A Modular Approach to Active Focus Stabilization for Fluorescence Microscopy

Fluorescent time-lapse experiments often suffer from focus drift, regularly rendering long measurements partially unusable. Frequently, this instability can be traced back to the specific mechanical components of the setup, but even in highly robust implementations z-drift occurs due to small temperature fluctuations which are hard to avoid. To resolve this issue, microscope manufacturers often offer their own interpretation of out-of-focus correction modules for their flagship instruments. However, self-assembled or older systems typically have to fend for their own or adapt their measurements to circumvent drift effects. In this manuscript, we propose a cost-efficient z-drift detection- and correction system that, due to its modular design, can be attached to any fluorescence microscope with an actuated stage or objective, be it in a custom or commercial setup. The reason for this wide applicability is specific to the design, which has a straightforward alignment procedure and allows sharing optics with the fluorescent emission path. Our system employs an infrared (IR) laser that is passed through a double-hole mask to achieve two parallel beams which are made to reflect on the coverslip and subsequently detected on an industrial sCMOS camera. The relative position of these beams is then uniquely linked to the z-position of a microscope-mounted sample. The system was benchmarked by introducing temperature perturbations, where it was shown to achieve a stable focus, and by scanning different positions while simulating a perturbation in the z-position of the stage, where we show that a lost focus can be recovered within seconds.

also opens up opportunities to mount it on other popular and conventional microscope systems. 66 In particular, the system is optimized for microscopes in which the optical infrared path and the 67 fluorescence path are shared, maximally eliminating reflections and noise. We show that our 68 method works well through experiments with beads and a varying temperature, and on COS-7 69 cells with manual z-dispositioning.

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The idea of this work is to use a detection technique similar to that of Binh et al. [11] to 72 automatically find the focus position for our microscope. This focus detection method uses 73 a collimated infrared laser to determine the sample position. In front of the laser, we put a 74 double-hole mask, which divides the collimated laser light into two parallel beams. These beams 75 are sent through the microscope body onto the sample, which is carried by a glass coverslip. It is 76 exactly this glass-sample interface that reflects the laser beams. The reflected beams are captured 77 by an industrial sCMOS camera.   two reflected laser spots. The horizontal distance between the detected spots is proportional to 80 the z-position of the cover glass. The bigger the distance between the two detected spots, the 81 further away is the sample from the zero of the z-position. Before starting an experiment, the 82 user can define the perfect focus position of the sample on the microscope. The distance between 83 the spots on that position determines the focusing distance. Control software then continuously 84 adjusts the z-position to retain or regain focus, even when it is lost, by tuning the distance between 85 the detected spots using a motorized stage or a mechanically actuated objective.

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The system has several advantages over existing approaches. Firstly, whereas other systems 87 [7, 9, 12] typically have a dedicated path for the z-tracking laser, our system is designed to 88 share their fluorescence path with the z-tracking laser path, which involves including dedicated 89 components to filter out spurious reflections on intermediary components (e.g., tube lens).

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Secondly, for the implementation given in Appendix A, the total price of the system comes to less 91 than Ä2000, a fraction of the cost of a commercial system. Thirdly, as the system focuses the two 92 beams on the glass-sample interface, it presents a relatively safe way of using infrared light as any

Infrared Beam Path
The infrared beam path starts from the infrared laser (the leftmost 117 component in Figure 3). This laser is a 3.0 mW, 830 nm infrared laser, chosen to avoid overlap 118 with the color channels used for fluorescence detection. This laser is mounted in a pitch/yaw 119 adapter which, with addition of an XY-mount and a rotation mount, allows for full freedom in 120 aligning the laser beam on a double-hole mask containing drilled holes that are 600 µm apart and 121 500 µm wide ( Figure 4 shows the mask and its dimensions).

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Next, two mirrors M1 and M2 guide the two beams toward the objective-sample interface while 123 a neutral density filter (ND1) reduces their power to less than 1 mW (safety category 2). Lenses 124 L1 and L2 are set up as a 2F relay. A 50/50 beam splitter (BS) passes half the light coming from 125 the laser into the direction of the microscope, while the reflected half is disposed in a beam dump.

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We have validated our z-drift correction system by visualizing the mitigation of temperature-154 dependent drift on a fluorescence microscope. As a sample, we have used sparsely dispersed 200 155 nm Tetraspeck beads. We use an UMPlanFI 50⇥ objective, with a numerical aperture of 0.80.

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The Spectra X light source is set to 50% at a band centered around 542 nm, a Chroma ZT561RDC When we repeat the same experiment with the focus detection-and correction system enabled, 166 Figure 9 shows that the images remain in focus. The focusing distance and z-position also stay 167 between the predefined desired bounds. Similar results were achieved with a UPlanSApo 10⇥ 168 objective with a numerical aperture of 0.40 and a working distance of 3.1mm (data not shown).

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To study the stability of our system on focus perturbations, we image COS-7 cells expressing

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We have described and built an automated out-of-focus detection-and correction system that is 197 compatible with any microscope that has an actuated stage or objective. This automatic focusing