RNA Detection

3. Use the “Freehand Selections” tool to draw a Region of Inter-
   est (ROI) around the object. This object ROI is automatically
   applied to the two channel images within the same stack.


4. For each image, measure the total integrated fluorescence den-
   sity and area (in pixels) within the object ROI by selecting
   Analyze>Measure.


5. For each image, measure the average background fluorescence
   by first drawing at least four ROIs in areas just outside the cell
   of interest (seeNote 18).


6. Measure the mean fluorescence density (per pixel) within each
   “background” ROI by selecting Analyze>Measure.


7. Compute the average of the mean fluorescence densities of the
   four background ROIs. Multiply this value by the area of the
   object ROI to obtain the total background fluorescence inten-
   sity within the object ROI.


8. Subtract the total background fluorescence intensity from the
   total integrated fluorescence density within the object ROI to
   obtain the total object fluorescence.

3.5 Quantifying the
Extent of Nonspecific
Opening

1. After quantifying fluorescence signals of MBs and IRDye® 800
   in water-in-oil emulsions and cells using the method described
   above, nonspecific opening of MBs can be determined as pre-
   viously described [18], according to the following equation:

%MBs opened¼

RcellRBUBBLES,CLOSED

RBUBBLES,OPENEDRBUBBLES,CLOSED

 100 %:

2.RCELLis the fluorescence ratio of living cells, andRBUBBLES,

CLOSEDandRBUBBLES,OPENEDare the fluorescence ratios of

aqueous bubbles prepared from water-in-oil emulsions of

unhybridized MBs (0% opened) and MBs hybridized with

excess complementary targets (100% opened), respectively

(seeNote 19).

3. Example images and resulting analysis that led us to conclude
   that the 2Me/PSLOOPMB is the most stable configuration
   among the different PS modified and non-PS modified MBs
   are shown in Fig.2 (seeNote 20).

3.6 Assessing the
Intracellular
Functionality of MBs

While 2Me/PSLOOPMBs exhibit the least nonspecific signals, it is

also important to confirm that they still retain the ability to hybrid-

ize to target RNA in living cells. This can be achieved by micro-

injecting excess fully complementary synthetic target RNA

molecules into cells previously microporated with MBs. If the

MBs are functionally active, injection of complementary target

RNA should lead to an increase in MB fluorescence.

250 Mingming Chen et al.