Project Description

Lindsay

Total Internal Reflection Fluorescence (TIRF) microscope on inverted Nikon Ti microscope

Imaging modalities

  • TIRF
  • Live cell imaging
  • DIC

Hardware Components

  • Nikon Ti motorized inverted microscope with Perfect Focus System
  • Nikon TIRF illuminator
  • Prior Proscan III linear-encoded motorized stage, excitation and emission wheels and shutters
  • Spectral Applied Research LMM-5 laser merge module with solid state lasers: 404nm (100 mW), 446nm (40mW), 515nm (50mW), 491nm (50mW), 561nm (50 mW) & 642nm (100mW).  Laser line selection is controlled by AOTF.
  • MetaMorph image acquisition software

  • Hamamatsu ImagEM EM-CCD camera (16 µm2 photodiode)

  • Hamamatsu D2 cooled-CCD camera (6.45 µm2 photodiode) for simultaneous imaging of two channels

  • Okolab 37°C, 5% CO2 microscope incubation enclosure
  • Lumencor SOLA light engine for fluorescence illumination
This instrument uses Class 3b lasers. All users must attend laser safety training.

Filter Spectra

Filters from Chroma

Illumination Setting Excitation TIRF Cube (ex/bs/em) Emission
404 / Qdot 404nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
632/60
446 446nm
none ZT442/514/561rpc ZET442/514/561M
470/24
491 491nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
525/50
515 515nm
none ZT442/514/561rpc ZET442/514/561M
535/30
561 RFP 561nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
632/60
561 TMR 561nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
600/50
642 642nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
700/75
FRET (CFP/YFP) 446nm
none ZT442/514/561rpc ZET442/514/561M
535/30
FRET (Cy3/Cy5) 561nm
ZET405/488/561/635X ZET405/488/561/635rpc ZET405/488/561/635M
700/75