Single-molecule fluorescence microscopy has become a popular tool for exploring structural changes and dynamics of biological systems. In our laboratory, we use single-molecule techniques to track conformational changes of immobilized nitric oxide synthase (NOS) and determine their relationship to catalytic activity. Properly immobilizing biomolecules like NOS on a glass surface requires careful attention to coverslip cleaning and preparation. There are many protocols available for cleaning glass coverslips, but these protocols are time-consuming and often use harsh conditions. Alternatively, commercially cleaned and passivated coverslips are available but are quite expensive. In this poster, we examine the possibility of using purchased pre-cleaned coverslips (Schott Nexterion) that come ready to be prepared for single-molecule measurements. We present figures and measurements of merit comparing the pre-cleaned coverslips with ozone cleaned coverslips; demonstrating the effectiveness of pre-cleaned coverslips for single-molecule fluorescence microscopy

Almail, Ali

Arnett, David C.

Grace, Emily

Gritters, Noah

English

NWCommons: Institutional Repository of Northwestern College

. Abstract:Single-molecule fluorescence microscopy has become a popular tool for exploring structural changes and dynamicsof biological systems. In our laboratory, we use single-molecule techniques to track conformational changes ofimmobilized nitric oxide synthase (NOS) and determine their relationship to catalytic activity. Properly immobilizingbiomolecules like NOS on a glass surface requires careful attention to coverslip cleaning and preparation. There are manyprotocols available for cleaning glass coverslips, but these protocols are time-consuming and often use harsh conditions.Alternatively, commercially cleaned and passivated coverslips are available but are quite expensive. In this poster, weexamine the possibility of using purchased pre-cleaned coverslips (Schott Nexterion) that come ready to be prepared forsingle-molecule measurements. We present figures and measurements of merit comparing the pre-cleaned coverslipswith ozone cleaned coverslips; demonstrating the effectiveness of pre-cleaned coverslips for single-moleculefluorescence microscopy.Introduction:Single-molecule fluorescence microscopy (SMFM) has become an indispensable tool in the biosciences by givinginsight into the kinetics and dynamics of biological molecules while leaving samples minimally perturbed (1). SMFM utilizesfluorescence, a process that occurs when a photon of light is absorbed by a conjugated fluorophore, which re-emits thephoton at a higher wavelength. The first detection of a single molecule using SMFM was reported by Tomas Hirshfield in1976 when he observed the presence of fluorescently labeled globulin proteins flowing past a detector (1). His success ledto the continued application of SMFM to many other structures such as enzymes, DNA, and other cellular structures.For SMFM to work properly, the coverslips used must be exceptionally clean because nonspecific molecular bindingcan interfere with the quantification of background noise (2,3). To prevent these interactions, surface passivation can beused. This process facilitates the specific binding of desired molecules to the surface. PEG passivation is very effective,and it has become a standard for SMFM, but the cleaning procedure can be very tedious, complicated, and involve harshconditions (2). One option is for labs to buy coverslips that have already been cleaned and passivated, but these are veryexpensive. A second, less expensive option is buying coverslips that have been cleaned but not passivated. However, somemay question the robustness, longevity, and consistency of this commercial cleaning. A third method is buying coverslipsthat have not been pre-cleaned and subjecting them to a quicker and easier cleaning procedure, such as ozone cleaning.Ozone cleaning involves using high powered UV light to generate ozone. The ozone reacts with contaminants on thecoverslip surface, forming volatile compounds that evaporate from the surface of the coverslip. This method can take aslittle time as 10-15 minutes, and then passivation can proceed. Here we explore the cleanliness and coverage quality ofpre-cleaned coverslips and ozone cleaned coverslips for SMFM applications.Materials and Methods:Pre-cleaned coverslips were purchased from Schott Nexterion (Louisville, KY). These coverslips were placedindividually in sterile 50mL falcon tubes and vacuum-sealed to preserve cleanliness. The lids on the falcon tubes wereturned very loosely, which allowed the air to be removed from the falcon tube. Two separate batches of these pre-cleanedcoverslips were used in this study. Also, a box of Platinum Line coverslips from ThermoFisher (Waltham, MA) wasprocured, and these are not pre-cleaned. For ozone cleaning, a UV Ozone Cleaner from Ossila (Sheffield, UK) was used.Three coverslips from each box were ozone cleaned prior to passivation, and another three coverslips from each boxwere solely passivated.The protocol for PEG passivation of glass coverslips was adapted from Gidi et al. (2). In a hood cleaned with acetone, asilicone mold form Grace Bio-Labs (Bend, OR) was placed onto a glass coverslip. The desired wells were washed with 110 μlof dry-acetone and emptied after each wash. Then the wells were airdried to remove any residual acetone. The coverslipwas placed in a desiccator apparatus and heated in an oven at 90°C for 5 minutes. To the desired wells, 99:1 25%v/v PEG-Silane and Biotin-PEG-Silane was added and then heated for 15 minutes at 90°C to facilitate an alcohol condensationbetween the silanol of the glass and the ethoxide of silane component of PEG. The desired wells were washed three timeswith Ambion water (Austin, TX).In this study, a confocal microscopy and avalanche photodiodes from ISS (Champaign, IL) were utilized, and theexperiments were carried out over a period of two months. To assess coverslip cleanliness, we compared untreatedcoverslips (coverslips right outside of the box) to their ozone cleaned counterparts from the two batches of SchottNexterion coverslips (pre-cleaned) and Platinum Line coverslips. The wells on the coverslips were washed with waterthree times and then filled with water and imaged at five different locations using the water objective. The area of theimages was 600 mm by 600 mm. The wavelength was set to 594 nm and the intensity was adjusted to achieve a power ofaround 630 nW when measured immediately before the laser entered the microscope. Utilizing the same settings andcoverslip categories, we compared how coverslip cleanliness related to coverage quality. The coverslips were passivatedaccording to the aforementioned protocol. The wells were then washed three times with fluorescence buffer, which iscomposed of 10 mL of 10X fluorescence buffer stock, 1 mL of 10 mg/mL BSA, and 89 mL of Ambion water mixed and runthrough a sterile 0.2 μm polyethersulfone membrane filter. The 10X fluorescence buffer stock is made of 1 M KCL and 0.5 MHEPES at a pH of 7.4. Then 110 mL of 100 nM solution of streptavidin-conjugated-Alexa Flour 594 from ThermoFisher wasadded, which binds biotin tagged molecules. This was allowed to incubate for fifteen minutes. Then the wells were washedthree more times with fluorescence buffer and filled with fluorescence buffer once more. Next, the coverslips were onceagain imaged at five different locations using the air objective. This was to determine the quality of the surface that hadbeen created. A patchy uneven spread of Alexa Flour 594 indicates a poor surface, while even coverage suggests higherquality surface passivation.Results:DiscussionTo assess the cleanliness of the coverslips, we compared the mean counts of untreated coverslips to their ozonecleaned counterparts. We concluded that ozone cleaning improved the cleanliness of Batch 1 Schott and Platinumcoverslips (Figure 1,2). However, cleanliness of coverslips did not necessarily correspond to the evenness of surfacecoverage when the coverslips was treated with PEG and incubated with streptavidin AF594. We found that ozone cleaningdid not impact the surface coverage of the Batch 1 and 2 coverslips but enhanced the surface coverage of Platinumcoverslips (Figure 3,4). Therefore, we conclude that Schott coverslips are adequate to use right of the box because theirbackground counts are low and their surface coverage is even. However, commercially uncleaned coverslips such as thePlatinum coverslips can be ozonated to improve the surface quality for single-molecule fluorescence microscopy, notingthat surface coverage was varied (Figure 3,4). Further directions would be to examine different coverslip cleaningprocedures that are cheaper, as ozone cleaning is expensive. Additionally, we could analyze how different surfacepassivation protocols, such as with BSA, might affect the evenness of the coverage on a coverslip.References and Acknowledgements1. Shashkova, S., & Leake, M. C. (2017). Single-molecule fluorescence microscopy review: shedding new light on old problems. Bioscience reports, 37(4), BSR20170031.https://doi.org/10.1042/BSR20170031 2. Gidi, Yasser & Bayram, Serene & Ablenas, Christopher & Blum, Amy & Cosa, Gonzalo. (2018). Efficient One-Step PEG-Silane Passivation of Glass Surfaces for Single-Molecule Fluorescence Studies. ACS Applied Materials & Interfaces. https://doi.org/10.1021/acsami.8b157963. Kudalkar, E. M., Deng, Y., Davis, T. N., & Asbury, C. L. (2016). Coverslip Cleaning and  Functionalization for Total Internal Reflection Fluorescence Microscopy. Cold Spring Harbor protocols, 2016(5), pdb.prot085548. https://doi.org/10.1101/pdb.prot085548We would like to thank Dr. Arnett for his guidance on this project. Support for this project was provided by the National Sci ence Foundation and Northwestern College.Figure 4: 600x600 µm area of PEG treated coverslips that are incubated with streptavidin AF594 that are either untreated or ozone cleaned. From left to right and top to bottom, the images correspond to areas from Platinum coverslips untreated and ozone treated, Batch 1 Schott coverslips untreated and ozone treated, Batch 2 Schott coverslips untreated and ozone treated.  These images are representative images from each category.Figure 1: 15x15 µm area of a variety of coverslip types that are ozone cleaned or untreated. From left to right: Platinum untreated, Platinum ozone cleaned, Batch 1 Schott coverslip untreated, Batch 1 Schott coverslip ozone cleaned, Batch 2 Schott coverslip untreated, Batch 2 Scott coverslip ozone cleaned. These images are representative images from each category and are standardized to the same count maximum.Comparing the different coverslip types that were untreated, both the Platinum coverslips and Batch 2 Schott coverslips had the lowest means andstandard deviations (Figure 1,2). For the ozone treatment, only the means of the Batch 1 Schott and Platinum coverslips were significantly different fromtheir untreated counterparts (Figure 1,2). Comparing how surface cleanliness improves surface coverage, ozone cleaning of platinum coverslipssignificantly reduced the coefficient of variation compared to its untreated counterpart (Figure 3,4). For the remaining coverslips, there was no significantdifference in the coefficient of variation between ozone cleaned and untreated coverslips (Figure 3,4). Ozone cleaning significantly increased the meancounts of Batch 1 and 2 Schott slides (Figure 3,4).Noah Gritters, Ali Almail, Emily Grace, David ArnettDepartment of Chemistry, Northwestern College, IowaFigure 2: Comparing fluorescence intensity from various coverslip types with and without ozone cleaning. The fluorescence intensity is measured by focusing on the surface of the slide. The mean counts, shown here, reflect the cleanliness of slide, with lower counts corresponding to a cleaner slide. * Indicates a significant difference from its counterpart in the same coverslip type (p<0.05). For each subcategory n=15 images from 3 slides.Figure 3: Comparing the evenness of surface coverage between different coverslip types. Surfaces were covered with streptavidin conjugated with AF594 following surface passivation with PEG-PEG Biotin. The mean refers to the brightness of an image area and the coefficient of variation corresponds to the differences in counts within an image area. Greater coefficient of variation indicates unevenness of coverage. * Indicates a significant difference between the means from the same coverslip type (p<0.05). *Indicates a significant difference between the coefficients of variation from the same coverslip type (p<0.05). Red error bars represent standard deviation of the coefficient of variation. For each subcategory n=15 images from 3 slides.

Using Pre-Cleaned Coverslips to Optimize Coverslip Preparation for Single-Molecule Fluorescence Microscopy

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Using Pre-Cleaned Coverslips to Optimize Coverslip Preparation for Single-Molecule Fluorescence Microscopy

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