## 2 Microscope Imaging Apparatus
### High-Resolution Digital Camera and Long-Distance Microscope Objective  
Although there are many options to record high-quality images under the conditions relevant to this study, the advantage of high-performance machine vision cameras or streamers, compared to dedicated, state-of-the-art high-speed cameras was discussed in the introduction (c.f. Section 1). For this work, we used a customized high-resolution digital camera equipped with a 12 Mpix CMOS sensor (AMS CMV12000), or 4096 \(\times\) 3072 pixels. The camera sensor features square, 5.5 \(\upmu\)m pipelined global shutter pixels, for image-side sensor dimensions of 22.5 \(\times\) 16.9 mm\({}^{2}\). The sensor counts 64 12-bit digitizers which can be operated at 8-, 10-, or 12-bit digital output, allowing the sensor to acquire at rates up to 330 frames per second in full-frame, 8-bit mode. The camera is connected to the computer system via fiber optics operating at 25 Gbit/s (SFP28 protocol), and streams the data directly to either the computer memory (RAM) or a dedicated high-speed storage system. As a result of limitations in camera front end electronics, the imaging system can effectively records full-frame images at acquisition rates up to 188 frames per second in 8-bit digital output mode, equivalent to 18.9 Gbit/s. A more practical metric to assess camera readout performance is the effective pixel throughput R\({}_{\text{pix}}\), which simply multiplies the selected pixel count Res\({}_{\text{img}}\) and acquisition frame rate F\({}_{\text{acq}}\) combination [16]:  
\[\text{R}_{\text{pix}}=\text{Res}_{\text{img}}\times\text{F}_{\text{acq}}, \tag{1}\]  
where Res\({}_{\text{img}}\) is the total number of pixels at which the camera can sustain the associated frame rate. The total number of pixels is calculated by summing all pixels or multiplying the horizontal (Res\({}_{\text{x}}\)) and vertical (Res\({}_{\text{y}}\)) sensor pixel counts. Under the present camera settings (full-frame, 8-bit digitization), the maximum effective pixel throughput is 2.37 Gpix/s. Such digital throughput approaches the performance of dedicated high-speed streaming cameras, or even that of entry-level high-speed cameras. Direct comparison is difficult, however, because high-speed cameras generally employ lower pixel count sensors than the 12 Mpix device used in this study. Although the exposure time is set by the LED or laser pulse, the camera was exposed for 100 \(\upmu\)s during the experiments. Such a short exposure time ensures the minimal amount of flare from the environment, but also minimizes the dark current and other exposure-related noises to be significant. The camera and illumination systems were synchronized by the camera frame output signal. This signal was used as input trigger to a 120 MHz arbitrary waveform generator configured in pulse mode. The delay between the frame acquisition output signal and the electrical pulse signal for the illumination was set to 1 \(\upmu\)s, and camera shutter and illumination jitter was below 5 ns, which is insignificant compared to the 100 \(\upmu\)s camera exposure time. The operating rate of the system was effectively determined by the camera acquisition frequency; which was set to 100 Hz for all the experiments reported in this manuscript. The long-distance microscope lens attached to the camera works in conjunction with the camera's sensor to provide the appropriate resolution at the object plane. We tested several state-of-the-art microscope objectives matching the requirements set for this work, and selected the Infinity K2-DistaMax because it offered the highest optical performance, certainly a result of the large optics used (2\({}^{\prime\prime}\) diameter). The microscope lens was equipped with the CF-3 objective, and set at 120 mm from the object plane, providing a magnification factor of 2.2\(\times\). Note that the object plane moved away from the microscope objective by 3.7 mm when VCSEL illumination was used. This is expected given that the microscope objective does not feature chromatic correction.