cell^TIRF Illuminator Applications 

 Total internal reflection fluorescence (TIRF) is employed to investigate events occurring at surfaces, an area that is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology. TIRF has enabled scientists to realize significant discoveries in both intact cells and in solution.

cell^TIRF for imaging at the surface.

In a majority of these studies, functionally relevant fluorophores bound to the surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional widefield fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often masked by the ambient fluorescence from a much larger population of nonbound molecules inhabiting the adjacent detection volume. Constraint of the optical field to a refractive index interface is the basis for all total internal reflection spectroscopy, spectrometry, and microscopy investigations. 

cell^TIRF for studying co-localization.

 cell^TIRF for studying co-localization. examples above, more than one type of molecule is of interest. cell^TIRF allows precise and automated adjustment of the angle of incidence for 4 separate lasers such that the evanescent plane of illumination extends for the same depth for each wavelength. This precision enables study of colocalization of multiple molecules of interest with confidence. For example, in  ecretory granules, it is possible to study the co-localization of structural membrane components, the molecular motors that mediate trafficking, and the granule contents as the secretory granule appears at the plasma membrane. As the membranes fuse and contents are released, the different components that are each labeled with a different fluorophore will remain in, or move out of, the planeof illumination. The cell^TIRF system ensures valid functional interpretation of image data by facilitating the exact same evanescent wave depth for all fluorophores. The 100X apochromatic objective with its 1.65 numerical aperture provides the maximum spatial resolution and optimal conditions for study co-localization by producing the thinnest optical slice at the surface of the specimen. The larger proportion of the back focal plane allows a wider range of supercritical angles of incidence within which TIRF will occur, 48 to 68 degrees. Not only does this make TIRF easier to attain but also gives additional control of a range of depths.

cell^TIRF for molecular studies.

 On featureless non-microscopic specimens to measure fluorophore concentrations or to record binding/unbinding equilibria and kinetic rates at a biological surface. Other applications include single molecule fluorescence experiments and model membranes, which have been constructed using the substrate for mechanical support. The technique is also useful for investigating the emission of fluorophores bound to surfaces. These and other experiments are designed to examine the chemistry and physics of interfaces themselves, and should continue to be the focus of TIRF microscopy studies in many diverse fields. Single molecule studies in particular benefit from TIRF’s enhanced signal-to-noise ratio of surface-bound molecules, without background emission from molecules in the adjacent solution. Olympus’s 150X TIRF objective provides ideal magnification for Nyquist sampling when using large pixel, high-sensitivity detectors commonly used in single molecule detection. High transmission down to 340nm makes it possible to perform TIRF microscopy with UV applications such as un-caging and/or photo-activation with the same lens.

 cell^TIRF for imaging living cells.

 Living cells in culture provide an excellent candidate for TIRF investigations. The technique enables selective visualization of contact regions between individual cells and the substrate, even in specimens where fluorescence from areas outside the surface would obscure important fluorescent information concerning adhesion points. Because illumination is restricted to the interface regions and does not penetrate the specimen bulk, living cells tend to survive longer under fluorescence observation using TIRF techniques. This feature enables microscopists to increase the length of observations and to perform time-lapse imaging for extended periods, often ranging from many hours to one or more days.

TIRF illuminator

Available laser Systems

405nm, 445nm, 473nm, 488nm, 515nm, 532nm, 561nm, 594nm, 640nm

Laser coupling

FC or FC8 connector

Field Number

FN11.8

Illuminator Body

Single Billet Aluminum

Safety

Integrated Interlock

Laser systems

Wavelengths

405nm, 445nm, 473nm, 488nm, 515nm, 532nm, 561nm, 594nm, 640nm

Power

Laser system available up to 100mW

Attenuation

Software control of laser intensity

Imaging Shutter

High speed 1msec TTL shutters

Safety

Integrated interlock

Epi-Fluorescence

Light source

100W Mercury, 75W Xenon, Metal halide.

Selection

TIRF/Brightfield or Brightfield only with 340nm transmission

Field number

FN22

TIRF Objectives

PLANAPON60XOTIRFM

PLANAPO 60X, NA 1.45, WD 0.1mm, Correction collar

APON60XOTIRFM

APO 60X, NA 1.49, WD 0.1mm, Correction collar

PLAPO100XOTIRFM

PLANAPO 100X, NA 1.45 WD 0.10mm

UAPON100XTIRF

UAPON 100X, NA 1.49 WD 0.09mm, Correction collar

AP0100XO-HR-SP

APO 100X, NA 1.65, WD 0.10mm

UAPON150XOTIRF

UAPO 150X, NA 1.45,WD 0.07mm,Correction collar

Immersion Oil

Type-F

Low Auto Fluorescence Immersion oil

High Refractive Index

RI 1.78 Immersion oil for use with AP0100XO-HR-SP 1.65NA 100X objective

Coverslips

High Refractive Index

RI 1.78 glass coverslips for use with AP0100XO-HR-SP 1.65NA 100X objective 

Compatible microscopes

IX71, IX81 and IX81-ZeroDrift