Bio-monomer preparation

A protein, BSA (Sigma-Aldrich, USA), was employed as the reactive monomer with RB incorporated (Avocado Research Chemicals, UK) as the photoactivator in the fabrication solution. All chemicals and reagents were of analytical grade. According to our previous point-scanning MPE experimental results, 20 mg/ml of BSA solution with approximately 2.0 mM RB is appropriate. Herein, the high-throughput microfabrication is a collective reaction, so the BSA in the fabricated solution and the RB concentration were increased to 150 mg/ml and 20 mM, respectively. Then, 30 μl of fabrication solution was confined in a small chamber created using a 40 μm thick adhesive tape as a spacer to separate a 0.17 mm cover slip and the microscope slide.

During fabrication, structures were created from the bottom (slide) to the top (slip) to prevent the incoming patterned excitation from being distorted by previously developed microstructures. Furthermore and according to our previous experiment, the two-photon absorption peak of RB is around 715 nm;

nevertheless, the range of the ti-sa ultrafast amplifier can be adjusted from 750 nm to 850 nm. The shortest laser wavelength of 750 nm was adopted and is sufficient for the TPC process.

4.3 Experimental results and discussions 4.3.1 System calibrations

Based on our experimental experience, control of the laser peak power and pulse number is a key parameter due to the efficiency limits of the TPC process.

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If the peak power is too low, MPE photochemistry in widefield excitation cannot be achieved; on the other hand, if the peak power of a single pulse is too high, damage to bio-samples, such as BSA, might occur within only a few pulses. The laser peak power of the ti-sa ultrafast amplifier is difficult to control evenly and precisely; hence, a suitable peak power level was initially chosen, and then the pulse number was controlled to accumulate an appropriate dose. Two mechanisms are implemented for precisely accumulating the global and local doses in the high-throughput MPE system, namely, the gated mode operation of the amplifier system and the DMD chip, respectively. Under gated mode operation, our LabVIEW program synchronizes with the amplifier system; hence, the global pulse number of the laser incident on the sample per layer can be selected. For an 8-bit DMD, the 0~255 gray levels represent different “on” state times, which means that it can act as a pulse selector to tune the laser pulse optional on or off in the fabrication area. In other worlds, the DMD gray level acts as an optical shutter that adjusts the local pulse number at every layer. For example, for an amplifier repetition rate of 10 kHz, a gray level of 255 represents 10,000 pulses per second, while a gray level of 128 refers to 5,000 pulses per second, and so on. Under the gated mode operation, the relationship between the normalized TPEF intensity and the laser pulse number at the fluence of 35 μJ/pulse is presented in Fig. 4-2(a). The correlation is approximately linear and nearly matches theoretical predictions. Figure 4-2(b) illustrates the TPEF intensity as a function of different DMD gray levels, with the gated mode operation controlling the global pulse number at 1000, and

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where the fluence of each pulse is also 35 μJ. Again, the relation is almost linear, thus providing further evidence that the DMD can reliably act as a second pulse selector to locally control the pulse number. Based on the two figures, our strategy for fabricating 3D gray-level BSA microstructures employs the gated mode operation to initially decide the global pulse number, and then locally assigns the pulse number via the gray-level DMD.

With the global and local pulse number selectors, the lateral resolution for fabricating crosslinked BSA structures requires clarification for further processing. According to the current adjusted magnification of the system, a fabrication area of 76 × 43 μm² corresponds to the 640 × 360 pixel number of the DMD chip, indicating that 1 pixel corresponds to roughly 120 × 120 nm² on the sample. Three stripe patterns with pixel numbers of 46, 23, and 14, with respective corresponding widths of around 5.5 μm, 2.7 μm, and 1.6 μm, were designed to examine the lateral resolution. However, since the beam profile is a Gaussian distribution and BSA photocrosslinking requires a threshold fabrication energy to initiate the TPC process, the actual fabricated sizes are smaller than the designed sizes. Figures 4-3(a) and 4-3(b) show that the fabricated widths of the stripes are approximately 2.3 μm and 1.1 μm, which correspond to the designed widths of 5.5 μm and 2.7 μm, respectively.

Experimental results indicate that both widths of the fabricated stripes are reduced approximately 60 %. However, the fabricated width of the smallest stripe corresponding to the designed size of 1.6 μm is also around 1.1 μm (data not shown here), which may result from the diffraction limit and/or system

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distortion. Consequently, the lateral fabrication resolution of the current system is around 1 μm.

The potential to separate layers vertically is dependent on the axial fabrication resolution of the system. According to our previous study [30], an axial imaging resolution of less than 3.4 μm can be provided as the maximum laser power is less than 40 mW. For fabrication, the TPP and TPC processes use photoinitiators to trigger reactions, and so an optical energy threshold to initiate the process is required, which can lead to the axial fabrication resolution being better than that of the imaging. Furthermore, the axial fabrication resolution is related to the material used; hence, an axial frication resolution of less than 2 μm can be achieved for BSA structures in the current system. Besides having the pulse selecting capability, the DMD can further enhance the axial resolution of the system. In the original temporal focusing setup, the pulsing beam is spatially dispersed via a grating in the meridian plane; then, the spatially dispersed frequencies go through a 4f setup to realize temporal focusing excitation. Due to this setup, the beam is focused as a line on the back-focal plane of the objective lens. The incorporation of the DMD into the system can also function as a grating. Therefore, the different frequencies of the pulsing beam can be spatially dispersed again in the sagittal plane. Consequently, the width of the line becomes wider on the back-focal plane. As increasing the beam coverage area on the back-focal plane, a larger NA of the system is utilized. Therefore, the axial resolution is improved. Moreover, the higher the chosen order of the DMD diffraction pattern is, the larger the NA is utilized.

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Figure 4-2 (a) Normalized TPEF intensity as a function of different pulse number under the gated mode operation. Fluence of each pulse is 35 μJ. (b) Relationship between the normalized TPEF intensity and the DMD gray-level with gated mode operation to control the global pulse number of 1000.

Figure 4-3 TPEF images and profiles of two stripes fabricated with the pixel numbers of (a) 46 and (b) 23, the fabricated widths of which are 2.3 μm and 1.1 μm, respectively. The profiles correspond to the red dash line in the TPEF images. Inset: corresponding 2D front-view bright-field images.

(a) (b)

(a) (b)

5 μm 5 μm

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4.3.2 Online imaging and inspection of BSA microstructures