Zeiss Laser TIRF microscope

Zeiss Laser TIRF microscope

– Total internal reflection microscope (TIRF)
Tatyana Sherstova using the high resolution TIRF microscope. Photo
The high resolution TIRF microscope. Photo: Per Henning/NTNU

The microscope is used to visualize fluorescent molecules and specimens (e.g. a cell membrane) close to the coverslip, due to the narrow excitation field of 100-200 nm. (Total Internal Reflection Fluorescence: TIRF). The technique can be combined with transmitted light observation techniques and atomic force microscopy (AFM).

 

The microscope

The Axio Observer.Z1 is an inverted motorized microscope with a touch pad, equipped for both TIRF microscopy and AFM. It is equipped with Definite Focus for adaptive focus control and a heater stage for live cell imaging.

 

Focus drives

  • Motorized objective nosepiece (z-step size 10 nm)

 

Objectives

 

Magnification NA Type Immersion Coverslip WD (mm)
10x 0.3 Plan Neofluar Ph1 Air 0.17 5.6
20x 0.5 Plan Neofluar Air 0.17 2.0
40x 0.75 Plan-Neofluar Air 0.17 0.71
100x 1.54 Alpha Plan-Fluar Oil 0.17 0.11

In addition, a 1.6 lense is located in the Optovar turret position

 

Stages and incubation accessories

  • Mechanical stage from Zeiss with laser-safety kit/cover
  • The baseplate scan stage from Bruker AXS with standard sample holder
  • A heater stage for the baseplate, including temperature controller
  • Perfusion accessories from Bruker AXS

 

Condenser lens

NA 0.55, air, working distance 26 mm

 

Lamps

  • Halogen: 12V-100W
  • Mercury arc lamp HXP 120W

 

Laser

TIRF multiline Argon-laser 458, 488, 514 nm. 25mW. Directed to the microscope via a fiber and coupled to the TIRF-1 manual slider.

 

Light for epi-fluorescence and transmitted light

  • HBO 100W Illuminator, self-adjusting
  • Halogen lamp: 12V – 100W

 

Filter cubes for epi-fluorescence

 

Name Excitation filter Emission filter Dichroic
Filter Set 51 BP 453 - 465 nm BP 470 - 500 nm 470 nm
Filter Set 52 BP 483 - 493 nm BP 500 - 550 nm 500 nm
Filter Set 53 BP 540 - 552 nm BP 550 - 600 nm 540 nm
Filter Set 74 HE BP 465 - 495 nm BP 510 - 536 nm 505 nm
BP 552 - 577 nm BP 588 - 645 nm 583 nm

 

Camera

Andor iXon DU 897_BV EMCCD from Andor Technologies

 

Software on the LSM computer

ZEN BLUE 2012 with the modules

  1. ZEN Module Measurements
  2. ZEN Module Multi Channel
  3. ZEN Module Image analysis
  4. ZEN Module Time Lapse
  5. ZEN Module Z stack
  6. ZEN Module Extended Focus
  7. ZEN Module Autofocus
  8. ZEN Module Colocalization

Andor Solis for Imaging, version 4.22, from Andor Technologies