All-optical microscope autofocus based on an electrically tunable lens and a totally internally reflected IR laser

Abstract

Microscopic imaging at high spatial-temporal resolution over long time scales (minutes to hours) requires rapid and precise stabilization of the microscope focus. Conventional and commercial autofocus systems are largely based on piezoelectric stages or mechanical objective actuators. Objective to sample distance is either measured by image analysis approaches or by hardware modules measuring the intensity of reflected infrared light. We propose here a truly all-optical microscope autofocus taking advantage of an electrically tunable lens and a totally internally reflected infrared probe beam. We implement a feedback-loop based on the lateral position of a totally internally reflected infrared laser on a quadrant photodetector, as an indicator of the relative defocus. We show here how to treat the combined contributions due to mechanical defocus and deformation of the tunable lens. As a result, the sample can be kept in focus without any mechanical movement, at rates up to hundreds of Hertz. The device requires only reflective optics and can be implemented at a fraction of the cost required for a comparable piezo-based actuator.

A micromirror array with annular partitioning for high-speed random-access axial focusing

“…Under a 2% settling time metric, the mean response time was measured to be 114 μs for a refresh rate of 8.75 kHz. Under a 10–90% settling metric for suitable comparison against alternative approaches to axial focusing, the mean response time was measured to be 64.8 μs for a refresh rate of 15.44 kHz, which is roughly two orders of magnitude faster than the current commercial optofluidic and liquid crystal-based varifocal systems 9 , 15 . Overall, these response measurements demonstrate operating speeds that match the 10 kHz benchmark achieved by galvanometer mirrors and that could be raised even further under optimized damping or drive shape conditions.…”

“…Currently, the most prevalent approaches to dynamic axial focusing achieve focus tuning by deforming or reorienting optofluidic 9 , 10 , elastomeric 11 or liquid crystal-based 12 lens components. While such technologies offer straightforward actuation mechanisms, their lagging performance capabilities are increasingly apparent relative to accompanying optical components, especially lateral scanning tools that are often used in conjunction with axial focusing for joint 3D scanning capabilities 1 .…”

Using time-lapse fluorescence microscopy to study gene regulation

“…When the sample deviates from the pre-determined focal plane, the reflected LED beam will shift on the sensor, triggering a feedback to move it to the focal position. Such feedback can either be done by physically moving the objective using a piezoelectric stage, or with an electrically tunable lens that can adjust the focal point with a current [22]. These autofocusing modules are accurate and fast, and since they are controlled internally by the microscope, they do not require any additional software.…”

VIEW-MOD: A Versatile Illumination Engine With a Modular Optical Design for Fluorescence Microscopy

“…It should be noted that large scale changes of the optical power of ETL3 will (de)magnify the image [17,34]. Other groups have used an ETL for axial control of the imaging plane by placing the ETL directly behind the detection objective [17,[34][35][36][37], however using ETL3 in a 4f system makes it easier to access and adjust as well as minimizes the (de)magnification effects [32,33]. We ensure that ETL3 lies flat (its optical axis is normal to the table) to avoid gravitational effects which would distort the Optotune lens.…”