Total Internal Reflection Fluorescence Microscopy: Nikon H-TRIF

Alias: 

TIRF

Primary Application: 

Acquisition of phenomena at the cell-coverslip interface, with excitation limited to a domain of ~100 nm in z.

Specifications

Microscope Stand: 

  • Inverted microscope Nikon Ti-E

Cameras: 

  • Hamamatsu Orca ER-C4742-0812AG CCD; 1344 (H) x 1024 (V); 6.45 µm/pixel
  • Andor Xion DU-897E EMCCD; 512 x 512; 16 µm/pixel

Fluorescence Acquisition Light Source: 

  • TIRF acquisition: Nikon LUN-4 laser diodes: 405 nm, 488 nm, 561 nm, 640 nm
  • Epifluorescence: Lumencor Sola SE LED (UV at 365 nm)

Flourescence Filter Sets: 

  • DAPI :  Chroma 49028-ET; Ex 395/25 nm, Em 460/50nm, DM 425nm
  • GFP : Chroma TRF49904-ET; Ex 488/10 nm, Em 525/50nm, DM 500 nm LP
  • RFP : Chroma TRF49909-ET; Ex 561/10 nm; Em 600/50 nm; DM 575 nm LP
  • GFP-RFP : Chroma TRF59904-ET: Dual Ex 488/10 nm & 561/10 nm; Dual EM 525/50nm & 600/50 nm
  • CY5 : Chroma 41008: Ex 620/60 nm; Em 700/75 nm; DM 660 nm LP

Objectives and calibrations :

Objectives Calibration Andor Xion
µm/pixel @ bin 1×1 µm/pixel @ bin 1×1
Nikon Plan Fluor 4x 0.13 NA 1,613 4,000
Nikon Plan Apo 10x 0.45 NA DIC N2 0,641 1,600
Nikon Plan Fluor ELWD 20x 0.45 NA Ph1 0,322 0,800
Nikon Plan Apo VC 20x 0.75 NA DIC N2 0,322 0,800
Nikon Plan Apo Lambda 40x 0,95 NA DIC N2 0,161 0,400
Nikon Apo TIRF 60x 1.49 NA oil TIRF 0,108 0,267
Nikon Plan Apo VC 60x 1.30 NA water 0,108 0,267
Nikon Plan Apo 100x 1.40 NA oil DIC N2 0,065 0,160

Other Nikon objectives available upon request.

Other components:

  • Perfect Focus System (PFS: automated focus maintenance)
  • Mightex Polygon 400 Photostimulator
  • MCL 100 µm Piezo-Z
  • 3D Tracking and Measurement analysis module on NIS Elements