Creating Biomedical Technologies to Improve Health

LABS AT NIBIB

Single molecule TIRF/Inclined illumination Microscope

This single molecule detection microscope is based on widefield illumination with the flexibility of tuning between TIRF and inclined illumination for different applications - enabling localization based imaging techniques including PALM and dSTORM. The TIRF illumination enables the monitoring of single molecule interactions on the glass coverslips or the imaging of biomolecules in a very thin layer in live cells (mostly on the basal plasma membrane). With the live cell environment system, it also allows single molecule tracking in live cells.

Donating Lab: Hari Shroff (NIBIB) and AIM internal development

Resolution: 20 nm in X,Y and 50 nm in Z after localization

Excitation wavelengths:  405 nm, 488 nm, 561 nm and 633 nm excitation laser lines

Detection channels: One EMCCD camera. Multiple emission bands are acquired sequentially. Currently available emission bands include DAPI, GFP, Cy3, Cy5, or similar fluorophores. Other emission bands can be easily accommodated.

Maximum field of view (1 z-stack): 89 x 89 x 20 microns. Typical FOV under single molecule imaging conditions is 15 x 15 microns.   

Objectives: 150X 1.49NA Oil (Olympus)

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).

Strengths:

  • Single molecule detection sensitivity in vitro and in vivo
  • TIRF or inclined illumination provides better signal to noise compared to normal widefield illumination.

Limitations:

Currently no 3D PALM and dSTORM capabilities, these may be added in the future.