Creating Biomedical Technologies to Improve Health

LABS AT NIBIB

Dualview Inverted Selective Plane Illumination Microscope (DISPIM)

The DISPIM is a dual light sheet microscope that acquires two orthogonal views of a sample. These two views can then be fused and joint deconvolved to generate a single volume with high isotropic resolution. Like other light sheet microscopes, the DISPIM provides rapid volume imaging with a low photodose and is therefore ideally suited for long term or high repetition rate imaging in samples where phototoxicity/photobleaching can be a problem. Unlike conventional light sheet microscopes, the joint deconvolution process provides good resolution in all directions.   

Donating Lab: Hari Shroff, NIBIB

Spatial resolution: 330nm in X,Y,Z

Temporal resolution: 200Hz framerate, 1-5Hz volumetric

Excitation wavelengths: 405nm, 488nm, 561nm, 637nm

Detection channels: 1 sCMOS camera per arm (2 arms). Emission bands can be acquired 1 at a time with full field of view or 2 at a time using a wavelength dependent image splitter. This latter approach reduces the field of view by half in X. 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): 330 microns x 330 microns x 4 millimeters

Objectives: 40X 0.8NA Water immersion (3.5mm working distance)

Sample specifications: Samples are mounted in a custom chamber in aqueous buffer (water, PBS, etc.), with no coverslip between the objectives and the sample (see diagram). The field of view is 330x330 microns. Imaging depth depends on the optical properties of the sample, however commonly imaged depths are <100 microns. Samples can be arbitrarily large in x-y as the microscope can stage scan the sample through the light sheets, acquiring 330x330 micron fields as it goes.   

Strengths:

  • Similar to other light sheet approaches, and in contrast to confocal microscopy, the only part of the specimen being illuminated is the plane being imaged. This dramatically reduces the photodose to the sample. DISPIM is ideal for imaging experiments in which photobleaching or phototoxicity is a critical issue – for example long time series requiring repeated imaging of the same sample.  
  • This is the fastest microscope currently available at AIM.

Limitations:

  • Depending on how much scattering and absorption occur in a sample, the depth to which we can acquire high quality images is typically limited to 50-100microns. The microscope itself however can accommodate much larger samples.
  • Resolution is adequate for tracking cells and larger subcellular organelles.

 

Reference:
Wu et al. Nature Biotechnology 31(11): 1032-1038, Nov 2013