Introduction to FAQ

Schematics of lgTIRFM in comparison with through-objective TIRFM. TIRF fiber optics Adapter and beam Conditioner (TAC) conducts light from the illuminator and couples the beam into TIRF lightguide. TIRF lightguide is a rectangular coverslip 22 x 40 x 0.15-0.17 mm. After entering the TIRF lightguide, the excitation beam reflects multiple times from its top and bottom surfaces and generates wide-area evanescent wave at the center of 22 x 40 mm coverslip. Sizes of the evanescent wave area are approximately 10 x 10 mm. lgTIRFM system is mounted at a 160 x 110 mm plate, which fits into windows of Ludl or Prior motorized translation stages; it also can be installed on the top of manual XY stages. lgTIRFM can be used for epi-fluorescence and other microscopy methods. It takes no time to switch between epi- and TIRF mode.

We offer TIRF lightguides/coverslips made of glass, silica, and sapphire. Glass and silica coverslips/TIRF lightguides are compatible with dry and water immersion objectives. For use with oil-immersion lenses, we offer sapphire coverslips at $45/ea. Sapphire, silica, and glass lightguides are reusable and with proper care can provide years of service. We offer selected low autofluorescence cover slips as TIRFM lightguides. At your own risk you can use coverslips from other suppliers. See Frequently Asked Questions for more details. We offer clean and chemically modified, sterile and sterilizeable coverslips with mounted open perfusion chambers. We also offer separate components – plain coverslips/TIRF lightguides, perfusion chambers, and mounting compound - along with assembly instructions.

Frequently Asked Questions

Q1. What are the advantages of lgTIRFM with respect to through-objective TIRFM?

A1. lgTIRFM exhibits better signal/noise and signal/ background ratios, because the excitation light, which generates the evanescent wave, escapes the lightguide from the opposite end without interfering with the emission channel (see schematics above) . In the case of through-objective TIRFM, the excitation beam travels through the objective, totally reflects at the interface between glass and water, and travels back into the objective (see the schematics). Scatter and reflections at multiple interfaces within the objective, and leaking of the excitation light through dichroic mirror and emission filter result in larger background and noise. In lgTIRFM, the excutation beam does not enter the emission channel. Similar advantage of prism-based TIRFM over through-objective TIRFM, has been documented in the literature [Ambrose WP, et al., Cytometry, 1999, 36, p. 224.]

Additional advantages include:

lgTIRFM is compatibile with dry, water- and oil-immersion objectives. Through-objective TIRFM is compatible only with specialized TIRFM objectives.

lgTIRFM is compatible with virtually any illuminator: LED, halogen, Xe- and Hg-arc lamps, and lasers. Available excitation wavelengths- 250-800 nm. Typically, through-objective TIRFM uses only lasers light sources.

lgTIRFM is factory-aligned, easy to install/uninstall system. lgTIRFM generates reproducible pattern of evanescent wave. In the case of through-objective TIRFM, the necessity of alignment results in irreproducible pattern and varioations of intensity of the evanescent wave between experiments.

lgTIRFM generates the evanescent wave at area of approximately 10 mm x 10 mm in the center of TIRF lightguide. Larger area of the evanescent wave (as opposed to small area in through-objective TIRFM) is convenient for panoramic imaging of a group of interacting cells. Then, without changing anything in TIRF system, you can change the objective to take a closer look at the object of interest. For microarray studies, this advantage of large area evanescent wave allows for simultaneous measurement of responses from multiple spots in the array. The latter feature is of paramount importance for normalization of TIRF signal, ratiometric measurements and monitoring of responses of internal controls printed in the same microarray. Typically, through-objective TIRFM provides only sub-millimeter size of the evanescent wave, which "travels" with the objective. You cannot use other than specialized TIRFM lenses to TIRF your specimen through objective.

Q2. What are disadvantages of lgTIRFM?

A2. At equal power of the excitation beam, in lgTIRFM the same amount of power is “diluted” over larger area than in through-objective TIRFM. To obtain equal intensity of the evanescent wave, lgTIRFM requires use of more powerful illuminators. Typically, this does not create a problem, because lgTIRFM is compatible with virtually any illuminator.

Q4. . Would rectangular (22x40), 0.17 mm coverslips purchased from an outside source (Fisher for example) work equally well on your system?

A4. Yes, coverslips purchased from other sources may work with lgTIRFM-170, if your application does not require the lowest background. If you do not intend to detect single molecules or employ other low-light applications, glass coverslips purchased from other sources may work as well as our selected low autoflurescence coverslips. Please remember that glass and silica coverslips are compatible with dry and water immersion objectives. For lgTIRFM with oil immersion lenses one should use sapphire coverslips. See also second paragraph in Introduction to FAQ.