Pico plus 5500 afm

For the atomic force microscopy (AFM) study, aliquots were taken from the incubated samples after 24 and 48 h of incubation and diluted to 3.5 µM with distilled water. The diluted sample was then drop-casted on a freshly cleaned muscovite mica substrate and kept at room temperature for drying. Upon drying, the sample was imaged immediately with a tapping mode of AFM using a Pico plus 5500 AFM (Agilent Technologies, United States) with a piezo scanner having a maximum range of 9 µm. The images were captured with a scan speed of 0.5 line/sec. For image processing, PicoView software was used. The images of cells treated with fibrils were taken using Asylum Research MFP-3D-BIO AFM (Oxford Instruments).

10 µl (conc. 2 mg/ml) of the nanoparticle solution was deposited on a freshly cleaved muscovite Ruby mica sheet 10 min after that the sample was dried under vacuum. AC-mode atomic force microscopy was performed by using a Pico Plus 5500 AFM (Agilent Technologies, Inc., Santa Clara, CA, USA) with a piezo scanner maximum range of 9 μm. Microfabricated silicon cantilevers of 225 μm in length with a nominal spring force constant of 21–98 N/m were used from nanosensors. Cantilever oscillation frequency was tuned into resonance frequency. The cantilever resonance frequency was 150–300 kHz. All the images (512 × 512 pixels) were captured with a scan size between 0.5 and 5 μm at the scan speed rate of 0.5 rpm. The images were processed by flattening using Pico view software (Molecular Imaging Inc., Ann Arbor, MI, USA).

A total of 5 μL of the samples (1 mM) were deposited onto a freshly cleaved muscovite Ruby mica sheet (ASTM V1 Grade Ruby Mica from MICAFAB, Chennai, India) for 5–10 min and then the sample was dried by using a vacuum dryer. An AAC mode AFM was performed using a Pico plus 5500 AFM (Agilent Technologies Santa Clara, CA, USA), using a piezo scanner, with a maximum range of 9 μm. Microfabricated silicon cantilevers of 225 μm in length with a nominal spring force constant of 21–98 N m⁻¹ from nanosensors were used. The cantilever oscillation frequency was tuned into resonance frequency. The cantilever resonance frequency was 150–300 kHz. The images (256 by 256 pixels) were captured with a scan size between 0.5 and 5 μm at a scan rate of 0.5 lines per s. The images were processed by flatten using Pico view1.4 version software (Agilent Technologies, Santa Clara, CA, USA). Image analysis was done through Pico Image Advanced version software (Agilent Technologies, Santa Clara, CA, USA).

The morphological features of proteins were analyzed applying samples (10 μl) on to freshly cleaved mica (ASTM grade ruby mica, 20 x 20 mm, 0.27–0.33 mm thickness; Mica Fab, Chennai, India) surface and imaging was done as described [30 (link)]. For analysis of the accumulation of co-aggregates on cell surface and its degradation by enzyme, the morphology of SH-SY5Y cells was analyzed in AFM. The cells were seeded on to glass cover slips at a density of 10⁶ cells/ml in a 6-well plate. The cells were treated with the test samples, viz., fibrin-Aβ42 co-aggregate preincubated with or without the plant enzyme or plasmin for 24 hr at 37°C. An equal volume of medium was added to control cultures and the cells were incubated for another 48 hr at 37°C. The analysis was performed using a Pico plus 5500 AFM (Agilent Technologies, USA) instrument. For imaging of protein samples a 9 μm piezoscanner and micro fabricated silicon cantilevers (225 μm in length) with a nominal spring force constant of 21–98 N/m were used. Cantilever oscillation frequency was tuned into 150–300 kHz resonance frequency. Imaging of cellular samples was done in liquid mode using 100 μm scanner and cantilevers of 450 μm length with a nominal spring force constant of 0.2 N/m. The resonance frequency was set at 13 kHz. Picoview 1.10.1(9995) software was used for image analysis.

AFM was performed using a Pico Plus 5500 AFM (Agilent Technologies, USA) with a piezoscanner having a maximum range of 9 μm. 10 μL of the samples were deposited on a freshly cleaved muscovite ruby mica sheet (ASTM V1grade ruby mica from MICAFAB, Chennai). After 30 minutes the sample was dried with a vacuum drier. The cantilever resonance frequency was 150–300 kHz. The images (256 pixels × 256 pixels) were captured using a scan size of between 0.5 and 8 μm at a scan speed of 0.5 lines/s. The length, height, and width of protein fibrils were measured manually using PicoView1.10 software (Agilent Technologies, USA).