Multicellular Optical Imaging

Phase-Sensitive Single Molecule Localization Microscopy (SMLM)
A simulated example of the new technique as described, imaging a stained microbead with the numerical approaches on the righ two images and standard imaging on the left.

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Randy Bartels

Jeff Fields

Reference No: 2021-017
Licensing Manager

Aly Hoeher

At a Glance

Researchers at Colorado State University have developed phase-sensitive SMLM that will increase imaging volume while remaining insensitive to aberrations and without sacrificing precision. The application of a novel optical imaging technique based on illumination intensity modulation called Coherent Holographic Image Reconstruction by Phase Transfer (CHIRPT) allows for expanded capabilities of localization-based super resolution microscopy. This new approach for SMLM enables super resolution studies to be taken from single cell preparations to multiple cells and other complex biological tissue environments.


Super-resolution (SR) imaging has evolved into a vital component of numerous biological investigations that rely on fluorescence microscopy. Emerging from the multitude of techniques that enable imaging beyond the diffraction limit, single molecule localization microscopy (SMLM) is routinely used to provide ~20 nm resolution in a myriad of samples.

The concept of SMLM is straightforward: given the diffraction-limited spatial impulse response of a microscope – the point-spread-function (PSF) – that is known to originate from a single molecular emitter, the precise location of an isolated fluorescent molecule can be determined by computing the centroid of a PSF measured from the molecule. Though the concept is simple, SMLM has been applied to stunning effect, leading to a range of discoveries so diverse and impactful that the Nobel Prize in Chemistry was awarded, in part, to the pioneers of SMLM in 2014.


Still, the development of SMLM is not complete. A variety of limitations restrict the range of investigations that can be carried out with SMLM. For example, the 3D volume over which SMLM can localize a fluorophore is restricted. There is a need to develop SMLM methods that will localize with <50 nm precision over volumes more than 100x greater than those provided by the current state of the art.


A new method for SMLM that increases the imaging volume >100x without sacrificing localization precision. To achieve large-volume SMLM, researchers employ a newly developed microscopic technique called Coherent

Holographic Image Reconstruction by Phase Transfer CHIPRT, which does not require image formation on a camera. CHIRPT enables holographic-like imaging of fluorescent molecules with a single-element detector by transferring the phase of spatially coherent illumination light to temporal modulations of the spatially incoherent fluorescent light emitted by a molecule. CHIRPT provides both intensity and phase information that will be jointly exploited to determine the location of isolated fluorescent emitters with precision and imaging volume exceeding those of camera-based SMLM methods.

Simulations and theoretical calculations (Cramér-Rao lower bound (CRLB)) show that information encoded in CHIRPT illuminations enhances localization precision even when a lower magnification objective lens is used, thereby enabling much longer working distances.

By leveraging CHIRPT, investigators demonstrate the first SMLM technique that determines the location of emitters within a volume in a specimen that exceeds 100x the limit of camera-based 3D SMLM. The approach retains high localization precision (<50 nm) with low NA illumination (0.5 NA) – enabling the application of SMLM to large tissue regions and complex multicellular cultures for the first time.

Figure 1 (below) illustrates the improvement in the depth of field (DOF) when utilizing the CHIRPT method compared to tight focusing at various wavelengths.

Figure 1. Improvement in CHIRPT depth of field compared to tight focusing at four wavelengths: 405 nm, 488 nm, 561 nm, and 647 nm. Parameters: n=1 and w=50µm.
  • Enables the application of SMLM to large tissue regions and complex multicellular cultures for the first time
  • Can determine the location of a molecular emitter over volumes approaching 100x the limit of camera-based 3D SMLM
  • Enhanced robustness to optical scattering, leading to higher image resolution
  • The ability to measure more fluorescent photons by combining signals from multiple detectors simultaneously
  • Multicellular culture imaging
  • Tissue engineering
  • Study of cellular interactions and movement across a volume

Last updated: January 2023