Efficient Bioimaging with Diaminodicyanoquinodimethanes: Selective Imaging of Epidermal and Stomatal Cells and Insight into the Molecular Level Interactions

The enhanced fluorescence emission of diaminodicyanoquinodimethanes (DADQs) in rigid and aggregated states holds great promise for bioimaging applications. This is demonstrated through their efficient application in epidermal and stomatal imaging with selective staining of cell walls and nuclei. Major advantages include the small quantities (a few nmols) of the fluorophore required, choice of DADQs soluble in water and organic solvents, and quick staining of the specimen in buffer-free state and in buffer medium. The molecular level interactions that enable staining are unraveled through isothermal calorimetry, infra-red spectroscopy and microscopy with energy dispersive X-ray spectroscopy analysis. It is proposed that DADQs with ionic or H-bonding functionalities bind to the polygalacturonic acid moieties in the epidermal layer; the former can bind also to nucleic acid polyanions. Fluorescence experiments explain the emission enhancement that enables the efficient imaging. DADQs are easy to synthesize, non-cytotoxic, and thermally, chemically and photo-stable, requiring no special storage conditions; preliminary experiments point to their potential utility in imaging different classes of cells.


Imaging of pea stomata with BT 2 in DMSO
Images of samples in the buffer medium stained by BT 2 in DMSO, without and with irradiation; staining of the inner guard cell wall and the response to light are clearly observed again. Imaging experiments carried out with leaves of dicotyledon plants showed that BT 2 is useful for a range of specimens.
Comparison of CLSM images Figure S3. Errors in the parameters determined for the DPZDQ / PGA -Na + are very large; the data and the fitting are therefore not meaningful.   Table S3. Atom % of Ca and K (with standard deviations) on the inner guard cell wall (IGCW) and epidermal cell junction (ECJ) regions (Figure 7a, b of main text, Figure S5) of fresh (in water) and stained epidermis layer of pea plant leaf, determined using EDX spectroscopy with FESEM; dyes used are BT 2 , DPZDQ, propidium iodide. Table S4. Atom % of Ca and K (with standard deviations) on the inner guard cell wall (IGCW) and epidermal cell junction (ECJ) regions, of epidermis layer of pea plant leaf maintained in water, MES buffer and MES buffer containing BT 2 .
In the MES buffer, Ca 2+ ions appear to leach out of the epidermis, and K + to move in. Relative changes of the spectral properties of BT 2 on adding PGA -Na + As the polymer: BT 2 mol ratio increases, the fluorescence intensity increases and begins to saturate above a ratio of ~ 200:1.

Autofluorescence analysis of pea epidermal cells
The autofluorescence of the pea epidermal cells was monitored under CLSM at different wavelengths, laser power and gain. The images recorded are shown in Figure S9. The contrasting images of stomata stained by BT 2 are shown in Fig. S10. It is seen that the autofluorescence images obtained even with significantly higher laser power and gain, are quite dull compared to those obtained with BT 2 based imaging.

Imaging of mammalian and bacterial cells
In order to demonstrate the generality of DADQs as florescence probes for bio-imaging, we have carried out the fluorescence imaging experiments with live DU145 human prostate cell lines (live mammalian cells) and cyanobacteria (fixed cells) using BT 2 and BBEDQ respectively.
DU145 cell line DU145 cell lines were stained by directly introducing 15 µl of the aqueous solution of the BT 2 . Cell lines were immediately washed with PBS and imaged in a CLSM. The dye molecules entered the cells and stained it completely (Fig. S11). Figure S11. CLSM images (fluorescence and overlay of fluorescence with bright field) of the stained DU145 cell lines using BT 2 . Scale bar: 20 µm.

Cyanobacteria cell
As BT 2 was found to be less efficient in this case, imaging was carried out using another DADQ derivative, 7,7-bis(benzylamino)-8,8-dicyanoquinodimethane (BBEDQ). S13 5 µl of 5 mM DMSO solution of BBEDQ was added on 0.2 ml of synechocystis pcc 6803 cells in buffer; the cells were imaged in a CLSM. The dyes entered into the cells and stained them completely.

Photostability studies
The dyes were excited at 488 nm and the fluorescence monitored. BT 2 is found to be very photo-stable; DPZDQ is slightly less stable.

Cytotoxicity assay
Cytotoxicity assay was carried out by Pondicherry Center for Biological Sciences, Pondicherry, India (http://http://pcbscience.webs.com/). 2 -4 × 10 5 HeLa cells were seeded in a 96-well cell culture plate with DMEM medium (Himedia) containing 1% anti-mycotic antibiotic (Himedia) and 10% FBS (Himedia), and incubated for 24 h in a CO 2 incubator at 37 o C. The cells were treated initially with the test sample (BT 2 , DPZDQ) in different concentrations (25, 50, 100, 250, 500 µg/mL) and incubated for 24 h. The medium was aspirated from the cells at the end of the treatment period. 0.5 mg/mL MTT and 1% PBS were ub ˚ 4 h. After incubation, the medium containing MTT was discarded from the cells. The crystals formed w v μL of DMSO; the absorbance at 570 nm was measured using a micro-plate reader to estimate the cell viability. IC 50 values of BT 2 and DPZDQ are found to be 1468 ± 345.4 µg/ml and 790.70 ± 98.73 µg/ml respectively