Application suite advanced fluorescence lite

The 20 µL of purified and re-suspended bacterial suspension (1.0 × 10⁷ CFU) was incubated with GP-dAuNPs@Ce6 (1.0 mg mL⁻¹, 200 µL) for 2 h in a shaking incubator (200 rpm) at 37 °C. The bacteria were harvested by centrifuging the mixture at 3381 × g for 5 min in Eppendorf (EP) tubes. The resulting bacteria were re-suspended and washed with PBS for three times. Then 10 µL of the washed bacteria solution was transferred onto a microscope slide covered by a coverslip, and then imaged by a confocal laser scanning microscope (CLSM, Leica, TCSSP5 II) with 30% power of diode laser. All fluorescence images were captured by CLSM with a × 64 oil-immersion objective and taken under the same optical conditions, and the same brightness and contrast were applied to the images by the microscope automatically. The processing and analysis of ROI were performed by the commercial image analysis software (Leica Application Suite Advanced Fluorescence Lite). Moreover, the distribution of GP-dAuNPs@Ce6 in the bacterial cells were confirmed by TEM (Philips CM 200).

The 20 µL of VNP or EC suspension with ~1.0 × 10⁷ CFU was purified and resuspended, followed by incubation with GP-ICG-SiNPs (15 mg/mL, 200 µL) in a shaking incubator at 200 rpm, 37 °C for 2 h. The constructed Trojan bacteria were harvested by centrifugation at 4508 × g for 10 min. Afterward, the harvested Trojan bacteria were resuspended again and washed with PBS buffer at least 3 times to remove excess GP-ICG-SiNPs or nonspecifically absorbed GP-ICG-SiNPs. To characterize the constructed Trojan bacteria, the solution containing Trojan bacteria of 10 µL was transferred onto a microscope slide and covered by a coverslip, followed by confocal laser scanning microscope imaging (CLSM, Leica, TCSSP5 II) with 30% power of diode laser. Of note, all fluorescent images were captured under identical optical conditions. The processing and analysis of the region of interest (ROI) were performed by the image analysis software (Leica Application Suite Advanced Fluorescence Lite). The morphology and structure of Trojan VNP and Trojan EC were characterized by TEM and SEM.

Image processing and analysis was conducted using Leica Application Suite Advanced Fluorescence Lite and ImageJ. Nuclear areas are expressed as a ratio of the average nuclear area in GFP marked clones to neighbouring cells (GFP negative). For each genotype over 50 cells were quantified from at least three egg chambers derived from separate animals. ANOVA was performed to check for significant differences between data groups. Significant differences were attributed for p<0.05.

The 20 µL of purified and re-suspended bacterial suspension (1.0 × 10⁷ CFU) was incubated with GP-Si-BPs (0.06 mM, 200 µL), GP-Si-Luc (0.06 mM, 200 µL), GP-Si-BPs (0.06 mM, 200 µL) + GP-Si-Luc (0.06 mM, 200 µL), Si-BPs (0.06 mM, 200 µL), Si-Luc (0.06 mM, 200 µL) and Si-BPs (0.06 mM, 200 µL) + Si-Luc (0.06 mM, 200 µL) for 2.5 h in a shaking incubator (250 rpm) at 37 ^oC. The bacteria were harvested by centrifuging the mixture at 3381 x g for 5 min in Eppendorf (EP) tubes. The resulting bacteria were re-suspended and washed with PBS for three times. Then 10 µL of the washed bacteria solution was transferred onto a microscope slide covered by a coverslip, and then imaged by a confocal laser scanning microscope (CLSM, Leica, TCSSP5 II) with 30% power of diode laser. All fluorescence images were captured by CLSM with a × 64 oil-immersion objective and taken under the same optical conditions, and the same brightness and contrast was applied to the images by the microscope automatically. The processing and analysis of ROI was performed by the commercial image analysis software (Leica Application Suite Advanced Fluorescence Lite). Moreover, the appearance of GP-Si-BPs in the bacterial cells were confirmed by TEM (Philips CM 200).

The numbers of motor neurons in anterior horns were manually counted using images of β3-tubulin-stained spinal cords visualised in Leica Application Suite Advanced Fluorescence Lite software. Motor neurons were defined as cells with cell body diameters of at least 15 µm showing positive immunostaining for β3 tubulin. Counts from the right and left anterior horns for each section were averaged, and then the average count from 3–6 sections used as a single measurement per animal for statistical analyses.

From ITC-treated A. thaliana, a rosette leaf was excised and immediately analyzed using an inversed Leica TCS SP5 confocal microscope with a 63× NA 1.2 apochromat water immersion objective. GFP was excited with a 488 nm argon-laser and emission was detected in the range 500–590 nm. Pinhole size was 1 airy unit. Pictures captured were of 1024 × 1024 formats with pixel size of 98.6 nm. For cancer cells, a microscopy chamber with transfected AY-27 cells grown over night was inspected with the same microscope and settings as described above with the following changes: emission detection range was 495–560 nm and pictures captured were in the format 512 × 512 with pixel size 60.2 nm. All data handling was performed using the software Leica Application Suite Advanced Fluorescence Lite. Apart from occasionally minor contrast-enhancements, all pictures were exported in an unedited raw format.