2.6 Microscope and
Imaging Software
- An inverted widefield fluorescence microscope. We use an
Olympus IX81 motorized inverted fluorescence microscope
equipped with a back-illuminated EMCCD camera (Andor), a
SOLA Light Engine (Lumencor, Beaverton, OR), and a
CoolLED pE-100 (740 nm, CoolLED pE-100). - 100NA 1.45 objective lens.
- Filter set for DAPI (D350/50, D460/50, 400dclp).
- Filter set for TMR (HQ545/30, HQ620/60, Q570lp).
- Filter set for CF640R (ET620/60x, ET700/75 m, T660lpxr).
- Filter set for Alexa®750 (HQ710/75, HQ810/90 m,
Q750lp). - FIJI software.
- MATLAB (Version R2014b 64-bit, MathWorks) software.
3 Methods
3.1 RBMB Synthesis 1. Hybridize Oligo1 and Oligo2: Combine equimolar quantities
of Oligo 1 and Oligo 2 in phosphate buffer in a low-retention
microcentrifuge tube so that the final concentration of each
oligo is 50μM and the total volume is at least 50μL.
- Incubate the oligos on a thermomixer at 37C overnight.
- Separate RBMBs from unhybridized oligos using a Superdex
200 or a Superdex 75 prep-grade column. - Concentrate purified RBMBs using a Microcon YM-10 centrif-
ugal device (seeNote 3). - Determine the final concentration of the RBMBs on a
spectrophotometer.
3.2 Cellular Delivery
of RBMBs
RBMBs are delivered into cells by microporation, a physical
method that previous studies show enables efficient cellular delivery
of oligo probes with high viability [5]. Specific procedures to
perform microporation are described below:- Seed cells in tissue culture flasks in MEM growth media with-
out phenol red or antibiotics and let them grow overnight. Be
cautious not to exceed 70% confluence on day 2 (seeNote 4). - Aspirate the media from the cells.
- Rinse the cells with 5 mL of 1PBS for about 2 min.
- Aspirate the PBS and add 1 mL of phenol red-free trypsin/
EDTA. - Following incubation of the cells in trypsin for several minutes,
add media to neutralize the enzyme (seeNote 5). - Gently resuspend the cells in media.
Ratiometric Bimolecular Beacons 235