Olympus releases new TIRF microscopy objectives
Internal Reflection Fluorescence Microscopy (TIRFM) can selectively
visualise processes and structures of the cell membrane, minimising
photo-damage and allowing cells to live longer.
The four new TIRFM objectives have magnifications between 60x and 150x, and feature extremely high numerical apertures (NA), including a world record NA of 1.65.
This is important because NA indicates the resolving power of a lens, so an objective with a larger NA will be able to visualise finer details than one with a smaller NA.
What is more, a high NA allows the laser light to attain the critical angle required for total internal reflection at the cover slip-medium interface, thus enabling a shallower laser light angle and reducing penetration depth of the evanescent field induced.
As a result, the evanescent field only excites fluorophores at the interface, so unlike standard widefield microscopy the light does not travel through specimens and damage to them is reduced.
In particular, with an NA of 1.65, the APO 100xOHR objective reduces the penetration depth of the evanescent field below 100nm, providing a very fine depth of focus for the precise observation of the cell surface and membrane-associated processes.
Olympus manages this through a high signal-to-background ratio and a z-resolution that is not even achieved by confocal microscopy.
Therefore, without any out-of-focus fluorescence, excitation only occurs in the plane of observation directly adjacent to the cover slip-medium interface.
Special highly refractive cover slips and immersion oil have to be employed however to match the extreme NA.
Two other objectives, PLAPON 60xO TIRFM (NA 1.45) and PLAPO 100xO TIRFM (NA 1.45), are designed for usage with conventional immersion oil and cover glasses.
Finally, the UAPO 150xO TIRFM (NA 1.45) with its high magnification has been especially developed for single molecule applications and is the only objective of its kind on the market.
It features a compensation collar for temperature and cover glass thickness, like the PLAPON 60xO objective.
"If you want to look at individual molecules you need the UAPO 150xO, whereas for general overview of cells you can use the PLAPON 60xO and PLAPO 100xO," Hitoshi Hatano, optical engineer for Olympus, told In-Pharmatechnologist.com.
"Obviously for very specialised detailed examination you need the APO 100xOHR, you can't find a similar objective on the market."
Olympus has a long tradition of providing commercial solutions for TIRFM, with its first commercial TIRFM illuminator and an objective with sufficiently high numerical aperture to enable objective-based TIR illumination released in 1998.
The series of TIRFM objectives, a range of lasers and several multi-port illuminators enable users to tailor systems for their own applications.
With a modular approach to TIRF imaging stations that encompasses all necessary components, Olympus aims to provide its customers with completely integrated systems.
