Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Summary

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Abstract

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Introduction

Progression through the bacterial cell cycle requires the coordination of many processes including membrane and cell wall biosynthesis, DNA replication and segregation, and cell division. To fully understand the complexity of bacterial cell biology, it is necessary to study these essential events; however, this is a non-trivial task since cell viability is compromised when key components of these pathways are mutagenized. Epifluorescence microscopy coupled with target-specific dyes is a powerful approach to probe these essential processes in wildtype and mutant bacterial strains.

Peptidoglycan-specific dyes include fluorescent antibiotics (vancomycin-FL, bocillin-FL) and fluorescent-d-amino acids (for example, 7-hydroxycoumarin-3-carboxylic acid-3-amino-d-alanine, HADA; 4-chloro-7-nitrobenzofurazan-3-amino- d-alanine, NADA; tetramethylrhodamine-3-amino-d-alanine; TADA). In Gram-positive bacteria, the use of sublethal concentrations of fluorescent antibiotic analogs to probe sites of peptidoglycan biosynthesis has been an effective strategy to reveal peptidoglycan insertion patterns^1,2,3,4. While fluorescent vancomycin labeling has been used to gain insights into the peptidoglycan insertion patterns in fixed Gram-negative bacteria⁵, the outer membrane generally provides a permeability barrier that prevents the use of fluorescent antibiotics as a probe for peptidoglycan biosynthesis in live cells. In contrast, short pulses of fluorescent-d-amino acids or d-amino acids with biorthogonal functional groups covalently label regions of recent peptidoglycan insertion in a wide range of living bacterial cells^6,7. Patterns of peptidoglycan insertion that have been observed with synthetic d-amino acids include punctate and septal (Escherichia coli and Bacillus subtilis), polar and septal (Agrobacterium tumefaciens and Listeria monocytogenes), septal only (Staphylococcus aureus), and apical (Streptomyces venezuelae)^6,7. These observations indicate that bacteria exhibit diverse patterns of cell wall biogenesis and that the use of synthetic d-amino acids as probes for examining growth patterning is a valuable strategy in many bacteria.

Dyes that label bacterial chromosomes include the deoxyribonucleic acid (DNA) specific minor groove binder (4,6-diamidino-2-phenylindole; DAPI) and high affinity cyanine dyes (Green and Orange; see materials list). DAPI staining of fixed cells assists in enumeration of bacteria from environmental samples⁸, whereas DAPI staining of live cells is used to indicate bacterial viability⁹. In contrast, cyanine dyes such as Orange and Green are frequently described as membrane impermeant "dead" cell stains to enumerate non-viable cells⁹. Remarkably, when these reagents are used to probe the morphology of the bacterial nucleoid during cell growth, DAPI, Orange, and Green were all shown to be membrane permeant and capable of labeling live cells¹⁰. In live E. coli cells, DAPI staining of DNA appears diffuse due to auto-fluorescence from the cytoplasm and repeated exposures of DAPI-stained cells to ultraviolet (UV) light perturbs the nucleoid structure¹⁰. Staining E. coli or B. subtilis with Orange reveals that this dye is membrane permeant and provides long-lasting fluorescence upon binding to DNA in live cells without impacting cell growth, DNA replication, or chromosome segregation¹⁰. These observations suggest that cyanine DNA dyes can be used to monitor the morphology of nucleoids during cell growth in many bacteria.

Phospholipid-specific stryl dyes such as N-(3-triethylammoniumpropyl) -4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide (4-64; see materials list) are cationic compounds and associate preferentially with negatively charged phospholipids such as cardiolipin and phosphatidylglycerol¹¹. Distinct patterns are observed when 4-64 is used to label the membrane of different bacteria. In Escherichia coli, 4-64 is enriched in the poles, in bands along the lateral wall, and in the division sites of late pre-divisional cells¹². In Bacillus subtilis, 4-64 labeling enables the visualization of lipid spirals¹³. In Agrobacterium tumefaciens, 4-64 labels the outer membrane and is observed in a characteristic "horseshoe" pattern in which the growth pole is devoid of labeling^14,15. These observations indicate that these bacteria exhibit heterogeneous lipid distributions due to the presence of lipid domains which contribute to cellular asymmetry. Changes in 4-64 labeling patterns such as the presence of diffuse labeling, blebs or vesicles, invaginations, or membrane shrinkage can be informative in characterizing mutants that impact the distribution or biosynthesis of lipids.

Beyond staining cells, determining the function of proteins participating in essential processes is necessary. The characterization of essential proteins is technically challenging because it is not possible to delete essential genes and study the phenotypic consequences. Thus, alternative approaches that deplete the protein have emerged. For example, an essential gene can be put under the control of an inducible promoter rather than its native promoter. Inducible promoters are responsive to small molecules such as; zinc¹⁶, isopropyl β-d-1-thiogalactopyranoside (IPTG)^{17,18,19,20,21}, arabinose²², vanillate^17,23, and xylose²³, thus the transcription of the target gene ceases and the protein of interest is depleted when the inducer is removed. Alternative approaches for depleting essential proteins of interest include synthetic riboswitches²⁴ which use small molecule-RNA interactions to hinder transcription of target genes, CRISPR interference^25,26 to block transcription of target genes, and inducible protein degradation^27,28 which uses peptide tags to target proteins for degradation by the ClpXP protease. Depletion strains provide only a short time for characterization before the cells lose viability, therefore, microscopic imaging of cells over time during protein depletion is a powerful approach for characterization. Indeed, microscopy of living bacterial cells has enabled researchers to gain insights into fundamental biological processes, including the mechanisms of cell shape maintenance, secretion, and compartmentalization²⁹.

A. tumefaciens is a bacterial plant pathogen³⁰ and natural genetic engineer^31,32. Thus, mechanisms related to pathogenicity, including host-pathogen interactions^33,34,35, secretion³⁶, and host transformation^30,31,37 have been extensively investigated. To design strategies that prevent A. tumefaciens mediated disease or enhance plant transformation, the processes essential for A. tumefaciens survival need to be better understood. The use of target-specific dyes and the recent development of a protein depletion strategy for A. tumefaciens¹⁸ provides a means to investigate essential processes.

Here, detailed protocols for microscopic analysis of wildtype, mutant, and protein depletion strains of A. tumefaciens are provided. The first two protocols describe how to prepare cells and label them with target-specific dyes. The third protocol provides step-by-step directions for preparing agarose pads (Figure 1) and imaging the bacterial cells (Figure 2, Figure 3, Figure 4). These protocols may also be suitable for other bacteria with additional adaptations to account for different media conditions, growth rates, oxygen requirements, and cell structures.

Protocol

1. Growth of A. tumefaciens Strains

1. Culturing A. tumefaciens strains
   1. Use a sterile wooden stick or pipet tip to inoculate 1 mL of ATGN growth media (see materials list for the recipe) with a single colony of the desired strain.
      NOTE: For A. tumefaciens depletion strains, the ATGN should contain 1 mM IPTG as an inducer to maintain biosynthesis of the essential protein.
   2. Grow the A. tumefaciens strains overnight in ATGN at 28 °C with shaking at 225 rpm.
   3. Measure the optical density of the cells at 600 nm (OD₆₀₀) using a spectrophotometer. Dilute the cell culture to an OD₆₀₀ = ~0.2 and continue to grow until OD₆₀₀ = ~0.6 is reached. For depletion strains, continue to grow with inducer until OD₆₀₀ = ~0.6 is reached.
   4. Use wildtype and mutant strains of A. tumefaciens at OD₆₀₀ = ~0.6 for target-specific staining and/or time-lapse microscopy (see sections 2 and 3.1). For depletion strains, wash out the inducer (see section 1.2.)
2. Removal of inducer for characterization of A. tumefaciens protein depletion strains
   1. Pellet 1 mL of exponential culture (OD₆₀₀ = ~0.4-0.6) by centrifugation at 7,000 x g for 5 min in a desktop centrifuge at room temperature.
   2. To wash, remove supernatant and resuspend the pellet in fresh media without inducer and pellet the cells as described above (1.2.1). Wash the cells a total of 3 times in fresh media.
   3. Resuspend the final pellet in fresh media and concentrate the cells to an OD₆₀₀ = ~0.8 for immediate staining with target specific dyes (section 2 and Figure 4B-D) or time-lapse microscopy (section 3.2 and Figure 4A). Alternatively, pre-deplete the cells for the desired amount of time by growing the cells in media without inducer prior to staining and imaging of the cells.

2. Target-specific Staining of A. tumefaciens Cells

1. Fluorescent d-amino acid cell wall labeling
   NOTE: FDAAs are non-toxic fluorescent d-amino acids which are readily incorporated into the A. tumefaciens peptidoglycan. This simple labeling procedure probes growth patterning in live cells⁶. Protocols for synthesis of four FDAAs are available³⁸.
   1. Pellet 500 µL of exponential culture (OD₆₀₀ = ~0.4-0.6) by centrifugation at 7,000 x g for 5 min in a desktop centrifuge. Resuspend the cell pellet in 100 µL of fresh media.
   2. Add 5 µL of 5 mM FDAA to washed and concentrated cells and incubate for 2 min in the dark.
      NOTE: Limit FDAA exposure to light. The time of incubation will vary depending on the growth rate of the strain. Typically, incubation times should be 5-10% of the doubling time⁶.
   3. Pellet the cells by centrifugation and wash cell pellets with phosphate buffered saline (PBS) three times.
   4. Resuspend pellet in ~50 µL PBS.
      NOTE: The volume of PBS used for resuspension may vary based on pellet size.
   5. Apply 0.8-1 µL of cells to an agarose pad and image using epifluorescence microscopy immediately (See sections 3.1 and 3.2).
      1. Alternatively, stop further label incorporation by fixing the cells with ice-cold 70% ethanol. Resuspend the cell pellet in 1 mL of ice-cold 70% ethanol and incubate on ice for 15 minutes.
      2. Collect cells by centrifugation and resuspend in a small volume of PBS for imaging at a later time. Store cell suspensions at 4 °C and image within 48 hours.
2. DNA staining with DAPI or Orange stain
   NOTE: To observe DNA structure, cells are labeled with either DAPI or Orange (dead cell stain). Although classically described as a "dead-cell stain", Orange is permeant, photostable, and does not affect the growth of bacterial cells, enabling live-cell DNA imaging¹⁰. In A. tumefaciens, Orange labeling works well in living cells and is suitable for time-lapse microscopy (Figure 3). In contrast, the ultraviolet (UV) light exposure necessary for imaging DAPI-stained cells is phototoxic (Figure 3) and thus DAPI staining is best suited for staining live cells for immediate imaging or staining fixed cells.
   1. DAPI staining of cells
      1. Pellet 1 mL of exponential culture (OD₆₀₀ = ~0.4-0.6) by centrifugation at 7,000 x g for 5 min in a desktop centrifuge.
      2. Optionally, to fix cells prior to staining, resuspend the cell pellet in 1 mL of 70% ice-cold ethanol. Incubate on ice for 10-15 min. Collect the cells by centrifugation as described in 2.2.1.1.
      3. Resuspend the cell pellet in 1 mL of PBS containing 1 µL of DAPI stock solution (1 mg/mL; see Materials Table). Mix gently by pipetting and incubate for 5 minutes in the dark.
      4. Pellet cells by centrifugation at 7,000 x g for 5 min and resuspend in 1 mL of PBS to remove excess DAPI. Wash the cells two more times and resuspend the pellet in 50 µL PBS or media.
      5. Apply 0.8-1 µL of cells to an agarose pad and image using epifluorescence microscopy immediately. See sections 3.1 and 3.2.
         NOTE: This protocol is not optimal for time-lapse microscopy to observe chromosome dynamics (Figure 3). Consider using Orange staining as an alternative (see section 2.2.2).
   2. Orange staining of live cells
   3. Pellet 1 mL of exponential culture (OD₆₀₀ = ~0.4-0.6) by centrifugation at 7,000 x g for 5 min in a desktop centrifuge. Resuspend cell pellet in 1 mL of PBS containing 1 µL of Orange stock solution (see materials list). Mix gently by pipetting and incubate for 5 min in the dark.
   4. Pellet cells by centrifugation and resuspend in 1 mL of PBS to remove excess Orange. Wash the cells two more times and resuspend the pellet in 50 µL PBS.
   5. Apply 0.8-1 µL of cells to an agarose pad and image using epifluorescence microscopy immediately. See sections 3.1 and 3.2.
      NOTE: This protocol works well with live cells and is suitable for time-lapse microscopy to observe chromosome dynamics (Figure 3). If it is not convenient to image immediately, cells may be fixed in ice-cold 70% ethanol (see section 2.2.1.2).
3. Membrane labeling
   NOTE: The lipophilic styryl fluorescent dye 4-64 has been used extensively to observe the membrane of bacterial cells. In contrast to many bacteria, 4-64 labeled A. tumefaciens cells do not label uniformly, but rather frequently exhibit a "horseshoe" pattern^14,15. Remarkably, the growth pole is nearly devoid of stain whereas the old pole is intensely labeled. Thus, 4-64 enables visualization of both cell growth patterns and membrane structure in A. tumefaciens.
   1. Add FM 4-64 to a final concentration of 8 µg/mL in 1 mL of exponential phase cell culture and incubate at room temperature for 5 minutes in the dark.
   2. Pellet labeled cells by centrifugation at 7,000 x g for 5 minutes in a desktop centrifuge and resuspend cell pellet in 1 mL of PBS. Wash the cells a total of three times to remove excess dye.
   3. Resuspend cells in a small volume of PBS and spot on an agarose pad to image immediately. (See sections 3.1 and 3.2)
      CAUTION: Cells must be imaged immediately to observe characteristic labeling patterns.

3. Imaging of A. tumefaciens Cells

1. Agarose pad preparation
   NOTE: Figure 1 contains an image sequence (panel A) and schematic (panel B) of a typical agarose pad prepared for time-lapse microscopy. Agarose pads are typically prepared as needed.
   1. 3.1.1. Use a coverslip as a guide and cut a 22 mm x 22 mm square of laboratory film (see materials list) by running a scalpel around the edges.
   2. Cut a square out of the center of the laboratory film leaving a ~2-5 mm border to serve as a gasket for the agarose pad. Discard the center cutout.
   3. Place the film gasket onto a glass slide (75 mm x 25 mm) cleaned with an ammonia and alcohol-free cleaner (see materials list) and heat until the film is slightly melted onto glass (top image in Figure 1A). To melt laboratory film, use the edge of a heat block set to 70 °C or melt lightly over a flame.
   4. Prepare agarose solution by mixing ~0.075 g agarose (see materials list) in 5 mL of media in a small flask. Heat the solution in a microwave with periodic swirling to mix until the agarose is dissolved and the solution is clear. Keep the agarose solution at 55-70 °C and use for construction of multiple agarose pads within 48 hours.
   5. Pipette media containing 1.2-1.5% agarose into the center of the gasket.
      NOTE: The volume of media is typically 50 - 60 µL but will vary based on gasket size. Adding media to a cold slide can cause the agarose to solidify too quickly, thus the slide should be kept on a warm surface. The edge of a heat block set to 70 °C works well. Water agarose or buffered agarose solutions such as phosphate buffered saline (PBS) can be used instead of media containing agarose for applications in which continued cell growth is not required. For imaging depletion strains, inducer can be present or absent in the media. Presence of inducer can be used as a control whereas the absence of inducer will reveal the depletion phenotype.
   6. Place a coverslip over the gasket to evenly distribute the agarose.
   7. Place slide on a cool, level surface to solidify for ~2 min. Avoid overdrying of the agarose pad as this will result in a wrinkled surface on the agarose pad and pooling of the cell suspension when applied to the surface.
   8. Carefully slide the coverslip off the agarose pad.
      Caution: Do not rush this step. The agarose pad can easily tear and an uneven agarose pad can make imaging difficult.
   9. Allow the agarose pad to air dry for 1-2 min at room temperature until the surface of the pad appears dry. Using a scalpel, remove a small strip of agarose to create an air pocket (middle image, Figure 1A); the air pocket is typically ~2 mm x 7-10 mm and A. tumefaciens cells tend to grow best near the air pocket (Figure 1C).
      NOTE: For imaging of fixed cells or under conditions where growth is not monitored, an air pocket is not necessary.
   10. Spot 0.8-1 µL of cells on the agarose pad and cover with a new coverslip. Gently place the coverslip over the top of the agarose pad to distribute the cells across the surface of the agarose pad.
   11. Seal the edges of the coverslip using melted VALAP (see Materials Table for recipe; Figure 1A, bottom image). Failure to seal along all the edges and corners of the coverslip can cause drying of the agarose pad, which will lead to the drifting of cells during imaging. NOTE: Sealing of the coverslip is only needed for long-term time-lapse imaging.

Figure 1: Agarose pad preparation. (A) Image sequence of the agarose pad preparation protocol. Image 1 is a slide with the laboratory film gasket. In image 2, the agarose pad and air pocket are visualized. Finally, image 3 shows the complete agarose pad with the cells under a coverglass and sealed with VALAP. (B) A schematic of an agarose pad for time-lapse microscopy is provided. Key features of the agarose pad are labeled on the schematic. (C) The air pocket promoted growth of A. tumefaciens on agarose pads. Images of wildtype A. tumefaciens cells grown on an agarose pad for 20 hours are shown. Images were taken at positions increasingly distant from the air pocket. The distance from the closest edge of the image to the air pocket is shown above each image. Scale bar = 10 µm. Please click here to view a larger version of this figure.

2. Imaging
   NOTE: Differential interference contrast, phase contrast, and epifluorescence imaging is performed with an inverted microscope equipped with hardware-based autofocusing, an automated stage, standard filters, an LED light source, 60X oil immersion objectives (1.4 NA) for phase contrast or differential interference contrast (DIC), and a 1K electron-multiplying charge-coupled-device (EMCCD) camera. The microscope should be placed in a temperature controlled room. Alternatively, a stage warmer or chamber can be used to maintain consistent temperatures during imaging.
   1. Epifluorescence microscopy
      NOTE: For fluorescence imaging of live cells, it is necessary to limit the number of image acquisitions and optimize the exposure time to minimize photobleaching and phototoxicity. For imaging of each dye, it is recommended to find an optimal exposure time which provides sufficient fluorescence detection but does not lead to photobleaching or phototoxicity. In Figures 2-4, exposure times of 200 ms were used for all fluorescence images. For the time-lapse sequences shown in Figure 3, images were acquired every 10 min for 3 h.
      1. Place immersion oil on the desired objective and place the inverted slide into the slide holder on the stage. Use the focusing knobs to bring the cells into focus.
      2. Acquire images in phase (or DIC) and the desired fluorescence filter.
         NOTE: The maximal excitation and emission wavelengths for the stains used in Figure 2, Figure 3, and Figure 4 are as follows: HADA (405/460), NADA (450/555), TADA (555/570), 4-64 (515/640), DAPI (360/460), Orange (547/570).
   2. Time-lapse microscopy
      NOTE: During time-lapse microscopy the following features are computer controlled: x, y, and z position, shutters, and fluorescence filters. A hardware-based auto-focus system is optimal to maintain focus during time-lapse imaging. Alternatively, a software-based auto-focusing loop can be used.
      1. Place immersion oil on the desired objective and place the inverted slide into the slide holder on the stage. Use the focusing knobs to bring the cells into focus.
      2. Optionally: Acquire multiple (x,y) positions. Randomly select 10 fields of cells in close proximity to the air pocket of the agarose pads.
      3. Set-up the time sequence acquisition to image in phase or DIC at the desired time interval.
         NOTE: For the time-lapse sequence shown in Figure 4, DIC images were acquired with 30 ms exposure every 10 min for 14 h. When using fluorescence imaging during time-lapse microscopy, adjust the acquisition interval and exposure time to minimize photobleaching and phototoxicity.

Results

Target-specific labeling of wildtype A. tumefaciens cells
In order to illustrate that cell morphology is not impacted by the wash steps or treatment with 1% DMSO (which is used to dilute the fluorescent dyes), cells were imaged directly from culture (Figure 2A, far left panel), after washing the cells by centrifugation as described in 1.2 (Figure 2A, left panel), or ...

Discussion

This protocol contains a series of procedures for the investigation of A. tumefaciens wildtype, mutant, and depletion strains. It is worth noting that all of the procedures listed in the protocol section can be readily adapted for other bacterial strains with additional modifications to account for growth media, temperatures, and growth rates.

The use of target-specific dyes is a valuable tool for providing a detailed characterization of cell-cycle events in bacterial cells. Here, pep...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacterial Strains
Agrobacterium tumefaciens C58	ATCC	33970	Watson B, Currier TC, Gordon MP, Chilton MD, Nester EW. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol 123:255-264.
Agrobacterium tumefaciens C58ΔtetRA::mini-Tn7T-GM-Ptac-ctrA ΔctrA			Figueroa-Cuilan W, Daniel JJ, Howell M, Sulaiman A, Brown PJB. 2016. Mini-Tn7 insertion in an artificial attTn7 site enables depeltion of the essentail master regulator CtrA in the phytopathogen Agrobacterium tumefaciens. Appl Environ Microbiol. 82:5015-5025.
Name	Company	Catalog Number	Comments
Media Components
ATGN Minimal Medium			To 1 L of sterilized water add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose. For plates, add 15 g Bacto Agar to 1 L of water and autoclave. Cool to 55 °C and add 50 ml 20X Buffer, 50 ml 20X Salts, 12.5 ml 40% glucose.
20X AT Buffer			Add 214 g/L KH2PO4 to water and adjust pH to 7.0 with sodium hydroxide. Autoclave.
NaOH	Fisher BioReagents	BP359
KH2PO4	Fisher Chemical	P288
20X AT Salts			Add 40 g/L (NH4)2SO4, 3.2 g/L MgSO4•7H2O, 0.2 g/L CaCl2•2H2O, and 0.024 g/L MnSO4•H2O to water. Autoclave.
(NH4)2SO4	Fisher Chemical	A701
MgSO4•7H2O	Fisher BioReagents	BP213
CaCl2•2H2O	Fisher BioReagents	BP510
MnSO4•H2O	Fisher Chemical	M114
Glucose	Fisher Chemical	D16	Prepare 40% stock in water. Filter sterilize.
Bacto Agar	Fisher BioReagents	BP1423	Add 15 g to 1 L of water when preparing plates.
Name	Company	Catalog Number	Comments
Optional Media Additives
Kanamycin	GoldBio	K-120	Prepare as a 100 mg/ml stock solution in water and filter sterilize. Use at final concentration of 200 µg/ml.
IPTG	GoldBio	I2481C5	Prepare as a 1 M stock solution in water and filter sterilize. Use at final concentration of 1 mM as needed for induction.
Name	Company	Catalog Number	Comments
Microscopy Materials
Microscope Slides	Fisherbrand	12-550D	25 X 75 X 1.0 mm. Clean with Sparkle glass cleaner.
Microscope Cover Glass	Fisherbrand	12-541-B	22 X 22 mm. No. 1.5. Clean with Sparkle glass cleaner.
Sparkle Glass Cleaner	Home Depot	203261385	Ammonia and alcohol free.
Ultra Pure Agarose	Invitrogen	16500-100	Add to water, PBS, or media to a final concentration of 1 - 1.5%. Melt in microwave and place on 70 C
PBS	Fisher BioReagents	BP399500	10X solution to be diluted to 1X with sterile water.
Parafilm	Bemis	PM-999	Laboratory film used as gasket in agarose pad preparation.
VALAP			Add equal weights of lanolin, parafin wax, and petroleum jelly to a conical tube. Heat tube in 70 °C dry, bead or water bath to melt and mix. Apply VALAP while still molten.
Lanolin Butter	SAAQIN	SQ-LAB-R1
Petroleum Jelly	Target Corp.	06-17644
Paraffin Wax	Crafty Candles	263012
Name	Company	Catalog Number	Comments
Target-specific dyes
DMSO	Fisher BioReagents	BP231-1	Use to dilute stock solutions of dyes as needed.
FDAAs (NADA, HADA,TADA)			FDAAs can be synthesized or acquired through agreement with Mike VanNieuwenhze (Indiana University). Prepare 100 mM stock solution in DMSO. Use at a final concentration of 5 mM.
DAPI	ThermoFisher Scientific	62247	Prepare 1 mg/ml stock solution in DMSO. Use at final concentration of 1 µg/ml.
SYTOX Orange Nucleic Acid Stain	Invitrogen	S11368	Stock concentration is 5 mM in DMSO. Use at final concentration of 5 µM.
FM4-64	Invitrogen	T3166	Prepare 8 mg/ml stock solution in DMSO. Use at final concentration of 8 µg/ml.
Name	Company	Catalog Number	Comments
Equipment
Dry bath	Sheldon Manufacturing, Inc.	52120-200
Metallic thermal beads	Lab Armor	42370-002
Epifluorescence microscope equipped with an EMCDD camera			Nikon Eclipse TiE equipped with a QImaging Rolera em-c2 1K electron-multiplying charge-coupled-device (EMCCD) camera is used in this work.