RNA Detection

2. Aliquot 0.2 mL of culture from each Erlenmeyer flask and
   transfer to a fresh microcentrifuge tube. Pellet the cells at
   4400 gfor 1 min at room temperature.


3. Remove the LB media and suspend the cells in 1 mL of imaging
   solution (seeNote 13).


4. Pellet the cells again at 4400gfor 1 min at room tempera-
   ture, remove the imaging solution, and resuspend the cells in
   1 mL of imaging solution.


5. Add 0.2 mL of cell suspension to poly-D-lysine coated 8-well
   glass chamber slides (Subheading3.3.1) and incubate the cells
   expressing SRB-2, DNB and tRNA scaffold (negative control)
   at 37C for 30–45 min to promote the adhesion of bacterial
   cells to the surface.


6. Gently wash the wells twice with 0.4 mL of imaging solution to
   remove unattachedE. colicells.


7. Finally add 300μL of imaging solution containing 1μM of the
   appropriate fluorogenic dye (RG-DN, TMR-DN, SR-DN, or
   TR-DN) and incubate the cells at 37C for 10 min before
   imaging (seeNote 14).
   Cells expressing SRB-2 aptamer should be incubated with
   SR-DN (seeFig. 5a). Cells expressing DNB aptamer can be
   incubated with one of RG-DN, TMR-DN, SR-DN, or TR-
   DN. (SeeFig. 5b). Cells expressing tRNA scaffold were incu-
   bated with RG-DN, TMR-DN, SR-DN, and TR-DN.

3.3.3 Imaging the
Aptamers

1. Place the 8-well slide with immobilizedE. coliin the pre-
   warmed incubation chamber at 37 C attached to the
   microscope.


2. Use the 100objective under bright-field illumination to
   focus on adherentE. colicells.


3. For the fluorescence illumination, a metal halide lamp and the
   following filter settings are used: for RG-DN: 470/30 nm
   excitation filter, 495 nm dichroic beam splitter, 525/30 nm
   emission filter; for TMR-DN and SR-DN: 560/40 nm excita-
   tion filter, 595 nm dichroic beam splitter, 630/60 nm emission
   filter; for TR-DN: 580/20 nm excitation filter, 595 nm
   dichroic beam splitter, 630/60 nm emission filter.


4. Focus on the well with cells expressing either SRB-2 or DNB
   incubated with the appropriate fluorogenic dye and determine
   the suitable exposure time for each dye such that one obtains
   highest fluorescent signal without saturating the pixels.


5. Acquire both bright-field and fluorescent illumination images.


6. Acquire the images of the bacteria carrying pET-tRNA plasmid
   by using exactly the same exposure time. This image will be
   used for comparisons.

296 Murat Sunbul et al.