous-wave laser (2 sets): 405nm, 488nm, 561nm, 640nm; Femtosecond pulse laser: 1040nm, adjustable 680-1300nm

Imaging Mode:

Wide-field fluorescence, bright-field, single-photon confocal, and two-photon imaging

Detectors:

Built-in (single-photon): 4 high-sensitivity GaAsP; External (two-photon): 4 NDD (ultra-sensitive), 2 spectral scanning PMT

Objectives:

CFI Plan Apo 10× (Glyc, NA=0.5, WD=5.5, 400-1300nm); CFI Plan Fluor 10× (Water, NA=0.3, WD=3.5, 400-1000nm); CFI Apo 20× (Water, NA=1.0, WD=2.8, 400-1300nm); CFI Apo 25× (Water, NA=1.1, WD=2.0, 400-1300nm); CFI Apo NIR 40× (Water, NA=0.8, WD=3.5, 400-1000nm); CFI Apo NIR 60× (Water, NA=1.0, WD=2.8, 400-1000nm); CFI Plan 100× (Water, NA=1.1, WD=2.5, 400-1000nm); CFI Plan Apo 25× (Silicone oil, NA=1.05, WD=0.55, 400-1000nm); CFI Plan Apo 40× (Silicone oil, NA=1.25, WD=0.30, 400-1000nm); CFI Plan Apo 2× (Air, NA=0.1, WD=8.5, 400-1000nm); CFI Plan Apo 4× (Air, NA=0.2, WD=20, 400-1000nm); CFI Plan Apo 10× (Air, NA=0.45, WD=4.0, 400-1000nm); CFI Plan Apo 20× (Air, NA=0.8, WD=0.8, 400-1000nm); CFI Plan Apo 60× (Oil, NA=1.42, WD=0.15, 400-1000nm); CFI Plan Apo 100× (Oil, NA=1.45, WD=0.13, 400-1000nm)

Resolution:

Confocal mode XY ≤ 150 nm, Z ≤ 300 nm (depending on emission wavelength)

Scan Resolution:

Maximum scan resolution ≥ 8192×8192 pixels

Scan Mode:

Galvo mode: 512×512 (4 fluorescence + 1 transmission channels), 2 fps; Resonant mode: 2048×16, 720 fps

Scan Zoom:

≥1–1000× (continuous)

Motorized Stage Movement Range:

±34 mm (X) and ±27 mm (Y)

Image Processing Software:

NIS-Elements analysis software with confocal super-resolution (1.5× XY, 1.7× Z), 3D reconstruction, structure measurement, fluorescence intensity analysis, co-localization analysis