Creating Biomedical Technologies to Improve Health

LABS AT NIBIB

Instant Structured Illumination Microscope (ISIM)/ Total Internal Reflection Fluorescence SIM (TIRF-SIM)

The ISIM is a fluorescence microscope that combines rapid image acquisition (100Hz) with a resolution roughly double that of a confocal microscope in all three dimensions. Unlike other structured illumination microscopes, the resolution is enhanced via hardware in a single frame acquisition rather than computationally processing multiple images, effectively making this an ‘instantaneous’ structured illumination microscope. Recently, the Shroff Lab has extended the capability of this microscope to rapid, super-resolution TIRF.   

Donating lab: Hari Shroff, NIBIB

Spatial resolution: 140nm lateral, 350nm axial (green channel)

Temporal resolution: Framerate up to 100Hz

Excitation wavelengths: 488nm, 561nm. Scheduled to add 639nm.

Detection channels: 1 sCMOS camera for detection, emission bands are acquired 1 at a time. Currently available emission bands include GFP and mCherry, or similar fluorophores. Other emission bands can be easily accommodated.

Maximum field of view (1 z-stack): 150 x 150 x 500 microns

Objectives: 60X 1.42NA Oil, 60X 1.2 NA Water, 60X 1.3NA Silicone, 100X 1.7NA TIRF

Sample specifications: The sample is imaged through a #1.5 (0.17mm thickness) glass coverslip in a standard inverted microscope geometry. Imaging depth depends on the optical properties of the sample, however commonly imaged depths are <50 microns (e.g. cultured cells). A temperature and gas environmental control chamber is available.   

Strengths:

  • Spatial resolution superior to that of a confocal microscope at substantially higher speeds.
  • Spatial and temporal resolution also available in TIRF.

Limitations:

  • Best for thin samples (<30 microns). Thicker samples induce progressively worse background and optical aberration.
  • Requires bright/photostable dyes for repeated imaging.

 

Reference:
York et al. Nature Methods 10, 1122-1126 (2013)