Introduction to TIRF Microscopy
Total Internal Reflection Fluorescence (TIRF) microscopy restricts excitation to a thin layer near the sample surface, often less than 100 nanometers. This technique dramatically improves signal-to-noise ratio for surface-proximal imaging. That performance depends on filters and dichroics that can handle excitation energy re-entering the emission path at high angles, something standard fluorescence optics aren’t built for.
An application note from Chroma Technology covers how evanescent field excitation works, what through-the-lens TIRF demands of optical filters, and how Chroma’s substrates, coatings, and cube hardware are built to meet those demands.
The Application Note covers these topics:
- How evanescent wave excitation restricts fluorescence to the sample surface
- The shift from prism-based TIRF to through-the-lens systems
- Why through-the-lens TIRF places extreme blocking demands on emission filters, exceeding OD 8
- Reflected wavefront distortion requirements for TIRF dichroics, and why they’re tighter than confocal
- How Chroma’s substrates, coatings, and TIRF-optimized filter cubes are built to meet those requirements
- Representative TIRF filter set configurations and their spectral performance
Target Audience for This Document
This application note was written for researchers and imaging core staff working with TIRF, single-molecule imaging, or other surface-proximal fluorescence techniques, particularly those specifying or troubleshooting filter sets for a through-the-lens TIRF system.
Read the document straight through for the full picture of how TIRF optics work, or keep it as a reference when specifying filters for a TIRF setup.
About Chroma Technology
Chroma Technology is a leading manufacturer and OEM supplier of high performance optical filters used in a wide range of applications, such as communications, inspection and metrology, astronomy, life sciences and health care, microscopy, calorimetry, machine vision, and Raman spectroscopy. Chroma offers an extensive selection of optical filter sets and filter accessories, as well as custom solutions tailored to customer-specific needs
For more information or a quotation for Chroma products, contact IL Photonics.