# TIRF light path questions

**URL:** <https://forum.microlist.org/t/tirf-light-path-questions/2050>\
**Category:** Theory\
**Created:** [January 27, 2023, 3:56pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050 "2023-01-27T15:56:03Z")\
**Posts on this page:** 4\
**Page:** 1

<div class="post-metadata">

**Author:** ![szh141](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/szh141/32/1110_2.png) [@szh141](https://forum.microlist.org/u/szh141)\
**Post date:** [January 27, 2023, 3:56pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050/1 "2023-01-27T15:56:03Z")

</div>

Hello all. I came across a TIRF light path diagram as below. Can anyone shed some light on the function/purpose of the following components?

QP, the quarter wave plate. I guess it’s to make the laser circularly polarized? What’s the purpose of a circularly polarized light for TIRF?

What’s the purpose of those two lenses before the 2D-Galvo mirrors?

The lens after the 2D-GM is to focus the laser into a spot on the ring of the objective back focal plane. Correct?

Thanks a lot!

![image](https://us1.discourse-cdn.com/flex020/uploads/microlist/original/2X/c/c617ba275f92986a8072ac1b493016fd36cf7a66.png)

---

<div class="post-metadata">

**Author:** ![talley](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/talley/32/3_2.png) [@talley](https://forum.microlist.org/u/talley)\
**Post date:** [January 27, 2023, 5:00pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050/2 "2023-01-27T17:00:09Z")

</div>

If you provide a reference/context to where you got that image, it would be helpful. But my guess is:

1. the QP is there to maximize excitation efficiency. Excitation efficiency depends on the relative orientation of the electric field (polarization) and the absorption transition dipole of the fluorophore (no excitation will occur if they are orthogonal). So, in theory, in a fixed sample, if the fluorophore isn’t tumbling freely, you could get “unlucky” with orientation and not get excitation. This is not uncommon in single-molecule applications (but I’m not sure how critical it is?)
2. i suspect the lenses are just relay lenses, (but we’d need to see the paper to tell you for sure)
3. yes, in all TIRF-like applications, you need to focus the laser spot on the back focal plane… but again, need to see the paper to tell you what that lens is doing

---

<div class="post-metadata">

**Author:** ![szh141](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/szh141/32/1110_2.png) [@szh141](https://forum.microlist.org/u/szh141)\
**Post date:** [January 27, 2023, 6:20pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050/3 "2023-01-27T18:20:08Z")

</div>

Thanks for the inputs. The paper isn’t very descriptive.

> **[Imaging Single-Vesicle Exocytosis with Total Internal Reflection Fluorescence...](https://link.springer.com/protocol/10.1007/978-1-0716-2209-4_12)**
>
> Total internal reflection fluorescence microscopy (TIRFM) provides extremely thin optical sectioning with excellent signal-to-noise ratios, which allows for visualization of membrane dynamics at the cell surface with superb spatiotemporal resolution....

---

<div class="post-metadata">

**Author:** ![szh141](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/szh141/32/1110_2.png) [@szh141](https://forum.microlist.org/u/szh141)\
**Post date:** [January 27, 2023, 6:20pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050/4 "2023-01-27T18:20:51Z")

</div>

It’s a book chapter. Page 157.