The measurement of dynamic biological processes in live cells has been fundamentally limited by slow data acquisition in three dimensions. Our aim is to develop new microscopy methods to enable real-time observation of fast, 3D biological phenomena.
One method which seeks to overcome this 3D speed limit is 3D Multi-resolution Microscopy (3D-MM). 3D-MM is achieved by the seamless hardware integration of 3D target-locking microscopy and two-photon laser scanning microscopy (2P-LSM). The target-locking module yields high precision data on nanoscale luminescence targets, while 2P-LSM yields a 3D volumetric image of the live cell environment, enabling the measurement of multiscale processes, such as viral infection.
The target-locking module effectively “locks” the probe in the focus of the objective lens using an optical detection modality which senses small deviations in the X, Y and Z position. These deviations are used to drive a 3D piezoelectric stage to move the sample to counteract any particle motions allowing the 3D-MM to track particles at diffusivespeeds up to 6 µm2/sec, with a temporal precision of 10 µsec and fixed particle precision down to 10 nm.
Movie 1: Real-time 3D tracking or target-locking. The particle at the center is “locked” in the focus of the objective lens using a fast piezoelectric stage and an optical feedback loop.
Movie 2: Real-time 3D-MM measurement of the landing of a 100 nm fluorescent nanoparticle of the surface of an NIH-3T3 fibroblast cell.