# Three-Dimension Resolution Enhanced Microscopy Based on Parallel Detection
## 2 Methods
### Experiment Setup  
The 3D-PRFED system set-up is depicted in Figure 4. Excitation is provided by a 640 nm laser (PDL828 Sepia II, PicoQuant, Berlin, Germany) with a pulse frequency of 80 MHz. For the sake of making sure that vortex modulated light can incoherently combine with hollow modulated light, a light path delay (\(\sim\)100 ps) was introduced between the p-polarization component and s-polarization component of excitation light at first. The excitation beam then passed a half wave plate before projecting into the SLM (X13139 LCOS-SLM, Hamamatsu Photonics K.K., Hamamatsu, Japan), which could adjust the angle of the polarization direction of excitation light and made sure that the direction of p-polarization component was parallel to the module direction of SLM. The p-polarization component of excitation light was modulated by the R part of SLM. A double pass through a quarter wave plate (QWP) rotated two polarization states by 90 degrees, which changed the original s-polarization into p-polarization that could be modulated by the L part of SLM, while the original p-polarization became s-polarization and would not be affected by the pattern in the L part. The lens L1 and mirror M4 and lens L2 and lens L3 were made up as 4f lens groups, which ensured that the R part and L part of SLM were conjugated with the x galvanometer. After the TriScanner, the excitation beam would continue to pass through a scan lens (f = 50 mm) and a tube lens (f = 200 mm) which were also constituted as a 4f lens group to ensure the x galvanometer was conjugated with the entrance pupil of OL. The conjugation relation can make sure that SLM is operating at the Fourier plane of OL. Finally, a 100x objective lens (OL, Apo TIRF, 100\(\times\)/1.49 NA, Nikon, Tokyo, Japan) was used to focus the excitation light on the sample plane. The TriScanner, used for deflecting excitation light to scan the sample and de-scanning the fluorescence signal from the sample into the fixed detector array, consists of three galvanometers, two deflecting mirrors (X and Y1) and one correcting mirror (Y2) as shown in Figure 4 inset (b). It should be noted that the mirror X close to the scan lens is conjugated with the entrance pupil of OL, which makes sure the SLM can be conjugated with the entrance pupil of OL. The correct mirror Y2 should make sure the defecting light from Y1 mirror is projected into the conjugate point of X mirror, which means that theoretically the correct mirror should be rotated double angle than Y1 deflection mirror. Figure 4: Schematic of 3D-PRFED setup. HWP: half wave plate; SLM: spatial light modulator; QWP: quarter wave plate; DM: dichromatic mirror; TriScanner: scanner module using 3 galvanometers; SL: scanning lens; TL: tube lens; OL: objective lens; M1 \(\sim\) M6: mirror; F: fluorescence filter; L: lens; PC: personal computer; DAQ: data acquisition card; APD: avalanche diode detector. inset: (**a**) the fiber bundle cross-section used as the detector array; (**b**) the triscanner sketch; (**c**) the modulated pattern on the SLM. The excited fluorescence (\(\sim\)670 nm) was collected by OL, relayed by TL and SL, descanned by the TriScanner, separated with the illumination light by a dichroic mirror (DM, ZT405/488/561/647 rpc, Chroma, Bellows Falls, VT, USA), and then passed through a fluorescence filter (FF660-Em02-B, Sernrock, Rochester, NY, USA) and focused by a lens (L4, f = 400 mm) onto a fiber bundle consisting of 19 fibers (125 \(\upmu\)m cladding diameter and 105 \(\upmu\)m core diameter) that transfer the fluorescence signal to 19 avalanche photodiodes (APD, SPCM-AQRH-16-FC, Excelitas Technologies, Waltham, MA, USA) respectively. The fiber bundle and 19 APDs are made up of our detector array. The whole system was controlled by a DAQ box (NI Card 6366, National Instruments, Austin, TX, USA) connected with a PC and operated by a LabView program.