Home › Cell Biology › A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors
Cell Biology JoVE (Open Access) Citable · DOI

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

DOI: 10.3791/53988-v
What you'll learn
  • ✓ Assemble and align a structured illumination TIRF microscope optical path
  • ✓ Calibrate polarization rotators and emission detection channels
  • ✓ Synchronize hardware and acquire multi-color super-resolution image stacks
Protocol

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Difficulty
advanced
Total time
~4–8 hours (initial setup and alignment); ~30–60 min per imaging experiment

Steps

1
Arrange and align excitation illumination path

Position and align laser lines, dichroic mirrors, and objective lenses to establish the TIRF excitation geometry. This step establishes the foundation for structured illumination pattern delivery to the sample.

▶ 00:54
2
Align polarization rotator and emission path

Configure polarization optics and adjust the emission filter turret and detector alignment to separate and collect fluorescence from multiple spectral channels with minimal crosstalk.

▶ 04:01
3
Synchronize system hardware and calibrate channels

Coordinate timing between lasers, spatial light modulators, and cameras; calibrate pixel registration and phase offsets for computational image reconstruction.

▶ 06:42
4
Acquire multi-colored super-resolution image sequences

Perform live-cell or fixed-sample imaging using the fully configured TIRF-SIM system to record high-speed, spatially structured frames for downstream reconstruction and analysis.

▶ 08:23
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