i-Diagnostics and TIRF Analytix – technologies empowering Apollo Program for Biodefense

Record-high Signal-to-Background Ratio

     Among TIRF geometries that include objective- and lightguide-TIRF, prism-based scheme, as mentioned above, ensures the closest profile to theoretically predicted exponential decay of the evanescent wave, the cleanest TIRF effect with the best signal-to-background ratio. In the case of through-objective TIRF, excitation and emission channels share the same optical elements; the intensity of undesirable stray light is large, and the quality of TIRF effect is compromised [43, 44]. In prism-TIRF, the excitation is naturally independent from the emission channel. This fact and the absence of additional auto-fluorescence, light-scattering and reflecting surfaces ensures the best signal-to-background ratio [42].

 

XY translation stages

TIRF Labs offers a broad range of prism-based TIRF systems configured for inverted and upright microscopes, with fixed and variable angles of incidence (contact TIRF Labs for more information). This web page describes only the most popular prism-TIRF systems. Our pTIRF systems are designed as an add-on accessories for inverted and upright microscopes. The photo above shows the puTIRF system installed into a K-frame window of a motorized XY translation stage of an inverted microscope. puTIRF is supplied on a platform of nested design, which also can be used with manual XY translation stages, round 4-inch diameter windows of microscopes, Gibraltar platforms, or rectangular windows with the footprint of 96-well SBS plate. Optional arm shown on the photo along Y axis provides travel of the excitation spot and EW together with objective. Contact us for details: info@tirf-labs.com.

 

pTIRF Systems are compatible with dry, water- and oil-immersion objectives

In pTIRF geometry total internal reflection occurs at the interface between a slide (or a coverslip) and water or aqueous solution, as shown in the schemes above.  The TIRF prism and slide are brought in optical contact by a droplet of refractive-index-matching fluid. For excitation light, the prism and the slide represent continuous optical medium. In the case of puTIRF system a thin layer of aqueous solution and an optical window separate the TIRF surface from the objective.

 

Embedded Microfluidic Channels and Fluidics Cartridge Create Planar Low-volume TIRF Flow Cell

An advanced microfluidic system embedded into pTIRF creates closed flow cell encompassing the TIRF surface and provides high share rates at small volumetric flow rates, which allows one to measure k-on and k-off rate constants with minimal amount of bioanalyte solution. Typically, 20-40 uL of bioanalyte is sufficient for measuring a kinetic sensogram. Alternatively to the embedded fluidics, one can use reusable fluidics cartridges as schematically shown in Figure below  panels 2 and 2a. Similar to the embedded fluidics, the cartridge creates 20-40 microliter flow cell around the TIRF surface. Virtually any shape of slide or cover slip with sizes larger than 20 mm can be used with the cartridge, including 1-inch x 3-inches slides (25 mm x 75 mm), half-slides ~25 mm x 38 mm, or round slides or cover slips with diameter larger than 20 mm. An external pump, or gravity flow (the latter is always by hand) can be used with TIRF fluidics system for kinetic experiments.

 

Precision Optical-Mechanical Design of pTIRF provides high reproducibility of TIRF measurements within one experiment and between different TIRF sessions. Figure of puTIRFM above, as mentioned, shows optional optical-mechanical arm installed at the microscope frame, which holds the collimator. The arm allows for relating the excitation spot with the center of the objective, so that the evanescent wave stays with the objective, if the specimen is moved along Y axis.

 

Silica Optics includes an adjustable collimator, TIRF prism, TIRF slides, and an optical window. The range of excitation wavelengths encompasses UV-Vis-Near IR 190-1000 nm. The size of the excitation spot can be adjusted in the range 0.1mm – 12 mm. For more information contact TIRF Labs at: info@tirf-labs.com.

 

pTIRF add-on accessory is the state-of-the-art, but robust system, which combines optical, mechanical, and fluidics modules. Typically, a researcher is capable of TIRFing with pTIRF after reading the Quick Start Guide. On-site training by TIRF Labs’ personnel is available as an option. TIRF Labs offers pTIRF systems equipped with closed flow cells or open perfusion chambers, designed for upright or inverted microscopes. For more information contact TIRF Labs at: info@tirf-labs.com.

 

The pTIRF systems are compatible with dry, water-, and oil immersion objectives, as mentioned, and can be used with 1-mm thick slides or 0.12-0.24 mm glass or silica coverslips as TIRF chips. TIRF Labs offers prism-TIRF with advanced microfluidics, which allows for operating with sub-microliter amounts of solutions. We also offer pTIRF systems for TIRFing specimens in Petri dishes, as shown in the long Figure above. Most of our pTIRF accessories are factory aligned systems: the angles of incidence are fixed to provide reproducible intensity of the evanescent wave. We also offer variable-angle pTIRF systems. However, even with fixed-angle pTIRF, one can decrease the depth of penetration using special optical traps that extinguish low angles of incidence, which results in a decreased penetration depth. For more information contact TIRF Labs at info@tirf-labs.com.