FIELD OF INVENTION
The present invention relates to the field of small molecules for staining of biological samples and imaging using fluorescence.
BACKGROUND OF THE INVENTION
Fluorescence based imaging is a prominent tool for the investigation of the structure and function of biological systems (Z. Liu, L. D. Lavis, E. Betzig, Mol. Cell. 2015, 58, 644). Issues related to cytotoxicity, photo-stability and emission quenching have limited the number of fluorophores that can be deployed in practical and efficient imaging applications. Fluorescent protein based probes are mostly expensive, and often suffer from low molar absorptivity, instability during sample fixation that may involve denaturants, potential interference with cell functions, and undesirable sensitivity to factors like temperature and pH (Z. Cai, Z. Ye, X. Yang, Y. Chang, H. Wang, Y. Liu, A. Cao, Nanoscale 2011, 3, 1974; E. S. Swenson, J. G. Price, T. Brazelton, D. S. Krause, Stem Cells 2007,25, 2593; J. Wiedenmann, F. Oswald, G. U. Nienhaus, IUBMB Life 2009, 61, 1029; T. J. Morikawa, H. Fujita, A. Kitamura, T. Horio, J. Yamamoto, M. Kinjo, A. Sasaki, H. Machiyama, K. Yoshizawa, T. Ichimura, K. Imada, T. Nagai, T. M. Watanabe, Sci. Rep. 2016, 6, 22342 (1-13)). Even though quantum dots are highly photo-stable and emissive, they are generally plagued by toxicity related issues (Z.-H. Li, J. Peng, H.-L.Chen, J. Nanosci. Nanotechnol. 2011, 11, 7521); nanoparticles based on small organic molecules and macromolecules are emerging as viable alternatives (Z. Tao, G. Hong, C. Shinji, C. Chen, S. Diao, A. L. Antaris, B. Zhang, Y. Zou, H. Dai, Angew. Chem. Int. Ed. 2013,52, 13002). Small molecule based fluorophores are relatively easy to synthesize and characterize, and afford the flexibility to incorporate desired functionality and interactions with the biological systems; however most are susceptible to aggregation-induced self-quenching of fluorescence emission. The limited classes of molecules that exhibit strong fluorescence in the aggregated/solid states (often called aggregation-induced emission enhancement) include tetraphenylethenes (Z. Zhao, J. W. Y. Lam, B. Z.

Tang, J. Mater. Chem.2012, 22, 23726), diphenylbutadienes (R. Davis, N. S. S. Kumar, S. Abraham, C. H. Suresh, N. P. Rath, N. Tamaoki and S. Das, J. Phys. Chem. C 2008, 112, 2137), hexaphenylsilole (Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Commun. 2009, 29, 4334) and diaminodicyanoquinodimethanes (DADQs). There have been previous reports on the strong fluorescence of DADQs in crystals (S. Jayanty, T. P. Radhakrishnan, Chem. Eur. J. 2004, 10, 791), nanocrystals (A. Patra, N. Hebalkar, B. Sreedhar, M. Sarkar, A. Samanta, T. P. Radhakrishnan, Small 2006, 2, 650), amorphous particles (C. G. Chandaluri, T. P. Radhakrishnan, Angew. Chem. Int. Ed. 2012, 51, 11849)and thin films (B. Balaswamy, L. Maganti, S. Sharma, T. P. Radhakrishnan, Langmuir 2012, 28, 17313; P. Srujana, T. P. Radhakrishnan, Angew. Chem. Int. Ed. 2015, 54, 7270); the critical role of specifically oriented aggregation in the fluorescence enhancement has been demonstrated recently (P. Srujana, T. Gera, T. P. Radhakrishnan, J. Mater. Chem. C 2016,4, 6510). DADQs are potential candidates for efficient bioimaging.
Stomatal imaging is critical for morphological and epidermal studies of plant species, understanding the stimuli responsive dynamics of inner/outer guard cell walls and related signal transduction pathways (S. Gilroy, M. D. Fricker, N. D. Read, A. J. Trewavas, Plant Cell 1991, 3, 333), and stomatal development issues like deposition pattern of callose in the guard cell wall (P. Apostolakos, P. Livanos, B. Galatis,CellMotil. Cytoskel. 2009, 66, 342). Small molecule based fluorophores such as propidium iodide, safranin, aniline blue, DAF-2DA, BCECF-AM, H2DCF-DA, calcoflour white and acridine orange have been popular choices for imaging epidermal constituents (R. Chitrakar, M. Melotto, J. Vis. Exp. 2010, 44, 1; M. G.Ajuru, B. E. Okoli, Int. J. Mod. Botany2012, 2, 115; P. Apostolakos, P. Livanos, T. L. Nikolakopoulou, B. Galatis, Ann. Bot. 2009, 104, 1373; . R. Puli, A. S. Raghavendra, J. Exp. Bot. 2012,63, 1349; O. Keech, E. Pesquet, A. Ahad, A. Askne, D. Nordvall, S. M. Vodnala, H. Tuominen, V. Hurry, P. Dizengremel, P. Gardeström, Plant Cell Environ. 2007, 30, 1523; Z. Chen, D. Liu, in Computer and Computing Technologies in Agriculture II, Vol. 3 (Eds: C. Zhao, D. Li), Springer, Boston, 2009, Ch. 63). Shortcomings of many of these probes include high cost and specialized storage

conditions like low temperature and protection from light (M. Jolicoeur, S. Germette, M. Gaudette, M. Perrier, G. Becard, Plant Physiol. 1998, 116, 1279; D. K. Gardner, M. Lane, in A Laboratory Guide to the Mammalian Embryo, (Eds: D. K. Gardner, M. Lane, A. J. Watson), Oxford University Press, New York, 2003, Ch. 3), the need to use non-aqueous solvents like DMSO which are toxic (J.Hebling, L. Bianchi, F. G. Basso, D. L. Scheffel, D. G. Soares, M. R. O. Carrilho, D. H. Pashley, L. Tjaderhane, CA. D. S. Costa, Dent Mater. 2015, 31, 399), carcinogenicity and mutagenicity (M. Nakamura, A. Awaad, K. Hayashi, K. Ochiai, K. Ishimura, Chem. Mater. 2012, 24, 3772); aggregation-induced quenching is a problem in most cases. In addition to low cost and convenient storage, the critical attributes of an ideal dye for fluorescence imaging of stomata include hydrophilicity to avoid binding to membranes, functionalities like ionic groups to selectively interact or bind with the cell wall, nucleus etc. and aqueous solubility to enable simple staining protocols.
There is a need in the art to identify and develop small molecule based bioimaging dyes, which are relatively low cost, efficacious, soluble in both organic and aqueous media, feasible for quick and easy sample preparation in water or buffer media, and high selectivity.
The present invention is covered by the provisional Indian patent application number 201741017444 dated 18-05-2017, which has subsequently been published in N. Senthilnathan, Ch. G. Chandaluri, T. P. Radhakrishnan, Sci. Rep. 2017, 7, 10583) on 21-08-2017.
SUMMARY OF THE INVENTION
In an aspect of the present invention, there is provided a method of staining of
walls of guard cells or walls of guard cells and walls and nuclei of epidermal cells, said
method comprising: (a) obtaining a composition comprising
diaminodicyanoquinodimethane derivative; and (b) placing plant tissue comprising guard cells in the composition comprising said derivative for a period of time; wherein said

method stains walls of guard cells or walls of guard cells and walls and nuclei of epidermal cells at different concentrations of said dye.
In an aspect of the present invention, there is provided a method of staining of walls and nuclei of guard cells and epidermal cells, said method comprising: (a) obtaining a composition comprising diaminodicyanoquinodimethane derivative; and; (b) placing plant tissue comprising guard cells in the composition comprising said derivative for a period of time; wherein said method stains walls and nuclei of guard cells and epidermal cells.
In an aspect of the present invention, there is provided a method of staining of walls of guard cells, said method comprising: (a) obtaining a composition comprising diaminodicyanoquinodimethane derivative and at least one buffer; and (b) placing plant tissue comprising guard cells in the composition comprising said derivative and buffer for a period of time; wherein said method stains walls of guard cells.
In an aspect of the present invention, there is provided a method of staining of animal origin cells, said method comprising: (a) obtaining a composition comprising diaminodicyanoquinodimethane derivative; and (b) placing animal origin cells in the composition comprising said derivative for a period of time, wherein said method stains animal origin cells.
In an aspect of the present invention, there is provided a method of staining of cyanobacterial cells, said method comprising: (a) obtaining a composition comprising diaminodicyanoquinodimethane derivative; and (b) placing cyanobacterial cells in the composition comprising said derivative for a period of time, wherein said method stains cyanobacterial cells.
This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1 depicts the (a) raw and (b) integrated thermograms from the isothermal
titration of sodium salt of Polygalacturonic acid (PGA-Na+) into 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) (BT2) in aqueous solution, fitting of the integrated thermogram is shown in (b), in accordance with an embodiment of the present invention.
Figure 2 depicts the FESEM image and EDX spectra of selected regions (ECJ: epidermal cell junction; IGCW: inner guard cell wall) of pea epidermis (a) placed in pure water, and (b) stained using BT2 solution in water (0.5 mL of 5 mM) for 2 min. (c) average Ca and K content in the IGCW region of the pea epidermis under different conditions, in accordance with an embodiment of the present invention.
Figure 3 depicts the FTIR spectra of 7,7-bis(piperazino)-8,8-
dicyanoquinodimethane (DPZDQ), Polygalacturonic acid (PGA) and DPZDQ+PGA mixture, in accordance with an embodiment of the present invention.
Figure 4 depicts the (a) variation of the intensity of the fluorescence emission of BT2 on adding PGA-Na+ in increasing mol ratios (the line is only a guide to the eye); (b) fluorescence emission spectra of BT2 in different forms: as microcrystalline solid, aqueous solution, aqueous solution with PGA-Na+ (200:1 ratio) and stain in pea epidermal layer (recorded in CLSM at the red points shown in the image in the inset), in accordance with an embodiment of the present invention.
Figure 5 depicts the CLSM images (fluorescence and overlay of fluorescence with
bright field) of the stained DU145 cell lines using BT2, in accordance with an
embodiment of the present invention.

Figure 6 depicts the CLSM images (fluorescence and overlay of fluorescence with bright field, different magnification) of the stained synechocystispcc 6803 cells by using 5 mM DMSO solution of 7,7-bis(benzylamino)-8,8-dicyanoquinodimethane (BBEDQ), (Scale bars represent 20 µM) in accordance with an embodiment of the present invention.
Figure 7 depicts the CLSM images of pea epidermis in buffer-free state, stained using (a – c) BT2 solution in water (15 µL of 0.05 mM), (d – f) BT2 solution in water (15 µL of 1mM), and (g – i) BT2 solution in DMSO (15 µL of 1mM); bright field images are shown in (a, d, g), and the fluorescence images at two magnifications in the remaining panels, in accordance with an embodiment of the present invention.
Figure 8 depicts the 3-D reconstructions (at different viewing angles) from CLSM Z-stack images of the pea epidermis stained using BT2 solution in water (15 µL of 1 mM), in (a, b) buffer-free state, and (c, d) in buffer medium, in accordance with an embodiment of the present invention.
Figure 9 depicts the CLSM fluorescence images (at different magnifications) of pea
epidermis kept in buffer medium (500 µL) and stained with BT2 solution in DMSO (15 µL
of 1 mM) under different conditions: (a, b) irradiated for 3 h and stained, (c, d) kept in
the dark for 3 h and stained, (e, f) stained
and irradiated for 3 h, in accordance with an embodiment of the present invention.
Figure 10 depicts the CLSM fluorescence images (at different magnifications) of pea epidermis kept in buffer medium (500 µL) and stained with BT2 solution in DMSO (15 µL of 1 mM) under different conditions: (a, b) irradiated for 3 h and stained, (c, d) kept in the dark for 3 h and stained, in accordance with an embodiment of the present invention.
Figure 11 depicts the CLSM images of stomata in the leaves of dicotyledon plants, (a) crown jasmine (Tabernaemontana divaricate), (b) paper rose (Bougainvillea glabra), and (c) thale cress (Arabidopsis thaliana), as well as a monocotyledon plant, (d) onion (Allium cepa) maintained in 500 µL buffer and stained using 15 µL aqueous solution of BT2 having concentrations, 5 mM (a-c) and 0.05 mM (d), in accordance with an embodiment of the present invention.

Figure 12 depicts the CLSM images of pea epidermis in buffer-free state, stained using 15 µL of 1.75 mM solutions of BT2 and DPZDQ in water and DMSO for 2 min, with and without washing, in accordance with an embodiment of the present invention.
Figure 13 depicts the molecular structure of the diaminodicyanoquinodimethanes derivatives, BT2, DPZDQ and BBEDQ, in accordance with an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and methods referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Definitions
For convenience, before further description of the present invention, certain terms employed in the specification, examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.
As used in the specification and the claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.
Functionally-equivalent processes and methods are clearly within the scope of the disclosure, as described herein.

The term “dicot” used herein refers to plants which at least are characteristic of having seeds with typically two embryonic leaves or cotyledons.
The term “monocot” used herein refers to plants which at least are characteristic of having seeds with typically one embryonic leaf or cotyledon.
The term “cyanobacterial cell” used herein refers generally to members belonging to archaea and cyanobacteria domain within the umbrella of prokaryotes.
The present invention relates to a method of staining of walls of guard cells, said
method comprising: (a) obtaining a composition comprising
diaminodicyanoquinodimethane derivative; and (b) placing plant tissue comprising guard
cells in the composition comprising said derivative for a period of time; wherein said
method stains wall of guard cells. In an embodiment, the
diaminodicyanoquinodimethane derivative is 7,7-bis(piperazine)-8,8-
dicyanoquinodimethane or 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-
toluene sulfonate) (M. Ravi, S. Cohen, I. Agranat, T. P. Radhakrishnan, Struct. Chem.
1996,7, 225). In a preferred embodiment, the diaminodicyanoquinodimethane derivative
is 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate). In an
embodiment the molar concentration of the diaminodicyanoquinodimethane derivative
used for staining of walls of guard cells is in the range of 0.025 mM to 5mM. In a
preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane
derivative used for staining of walls of guard cells is in the range of 0.03 mM to 2mM. In a
more preferred embodiment, the molar concentration of the
diaminodicyanoquinodimethane derivative used for staining of walls of guard cells is 0.05mM. In an embodiment, the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) substantially comprises water. In an embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period in the range of 30 seconds to 5 minutes. In a preferred embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane

bis(p-toluene sulfonate) for a time period of about 2 minutes. In an embodiment, the tissue is rinsed to remove excess dye. In an embodiment, the plant tissue is a dicot.
The present invention relates to a method of staining of walls of guard cells and walls and nuclei of epidermal cells, said method comprising: (a) obtaining a composition comprising diaminodicyanoquinodimethane derivative; and (b) placing plant tissue comprising guard cells in the composition comprising said derivative for a period of time; wherein said method stains wall of guard cells and walls and nuclei of epidermal cells. In an embodiment, the diaminodicyanoquinodimethane derivative is 7,7-bis(piperazine)-8,8-dicyanoquinodimethane or 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate. In a preferred embodiment, the diaminodicyanoquinodimethane derivative is 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate). In an embodiment the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of walls of guard cells and walls and nuclei of epidermal cells is in the range of 0.2 mM to 5mM. In a preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of walls of guard cells and walls and nuclei of epidermal cells is in the range of 0.5 mM to 2mM. In a more preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of walls of guard cells and walls and nuclei of epidermal cells is 1mM. In an embodiment, the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) substantially comprises water. In an embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period in the range of 30 seconds to 5 minutes. In a preferred embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period of about 2 minutes. In an embodiment, the tissue is rinsed to remove excess dye. In an embodiment, the plant tissue is a dicot
The present invention relates to a method of staining of walls and nuclei of guard cells and epidermal cells, said method comprising: (a) obtaining a composition

comprising diaminodicyanoquinodimethane derivative; and (b) placing plant tissue
comprising guard cells in the composition comprising said derivative for a period of time;
wherein said method stains walls and nuclei of guard cells and epidermal cells. In an
embodiment, the diaminodicyanoquinodimethane derivative is 7,7-bis(piperazine)-8,8-
dicyanoquinodimethane or 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-
toluene sulfonate). In a preferred embodiment, the diaminodicyanoquinodimethane
derivative is 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate).
In an embodiment the molar concentration of the diaminodicyanoquinodimethane
derivative used for staining of walls and nuclei of guard cells and epidermal cells is in the
range of 0.2 mM to 5mM. In a preferred embodiment, the molar concentration of the
diaminodicyanoquinodimethane derivative used for staining of walls and nuclei of guard
cells and epidermal cells is in the range of 0.5 mM to 2mM. In a more preferred
embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative
used for staining of walls and nuclei of guard cells and epidermal cells is 1mM. In an
embodiment, the composition comprising 7,7-bis(piperazinium)-8,8-
dicyanoquinodimethane bis(p-toluene sulfonate) substantially comprises water. In an embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period in the range of 30 seconds to 5 minutes In a preferred embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period of about 2 minutes. In an embodiment, the tissue is rinsed to remove excess dye. In an embodiment, the plant tissue is a monocot. In another embodiment, the plant tissue is a dicot.
The present invention relates to a method of staining of walls of guard cells, said
method comprising: (a) obtaining a composition comprising
diaminodicyanoquinodimethane derivative and at least one buffer; and (b) placing plant tissue comprising guard cells in the composition said derivative and buffer for a period of time; wherein said method stains walls of guard cells. In an embodiment, the

diaminodicyanoquinodimethane derivative is 7,7-bis(piperazine)-8,8-
dicyanoquinodimethane or 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-
toluene sulfonate). In a preferred embodiment, the diaminodicyanoquinodimethane
derivative is 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate).
In an embodiment the molar concentration of the diaminodicyanoquinodimethane
derivative used for staining of walls of guard cells is in the range of 0.05mM to 5mM. In a
preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane
derivative used for staining of walls of guard cells is in the range of 0.5mM to 2mM. In a
more preferred embodiment, the molar concentration of the
diaminodicyanoquinodimethane derivative used for staining of walls of guard cells is 1mM. In an embodiment, the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) substantially comprises DMSO. In an embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period in the range of 30 seconds to 5 minutes. In a preferred embodiment, the plant tissue is placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period of about 2 minutes. In an embodiment, the tissue is rinsed to remove excess dye. In an embodiment, the plant tissue is a dicot. In an embodiment, the buffer is 10mM MES-KCl solution. In an embodiment, the pH of the buffer solution is about 7.0.
The present invention relates to a method of staining of animal origin cells, said
method comprising: (a) obtaining a composition comprising
diaminodicyanoquinodimethane derivative; and (b) placing animal origin cells in the
composition comprising said derivative for a period of time, wherein said method stains
animal origin cells. In an embodiment, the diaminodicyanoquinodimethane derivative is
7,7-bis(piperazine)-8,8-dicyanoquinodimethane or 7,7-bis(piperazinium)-8,8-
dicyanoquinodimethane bis(p-toluene sulfonate). In a preferred embodiment, the
diaminodicyanoquinodimethane derivative is 7,7-bis(piperazinium)-8,8-

dicyanoquinodimethane bis(p-toluene sulfonate). In an embodiment the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of animal origin cells is in the range of 0.05mM to 5mM. In a preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of animal origin cells is in the range of 0.5mM to 2mM. In a more preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of animal origin cells is 1mM. In an embodiment, the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) substantially comprises water. In an embodiment, the animal origin cells are placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period in the range of 1 minute to 5 minutes. in a preferred embodiment, the animal origin cells are placed in the composition comprising 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate) for a time period of about 2 minutes. In an embodiment, the animal origin cells are rinsed to remove excess dye. In an embodiment, the animal origin cells are of mammalian origin. In an embodiment, the animal origin cells are of non-mammalian origin. In an embodiment, the animal origin cells are of primate origin. In a preferred embodiment, the animal origin cells are of human origin. In a more preferred embodiment, the animal origin cells are carcinogenic human origin. In an embodiment, the dye enters the cells and stain the cell completely. In a preferred embodiment, the cells are live cells.
The present invention relates to a method of staining of cyanobacterial cells, said
method comprising: (a) obtaining a composition comprising
diaminodicyanoquinodimethane derivative; and (b) placing cyanobacterial cells in the
composition comprising said derivative for a period of time, wherein said method stains
cyanobacterial cells. In an embodiment, the diaminodicyanoquinodimethane derivative is
7,7-bis(benzylamino)-8,8-dicyanoquinodimethane (C. G. Chandaluri, T. P.
Radhakrishnan. Opt. Mater. 2011, 34, 119). In an embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of cyanobacterial cells

is in the range of 1mM to 10mM. In a preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of cyanobacterial cells is in the range of 3mM to 7 mM. In a more preferred embodiment, the molar concentration of the diaminodicyanoquinodimethane derivative used for staining of cyanobacterial cells is 5mM. In an embodiment, the cyanobacterial cells are placed in the composition comprising 7,7-bis(benzylamine)-8,8-dicyanoquinodimethane for a time period in the range of 1 minute to 5 minutes. In a preferred embodiment, the cyanobacterial cells are placed in the composition comprising 7,7-bis(benzylamine)-8,8-dicyanoquinodimethane for a time period of 2 minutes. In an embodiment, the cyanobacterial cells are rinsed to remove excess dye. In an embodiment, the dye enters the cells and stains the cell completely.
The present invention relates to a method of staining of guard cells, epidermal cells, cyanobacterial cells, and cells of animal origin as substantially described herein, wherein said stained tissue or cells can be visualized using fluorescence response.
The diaminodicyanoquinodimethane based dyes described in the present invention are low cost compared to other small molecule dyes.
The diaminodicyanoquinodimethane based dyes described in the present invention are efficacious in low molar concentrations compared to other small molecule dyes.
The diaminodicyanoquinodimethane based dyes described in the present invention are non-toxic.
The diaminodicyanoquinodimethane based dyes described in the present invention are non-carcinogenic.
The diaminodicyanoquinodimethane based dyes described in the present invention are easy to prepare and store.
The diaminodicyanoquinodimethane based dyes described in the present invention are soluble in organic and/or aqueous media.

The diaminodicyanoquinodimethane based dyes described in the present invention are photostable.
The diaminodicyanoquinodimethane based dyes described in the present invention are thermostable.
EXAMPLES
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Example 1
Isothermal titration calorimetry
PGA in the leaf cell walls has both ionic (carboxylate) and neutral (carboxylic acid) sites. The well-known ‘egg box model’ envisions Ca2+ ions stitching the PGA chains together through the carboxylate groups (G. T. Grant, E. R. Morris, D. A. Rees, P. J. C. Smith, D. Thom, FEBS Lett. 1973, 32, 195).The strong binding between PGA-Na+ and BT2 is demonstrated by ITC experiment. BT2 in aqueous medium was titrated against the sodium salt of PGA (PGA-Na+), also dissolved in water. The thermograms recorded are shown in Figure 1; data analysis shows that the heat changes follow a simple binding model with an equilibrium constant of 1.33×106 dm3 mol-1 and enthalpy and entropy changes of -5.97 kJ mol-1 and 97.1 J mol-1 K-1. The molar ratio for binding is found to be 1.75, close to that expected for the two piperazinium sites of BT2 locking with the negatively charged carboxylate sites on the PGA polyanion. Figure 1a shows the raw thermogram from the isothermal titration of PGA-Na+ into BT2 in aqueous solution while Figure 1b shows the integrated thermograms from the isothermal titration of PGA-Na+ into BT2 in aqueous solution.

Example 2
Microscopy and elemental analysis
The epidermal layer placed in pure water, as well as in aqueous solution of BT2 and subsequently washed, was imaged in an FESEM, and EDX spectra recorded at several points in the relevant regions; the images and representative spectra are shown in Figure 2a, b. A clear reduction in the Ca content is seen in the samples treated with BT2, with the effect being enhanced with higher concentration of the dye; changes in the K content are not significant. DPZDQ has practically no impact as expected. Experiments with propidium iodide show effects similar to BT2. Parallel trends are seen in the epidermal cell junction as well. Figure 2a shows the FESEM image and EDX spectra of selected regions (ECJ: epidermal cell junction; IGCW: inner guard cell wall) of pea epidermis placed in pure water. Figure 2b shows the FESEM image and EDX spectra of selected regions (ECJ: epidermal cell junction; IGCW: inner guard cell wall) of pea epidermis placed in BT2 solution in water (0.5 mL of 5 mM) for 2 min. figure 2c shows the average Ca and K content in the IGCW region of the pea epidermis under different conditions.
Example 3
Spectroscopy
In order to probe the possible molecular level interactions that could lead to the DPZDQ staining, FTIR spectra of DPZDQ, PGA and the mixture of the two prepared by grinding the solids together was recorded (Figure 3). The prominent peak due to the carbonyl stretch vibration in PGA at ~ 1751 cm-1 is found to diminish significantly in the mixture; this is likely to be a consequence of the H-bonding interaction with the piperazine moieties in DPZDQ. It is noticed also that the broad peaks due to N-H stretch vibration in DPZDQ centered around 3420 cm-1 and O-H stretch vibration in PGA around 3510 cm-1, transform to a relatively narrower peak at 3415 cm-1 in the mixture, which again is suggestive of a well-defined H-bonding situation. Fluorescence experiments were

conducted to probe the origin of the strong emission that leads to efficient imaging. As the polymer to BT2mol ratio increases when PGA-Na+ is added to BT2, the fluorescence intensity increases and begins to saturate above a ratio of ~ 200:1 (Figure 4a); the Wemains nearly constant at ~ 535 nm as in the pure aqueous solution (Figure 4b). The emission spectrum recorded on the CLSM corresponding to the fluorescence image of the stomatal guard cell stained with BT2 (Figure 4b) shows a ^max ~ 537 nm with a shoulder at ~ 525 nm. The blue shifted peak is indicative of the presence of neighboring zwitterionic BT2 molecules, and their local field effects (P. Srujana, T. P. Radhakrishnan, Angew. Chem. Int. Ed. 2015,54, 7270). This is supported by the emission spectrum of BT2 in the microcrystalline solid with a ^max at ~ 522 nm (Figure 4b).
Example 4
Staining of DU145 human prostate cell line
DU145 cell line was stained by directly introducing 15 µl of the aqueous solution of the 1 mM BT2. The cell line was immediately washed with PBS and imaged in a CLSM. It was observed that the dye molecule enters the cells and stains it completely (Figure 5).
Example 5
Staining of cyanobacteria
BT2 was found to be less efficient in staining of cyanobacteria. Staining and imaging was carried out using another DADQ derivative,7,7-bis(benzylamino)-8,8-dicyanoquinodimethane (BBEDQ). 5 µl of 5 mM DMSO solution of BBEDQ was added on 0.2 ml of synechocystispc 6803 cellsin buffer; the cells were imaged in a CLSM. It was observed that the dye enters the cells and stains them completely (Figure 6).
Example 6
Staining of pea epidermis using BT2

Very small quantities of BT2 as aqueous solution (0.05mM or 1mM) or as DMSO solution (1mM) was spread directly on the epidermal layer of pea leaf placed on a microscope slide, kept for ~ 2 min, washed briefly to remove the excess dye, protected with a cover slip and imaged directly in a CLSM. Processes like incubation for extended time are not required, and the amount of dye used is 0.75 – 15 nmol. When the lower concentration (0.05mM) of BT2 in water is used, only the inner wall of the guard cells are stained (Figure 7a-c); with the higher concentration (1mM), walls of the guard cell, as well as walls and nuclei of the epidermal cells are stained clearly (Figure 7d-f). BT2 in DMSO at a concentration of 1mM enters the guard cell and stains its nucleus as well (Figure 7g-i).
The 3-D view constructed using Z-stack images (Figure8a, b) shows staining of the nucleus. Interestingly, the staining by BT2 is found to persist in samples which were washed rigorously and repeatedly.
Imaging experiments were also conducted on pea leaf epidermal layer kept in 500 µL of a buffer solution in which BT2 solution in water (0.05mM) was added earlier.It may be noted that the effective concentration of BT2 in this case is only 29µM. After allowing the dye adsorption (for about 10 min), the sample was taken out, washed and placed on a microscope slide, protected with a cover slip and imaged in a CLSM.As seen in Figure 9a-b, when the sample is irradiated for about 3 hours prior to staining, the guard cells are open and only the inner guard cell wall is stained. A seen in Figure 9c-d, when the sample is kept in dark for about 3 hours prior to staining, the guard cells are closed. As seen in Figure 9e-f, guard cells are open when irradiated in the presence of dye, which show that the staining process does not affect stomatal opening. Figures 8c, d show the 3-D view of the open inner guard cell wall. Figure 10a-b shows the images of samples in buffer medium stained by BT2 in DMSO irradiated for 3 hours prior to staining. Figure 10c-d shows the images of samples in buffer medium stained by BT2 in DMSO kept in dark for about 3 hours prior to staining. It can be clearly seen that opening and closing of guard cells is preserved even in the presence of dye. As the buffer helps to keep the cells alive,

staining with BT2 is of potential interest in live-cell studies that target the dynamics of the cell wall or related phenomena.
Example 7
Staining and imaging using diaminodicyanoquinodimethane based dyes in dicots, and onion
Imaging experiments were also carried out with leaves of dicotyledon plants such as crape jasmine (Tabernaemontana divaricate) (Figure 11a), paper rose (Bougainvillea glabra) (Figure 11b), thale cress (Arabidopsis thaliana) (Figure 11c), as well as a monocotyledon plant onion (Allium cepa) (Figure 11d), each of which show that BT2 is useful for various specimens (BT2 concentration of 5mM in Figure 11a-c, and 0.05mM concentration in Figure 11d).
Experiments were conducted also with DPZDQ to explore the general utility of DADQs in imaging. Even though DPZDQ has limited solubility in water, good quality images could be obtained using aqueous and DMSO solutions (Figure 12). Pea epidermis was stained for 2 minutes using 1.75mM DPZDQ in water or DMSO, with or without washing and imaged.
Example 8
Comparison of commercial dyes vs. BT2 for stomatal imaging
Table 1 below depicts the comparison of commercially available dyes in staining of stomata vs. BT2 as disclosed in the present invention. As seen from Table 1 below, BT2 at substantially lower concentrations and incubation time, effectively stains stomatal cells and presents a superior alternative to dyes known in the art.

#DAF-2DA: 2′,7′-dichlorofluorescein diacetate; BCECF-AM: 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceinacetoxymethyl ester; H2DCF-DA: 4,5-diaminofluorescein diacetate; PI: propidium iodide
References:
1. Apostolakos, P.; Livanos, P.; Galatis, B. Cell Mobil. Cytoskele. 2009, 66, 342-349.
2. Apostolakos, P.; Livanos, P.; Nikolakopoulou, T. L; Galatis, B. New Phytol. 2010, 186, 623-635.
3. Puli, M. R.; Rajsheel, P.; Aswani, V.; Agurla, S.; Kuchitsu, K.; Raghavendra, A. S. Planta 2016, 244, 831-841.
4. Agurla, S.; Gayatri, G.; Raghavendra, A. S. In Plant Nitric Acid Methods and Protocols; Gupta, K. J.,Eds.; Humana Press: New York, 2016; Vol. 1424, pp 49-56.
5. Puli, M. R.; Raghavendra, A. S. J. Exp. Bot.2012,63, 1349-1356.
6. Kong, Z.; Hotta, T.; Lee, Y.-R. J.; Horio, T.; Liu, B. Plant Cell 2010, 22, 191-204.
7. Hachez, C; Ohashi-Ito, K.; Dong, J.; Bergmann, D. C. Plant Physiol. 2011, 155, 1458-1472.
8. Chitrakar, R.; Melotto, M. J. Vis. Exp. 2010, 44, 2-5.
9. Tripathi, S.; Mondal, A. K. Int. J. Sci. Nat. 2012, 3, 788-798.

10. Ajuru, M. G.; Okoli, B. E. Int. J. Mod. Botany, 2012, 2, 115-119.
11. Tripathi, S.; Mondal, A. K. Int. J. Life Sci. Biotechnol. Pharma Res. 2012, 1, 104-113.
12. Mitra, S.; Maiti, G. G.; Maity, D. Adansonia 2015, 37, 139-147.
Overall, the present invention provides a novel method of staining of guard cells using diaminodicyanoquinodimethane based dyes (BT2, DPZDQ and BBEDQ) (Figure 13), which are cheap, thermo and photostable, and effective at significantly lower concentrations, and require substantially less incubation time. The methods described herein are non¬toxic to cells and can be used in live cell imaging to study physiology in real-time.

I/We claim:
1. A method of staining of walls of guard cells or walls of guard cells and walls and nuclei
of epidermal cells, said method comprising:
a. obtaining a composition comprising diaminodicyanoquinodimethane
derivative; and
b. placing plant tissue comprising guard cells in the composition comprising
said derivative for a period of time;
wherein said method stains walls of guard cells or walls of guard cells and walls and nuclei of epidermal cells at different concentrations of said dye.
2. A method of staining of walls and nuclei of guard cells and epidermal cells, said
method comprising:
a. obtaining a composition comprising diaminodicyanoquinodimethane
derivative; and
b. placing plant tissue comprising guard cells in the composition comprising
said derivative for a period of time;
wherein said method stains wall and nuclei of guard cells and epidermal cells.
3. A method of staining of walls of guard cells, said method comprising:
a. obtaining a composition comprising diaminodicyanoquinodimethane
derivative and at least one buffer; and
b. placing plant tissue comprising guard cells in the composition comprising
said derivative and buffer for a period of time;
wherein said method stains walls of guard cells.
4. A method of staining of animal origin cells, said method comprising:
a. obtaining a composition comprising diaminodicyanoquinodimethane derivative; and

b. placing animal origin cells in the composition comprising said derivative for a period of time; wherein said method stains animal origin cells.
5. A method of staining of cyanobacterial cells, said method comprising:
a. obtaining a composition comprising diaminodicyanoquinodimethane
derivative; and
b. placing cyanobacterial cells in the composition comprising said derivative
for a period of time,;
wherein said method stains cyanobacterial cells.
6. The method as claimed in claims 1-5, wherein said diaminodicyanoquinodimethane derivative is selected from the group consisting of 7,7-bis(piperazinium)-8,8-dicyanoquinodimethane bis(p-toluene sulfonate), 7,7-bis(piperazine)-8,8-dicyanoquinodimethane, and 7,7-bis(benzylamino)-8,8-dicyanoquinodimethane.
7. The method as claimed in claim 1, wherein the molar concentration of said derivative is in the range of 0.025 mM to 5mM, preferably in the range of 0.03 mM to 2mM, and more preferably 0.05mM for staining of walls of guard cells; and wherein molar concentration of the said derivative is in the range of 0.2mM to 5mM, preferably in the range of 0.5mM to 2mM, and more preferably 1mM for staining of walls of guard cells and walls and nuclei of epidermal cells.
8. The method as claimed in claims 2 to 4, wherein the molar concentration of said derivative is in the range of 0.05mM to 5mM, preferably in the range of 0.5mM to 2mM, and more preferably 1mM.

9. The method as claimed in claim 5, wherein the molar concentration of said derivative is in the range of 1mM to 10mM, preferably in the range of 3mM to 7mM, and more preferably 5mM.
10. The method as claimed in claims 1 to 5, wherein the composition substantially comprises a solvent selected from the group consisting of water, and DMSO.