Protocol for subcellular targeted microthermal protein damage in cells cultivated on plasmonic nanosilver-modi ed surfaces

Martin Mistrik (  martin.mistrik@upol.cz ) Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Zdenek Skrott Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Petr Muller Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Ales Panacek Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic Lucie Hochvaldova Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic Katarina Chroma 1Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Tereza Buchtova Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Veronika Vandova Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Libor Kvitek Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic Jiri Bartek (  jb@cancer.dk ) Danish Cancer Society Research Center, Copenhagen, Denmark


Introduction
Exposure of cells to elevated temperatures is used in research on protein thermal stability pro ling, thermal therapies, treatments of accidental burns, and proteinopathies involving an accumulation of defective proteins. At the cellular level, the thermal damage primarily impairs proteins, causing their unfolding, aggregation, amyloidogenesis, and denaturation, phenomena implicated in various pathologies 1 . Studying responses to thermal damage of proteins on the level of a single living cell or even subcellular level represents a signi cant challenge due to the lack of available methods allowing precise and fast delivery of the heat to the target structure at the micrometer scale.
The emerging eld of plasmonic nanoparticles (NPs) has opened a new way for localized thermal therapy due to the e cient and tunable photothermal properties. When illuminated by light, free electrons localized on the nanoparticle surface become excited, and the local electron cloud is asymmetrically distributed over the whole nanoparticle. This distribution produces a coulombic restoring force between positively charged nuclei and negatively charged electrons from the conduction band, which leads to collective oscillation of the electron cloud on the particle surface called localized surface plasmon (LSP). The localized surface plasmon resonance (LSPR) takes place if the frequency of the incident light matches with the frequency of LSP oscillation. 2,3 Absorption of light by nanoparticles may be nonradiatively relaxed and simultaneously converted to heat energy. Silver nanoparticles can be easily tailored to possess an intense SPR band at a suitable wavelength region, which enables them to produce heat after the irradiation with the appropriate laser. Plasmon NPs convert energy from the light to heat immediately and e ciently, allowing localized heating of the surrounding environment. [4][5][6] Here, we adopted the NPs technology for direct focusing of the heat to the individual cells within a micrometer scale. The method is based on modi ed microscopic cell culture plates, pre-coated by a layer of anisotropic silver NPs allowing excitation through targeted irradiation by conventional lasers used in the laser scanning microscopes (LSM) and allowing controllable heating. The deposition of NPs with suitable plasmonic properties on the cultivation surface is based on the layer-by-layer self-assembly technique, which facilitates the binding of negatively charged silver NPs using positively charged thin polymeric lm deposited on the surface of the cultivation plate ( Figure 1). 10. Next day seed the desired cell line (cell line with GFP-tagged protein, such as HSP70, is suitable for direct monitoring of heat shock response of targeted cell) using standard cell culture protocol. Between 80.000 and 100.000 cells per well (depending on cell type) is suitable for optimal con uency for a 24-well plate.

Reagents
11. Next day put the well plate with cells to the prewarmed (35°C-37°C) confocal microscope and wait about 15 minutes to allow temperature equilibration.
12. Visualize the cells expressing GFP reported using a blue laser (e.g. 488) and appropriate lters.
13. Set carefully the focus of the objective to the plasmon layer by nding the interface between the cell body and the bottom of the well surface. Alternatively, the plasmon layer can be visualized by the transmission light mode.
14. Activate the plasmon layer by 561 nm solid-state laser (or another appropriate laser with a similar wavelength). The amount of emitted heat is regulated by the laser power, pixel dwell time, and the number of irradiation cycles. The appropriate setup must be determined experimentally by the user as there are inevitable differences between various microscopes and lasers.
15. To target heat damage to the de ned subcellular region, two approaches can be employed. First, the FRAP-like experiment can be performed. The de ned region of interest (ROI) is "bleached" (means exposed to) by 561 nm laser and the cell (with the GFP reporter) is visualized by 488 nm laser. For our FRAP-like experiments where irradiation ROI was pre-de ned, the pixel dwell time was xed at 100 µs, and laser power between 5% to 20% for Alpha Plan-APOCHROMAT 40x water immersion objective. The second approach is based on collinear laser stripes (optimally 16-32 stripes per eld). The plasmon layer is activated by 561 nm laser in the pattern of collinear stripes. The pixel dwell time was xed at 709 µs, and the total irradiation time was 0.85s for one irradiation cycle resulting in 32 colinear stripes across the one microscopic eld. See also ref 7 for more details for setting up the laser stripping approach in LSM.

Troubleshooting
Step 2: stir vigorously during the whole reaction; add hydrazine to the solution, after the reduction via borohydride is completed (colour change to light-yellow) Seep 4, 6, 8: do not shake or rotate the plates during the adsorption of the layers, the surface must stay still during functionalization Step 7: wash the wells properly with distilled water, free-PDDA or other impurities might cause aggregation of AgNPs Step 13: The focus on the plasmon layer must be properly set otherwise the effectiveness of heat emission will be severely compromised. Set the focus in the transmission light mode.  Figure 1 Schematic representation of the concept of microthermal damage in icted on cellular proteins. The cell culture plate surface is modi ed by a thin polymeric lm for the e cient binding of plasmonic silver NPs. Plasmon NPs convert energy from light (laser) immediately and e ciently to heat, enabling direct focusing of the heat on subcellular regions.

Figures
Page 9/9   Demonstration of microthermal damage induction by the FRAP-like approach. The Microheated region was de ned as ROI and exposed to 561 nm laser. Heat damage is accompanied by the immediate accumulation of HSP70-GFP within the target ROI region (marked by arrows).