Non-mechanical Refocusing in Microscopy
The brain’s neurons are connected in 3D, and that’s challenging to study with laser scanning microscopes that natively look at one depth at a time. Existing methods of changing a microscope’s focus aren’t fast enough to catch neuronal dynamics happening on the millisecond timescale across the millimeter length-scale of neuronal connectivity.
Making things even more challenging, some neuroscience researchers are moving toward low-magnification, large-NA objectives for higher resolution over a larger field of view. Some of these microscope objectives are heavy, so they’re hard to mechanically refocus. They also tend to have large back apertures, some in excess of 30 mm in diameter, making them difficult to refocus with technologies such as liquid lenses.
To solve this problem, we took advantage of a technology we’d previously developed for 2D beamsteering, and applied it to flexible axial refocusing. Using liquid crystal polarization grating lenses (LCPG lenses) in combination with controllable liquid crystal (LC) switches, we were able to show focus changes of more than 500 micrometers in less than 40 microseconds in a multiphoton microscope.
Remote Focusing with Switchable PG Lens Stacks
|Focal Plane Change||
- Machine Vision
- Remote Focusing
Although the technologies used for recording brain activity are improving, the crucial ability to precisely stimulate and record 3D populations of neurons in a behaving animal has remained elusive until recently. A major obstacle is the inability to change focus on the sub-millisecond timescale required to catch individual action potentials.
It was this problem that motivated our collaborator, optogenetics researcher Prof. Darcy Peterka, to approach BNS about a solution for fast z-focusing that would make use of our high-speed liquid crystal polarization grating lenses.
Current strategies for changing the focus of multiphoton microscopes have disadvantages that are particularly noticeable when working at very fast timescales, or with modern microscope objectives whose back apertures can greatly exceed 25 mm diameter.
The PG lens stack is based on the switchable liquid crystal polarization grating (LCPG) technology developed by BNS in collaboration with North Carolina State University. LCPGs are very high-efficiency diffraction gratings (we usually achieve at least 99.5% efficiency at our target wavelength) whose polarity depends on the handedness of the circular polarization of the input beam. In combination with liquid crystal (LC) switchable waveplates to control the polarization of the input beam, LCPGs can produce fast, high-efficiency deflection.
We built LCPGs with concentric rather than linear grating patterns so that they would behave as switchable lenses when coupled with LC switches. The concentric grating pattern acts as a lens similarly to a Fresnel zone plate, but without its physical discontinuities between zones. The polarity of the lens (positive or negative focal length) depends on the handedness of the input beam’s polarization. By cascading multiple LCPG lens stages, we can increase the number of available focal lengths.
As with linear LCPGs, we can build PG lenses with clear apertures of more than 100mm in diameter. The number of achievable focal planes scales with the number of LC switch/PG lens stages as 2N.
For Dr. Peterka’s setup we built a two-stage PG lens stack with 30mm clear aperture. We chose the PG focal lengths to provide a >500um focus jump when used with a low-magnification objective.
In Dr. Peterka’s multiphoton microscope, we were able to use the PG lens stack to show remote focusing over >300 um (20x objective), in less than the time required for a resonant scanner line.
We measured the PG lens stack’s switching speed to be <40 μs in the fast direction and <3 ms in the LC’s slower relaxation direction. Note that this speed is independent of the amount of refocus or of the diameter of the microscope objective’s back aperture. This fast response is comparable to the time required for a single line of a resonant scanner. The focus change happens smoothly and non-mechanically, with no hysteresis or ringing.
- 40 µs
fast direction switching speed
- 3 ms
relaxation direction switching speed
- 1 J/cm2
approximate value of pulsed damage threshold
Benefits of PG Lens Remote Focusing
PG lenses are an excellent choice for imaging systems that need fast nonmechanical refocusing over large clear aperture.
- Low size, weight, and power
- <40 μs fast direction; <3 ms slow direction
- Robust non-mechanical operation
- Large apertures possible (>100 mm)
- High diffraction efficiency (>99%)
- Multiphoton microscope integration demonstrated
- Demonstrated in VIS to MWIR
- Broadband systems possible
Remote Focusing in a Two-photon Microscope
2P microscope images courtes of Darcy Peterka, Columbia University