Felh0550

Optical measurement, including the confocal imaging, the fluorescence spectrum, and antibunching experiments, was based on our home-built confocal microscope, as shown in Fig. S15. A continuous-wave 488 nm laser was used for excitation. The laser was focused onto the sample using a high-numerical-aperture (NA = 0.95, Olympus) objective lens. The FWHM of the focal spot is 339 nm (Fig. S16). A polariser combined with a half-wave plate was used to control excitation power. For PL mapping and position, an X-Y-Z piezoelectric stage (PI instrument) was used. The collected fluorescence was filtered using a 500 nm dichroic mirror and an additional long-pass filter (Thorlabs FELH0500 or FELH0550). The signal was split by a beam splitter in the ratio of 30:70, and coupled into a grade-index fiber. One part of the signal was directed into a spectrometer (Princeton instruments) for collecting PL spectra, while the other part was directed into the two avalanche photodiodes (Excelitas, Dark count rate: around 80 Hz; Quantum efficiency: around 70% at 650 nm) for autocorrelation measurements. The fibre aperture serves as a confocal pinhole. Antibunching measurements were done using a time-correlated single-photon counting module (PicoHarp 300, PicoQuantum). The g²(t) data were not corrected for background luminescence.

Figure 1A shows a scheme of the in-house-developed fluorescence optical tweezers (FOT) based on the Olympus IX71 inverted biological microscope (Olympus, Hamburg, Germany). A strong optical trap is generated by a Nd:YAG 1064 nm laser (maximum output power 4W), and the Galvano-mirror XY scanning system enables control of the position of the trap in the sample. In the presented setup, we used two different kinds of cameras. Camera 1 (CAM1) is a fast and precise CMOS camera (MC1362, Mikrotron GmbH, Unterschleißheim, Germany) used in the calibration process [22 (link)] and in imaging samples during trap manipulation, as shown in Figure 1B. Second is the low-noise and high-sensitive camera 2 (CAM2, IRIS9, Photometrics, Tucson, AZ, USA), which is part of the fluorescence detection system, as shown in Figure 1C. Instead of the popular mercury arc lamps, in our setup, we used high-power LED with a peak wavelength of 490 nm. The set of fluorescence filters consists of excitation filter Exc.F (FESH0500, Thorlabs, Mölndal, Sweden), emission filter Em.F (FELH0550, Thorlabs), and dichroic mirror DM2 (MD499, Thorlabs). The FOT enables the forming of cellular spheroids and real-time fluorescence imaging.