Nikon TIRF microscope

Profile: Combined TIRF and epifluorescence setup for (live) imaging with a great variety of wavelangths.

The Nikon TIRF system is an inverted and fully automated Ti microscope with both widefield fluorescence and brightfield illumination and with objective TIRF illumination. A Nikon Perfect Focus System (PFS3) is included, that continuously determines the distance to the coverslip, readjusting it if required, for instance because of thermal drift. For life cell imaging of mamalian cells, the system is equipped with an on-stage incubation chamber from TokaiHit, controling temperature, CO2-concentration and humidity.

TIRF is short for Total Internal Reflection Fluorescence (Microscopy): By directing a laser beam under a very shallow angel towards the coverslip on which cells adhere (and on the condition that the refractive index of the medium on that side is lower than that of glass and oil), the light will not pass through the coverslip but be totally reflected. But if the light is totally reflected, what should be the use of such an illumination? In fact even though the beam does not reach the specimen side of the coverslip, an ultra thin (around 100 to 200 nm) "evanescent electromagnetic field" of light does exist there, able to excite fluorochromes. That means that in a TIRF microscope all fluorescently labelled structures of adhering cells as close as 100 to 200nm from the coverslip will be visible, everything further afar will stay jet black. And in this region the cell membrane and the adjacent cytoskeleton are located and can be studied without being affected by extra-focal blur. But why not use a confocal microscope? It would be able to show any image plane, not only the one closest to the coverslip. That's true but firstly a confocal could not selectively illuminate such a narrow slice (but rater something in the order of 300 to 400nm) and secondly a TIRF microscope does not need a confocal pinhole for imaging but is able to visualize the region of the cell membrane in total with a high sensitive EMCCD camera. These cameras are much more sensitive than photomultipliers that are used as light detectors in confocal microscopes. In the end, a TIRF microscope is limited to showing the region of the cell membrane but it can do so in much better quality and with much higher frame rates than a confocal microscope could do.

Nikon TIRF Microscope

Objectives:

  • Nikon Plan Fluor 10x NA 0.3 (working distance 16mm), corrected for 0.17mm coverslips (= type #1.5).
  • Nikon S Plan Fluor ELWD 20x NA 0.45 (working distance 8.2 - 6.9mm) adjustable for glas and plastic bottom dishes with a thickness between 0 and 2mm).
  • Nikon S Fluor 20x NA 0.75 (working distance 1.0 mm), corrected for 0.17mm coverslips (= type #1.5).
  • Nikon S Fluor 40x NA 1.30 oil immersion objective (working distance 0.22 mm), corrected for 0.17mm coverslips (= type #1.5).
  • Nikon Plan Apo VC 60x NA 1.40 oil immersion objective (working distance 0.13 mm), corrected for 0.17mm coverslips (= type #1.5).
  • Nikon Apo TIRF 60x NA 1.49 oil immersion objective (working distance 0.12mm) adjustable for glas coverslips with a thickness between 0.13 and 0.21mm), temperature adjustment for 23° and 37°C.
  • Nikon Plan Apo λ 100x NA 1.45 oil immersion objective (working distance 0.13mm), corrected for 0.17mm coverslips (= type #1.5).
  • Nikon Plan Fluor 100x NA 1.30 oil immersion objective (working distance 0.16mm), corrected for 0.17mm coverslips (= type #1.5).

Please note: not all the objectives are included in the system permanently. Some are only given to the users on request.

Cameras

  • Andor iXon Ultra DU-897U single photon detection EMCCD camera (for TIRF). This camera offers a resolution of 512 x 512 pixels with a gigantic on-chip pixel size of 16 x 16µm and a pixel well capacity of 180,000 electrons. The chip is cooled down to -100°C and 56 full frames can be read out per second.
  • Hamamatsu Ocra AG (Model C4742-80-12AG - for wide field epifluorescence). The resolution of this camera is 1344 x 1024 pixels at a physical pixel size of 6.45 x 6.45μm. Pixel well capacity is 18.000 electrons. The chip is cooled down to -30°C.

Lasers for TIRF

  • 405nm
  • 488nm
  • 561nm
  • 640nm

Filters

For widefield epifluorescence imaging the system is equipped with separate switchable excitation- and emission-filters. The dichroic mirrors can also be switched independently.

Excitation Filters (center wavelength/bandwidth):
  • 340/26
  • 387/11
  • 390/18
  • 430/24
  • 470/24
  • 500/20
  • 550/32
  • 565/55
  • 575/25
Emission Filters (center wavelength/bandwidth):
  • 470/24
  • 510/30
  • 525/30
  • 535/30
  • 641/75
  • 535/30
  • 593/40
  • 700/70
  • 795/50
Filter Cubes
bs = beam splitter ex = excitation filter em = emission filter
  • bs 409|em 510/84
  • tripple bs: CFP/YFP/mCherry|no ex|no em
  • bs 495|em 523/45|no ex
  • bs 595|em 630/60
  • dual bs 488+561|no ex|em 523/45+610/60
  • dual bs 488+561|no ex|em 560/100+780/240
more filter cubes are available

 

Bearbeiter: Christian Ackermann

Latest Revision: 2017-02-23

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