Results and Discussion

Chapter II. Mitochondria Localization Induced Self-assembly of Peptide Amphiphiles for Cellular

2.3. Results and Discussion

2.3.1. Self-assembly and mitochondrial accumulation of Mito-FF. The Mito-FF molecular design consists of Phe-Phe-Lys (FFK) tripeptide as a backbone, synthesized via standard solid-phase synthesis (SSPS) (Experimental; Fig. 2.5-1). The N-terminus of FFK was conjugated with pyrene butyric acid, which not only serves as a fluorophore but also increases the propensity for self-assembly by enhancing hydrophobic and π-π interactions, and the amine group of the lysine side chain was conjugated to TPP for targeting mitochondria (Fig. 2-2a). Mito-FF self-assembled into nanofibers with a width of 9.6 ± 1.1 nm averaged over 100 nanofibers (in triplicate) for the individual fibers and a length of several micrometers in phosphate-buffered saline (PBS) (Fig. 2-2b). The CAC was determined to be ~60 µM by examining the excitation spectra of pyrene (Fig 2-2c), further confirmed by performing surface tension analysis (Fig. 2-2d). Fourier transform-infrared analysis (FT-IR) indicated an amide I band near 1637 cm^-1, indicating the presence of a β-sheet structure confirmation (Fig. 2-2e). Spectrofluorometric analysis exhibited intense sky blue emission at 450 nm in PBS above the CAC, indicative of pyrene excimer formation in the aggregation state. However, the emission was significantly reduced to a pale blue color in methanol and in PBS below the CAC, giving emission in the range of 370–410 nm reflective of pyrene in its non-aggregated state (Fig. 2-2f).

Intra mitochondrial assembly (above CAC)

Cell Apoptosis

Membrane disruption No peptide assembly

(below CAC) Cell entry by energy

independent pathway

N

Leakage of mitochondrial content (Protein, Apoptotic bodies, Matrix) Mito-Accumulation

Page 24 of 106 Figure 2-2. (a) Structural design of the mitochondria-targeting peptide amphiphile, Mito-FF, which is equipped with a pyrene group for fluorescence detection inside cells and TPP for targeting of mitochondria. (b) TEM image of Mito-FF fibril in water (scale bar, 50 nm) inset; diameter distribution of Mito-FF fibers showing average diameter of 9.6 ± 1.1 nm. (c) Plot of I344/I339 ratio of pyrene excitation in PBS as a function of Log [Mito-FF]. (d) Plot of Mito-FF surface tension as a function of Log [Mito- FF]. The CAC was indicated by the sudden drop of surface tension value calculated from the contact angle. (e) FT-IR spectra of Mito-FF fibril showing the beta sheet conformation. (f) Emission Spectra for Mito-FF showing the excimeric emission above the CAC (sky blue line) and lack of excimer below CAC.

2.3.2. Cellular Internalization. To analyses the cellular uptake and mitochondrial localization, Mito- FF were incubated in HeLa (Human cervix cancer) cell line, pre-labelled with MitoTracker Red and analyzed using confocal microscopy. Confocal microscopic imaging showed a good overlap between the red fluorescence of Mito Tracker Red and the blue fluorescence of Mito-FF, indicating that Mito- FF internalize and locate inside the mitochondria of the cell (Pearson’s Coefficient (Rr) was calculated as +0.8)¹² (Fig. 2-3a). Cellular uptake analysis at 4 ^oC showed similar uptake and co-localization inside mitochondria as that of 37 ^oC suggesting that the peptide enters inside the cell via an energy-independent pathway (Fig. 2-3b). Although the assembled structure and high molecular weight molecules generally are not capable of cellular uptake and require a complex energy-dependent mechanism such as endocytosis, Mito-FF overcomes this barrier because it diffuses through the plasma membrane and is readily internalized.

0.8 1.2 1.6 2.0

1.2 1.3 1.4

I344/I339

Log (C) ( ) CAC = 60 M

a b

d e f

400 450 500 550 600 0

50 100 150 200 250

Monomer

Intensity (a.u)

Wavelength (nm) Excimer

2000 1800 1600 1400

0.00 0.04 0.08 0.12

1735 cm-1

1637 cm-1

Intensity (a.u)

Wavenumber (cm-1)

1675 cm-1

c

7 8 9 10 11 12 0

10 20 30 40

Populations

Diameter (nm)

50 nm

Page 25 of 106 Figure 2-3. Mitochondrial co-localization of Mito-FF measured with Mito Tracker Red FM shows high localization inside mitochondria (a) at 37 ^oC (scale bar, 2 μm) and (b) 4 ^oC (scale bar, 5 μm).

2.3.3. Intra-mitochondrial assembly. The mitochondrial accumulation of Mito-FF was analyzed to determine whether the effective concentration inside the mitochondria could exceed the CAC required for self-assembly (Experimental; Fig. 2.5-2, 2.5-3). Mito-FF was incubated with HeLa cells for 3 h and its uptake was measured by monitoring the emission spectra of Mito-FF in the cell lysate (Fig. 2-4a- c).¹³ Our calculations showed that the mitochondrial accumulation of Mito-FF increased in accordance with the original concentration in the culture medium, i.e., 3.1±1 mM and 11.2±2.6 mM for external concentrations of 5 μM and 10 μM respectively, suggesting that the self-assembly of Mito-FF inside the mitochondria could readily occur as a result of the high local concentration (Fig. 2-4d). We envisioned that intracellular self-assembly of Mito-FF would result in excimeric emission from the pyrene, similar to the behavior of Mito-FF in the bulk solution (Fig. 2-2f). To study the excimeric emission of Mito-FF in HeLa cells, we performed a two-photon microscopic analysis of HeLa cells pretreated with Mito-FF at a two-photon excitation wavelength of 780 nm. The emission analysis through different band-pass (BP) filters for wavelengths 420-480 nm (for detection of excimeric emission) and 380-420 nm (for detection of monomeric emission) showed different intensities of emission. The 420-480 nm filter provided much higher-intensity emission, which indicates that the Mito-FF fibrils formed inside mitochondria, giving rise to excimer emission from the pyrene in the aggregated state (Fig. 2-4e).

Mito-FF Mito tracker Merge

37 oC4 oC a

b

2 μm 2 μm 2 μm

5 μm 5 μm 5 μm

Page 26 of 106 Figure 2-4. (a) Emission spectra of Mito-FF in Buffer/MeOH mixture (1:1) recorded for the generation of calibration plot. (b) Calibration plot of Mito-FF from the emission spectra. (c) Emission spectra of HeLa cell lysate (1:1 Buffer/MeOH mixture) after 3 h treatment with Mito-FF. (d) Analysis of mitochondrial accumulation of Mito-FF in HeLa cells after 3 h of incubation. (e) Two photon microscopic analysis of Mito-FF showing reduced emission through filter of wavelength 380-420 nm, representing monomeric emission and intense emission through 420-480 nm filter indicating excimeric emission confirming that the self-assembly of Mito-FF could happens inside the mitochondria of living cells.

To further study the fibril formation inside the mitochondria, another designer peptide was synthesized: Mito-FF-NBD, where pyrene was replaced with the environmentally sensitive dye, 4- nitro-2, 1, 3-benzoxadiazole (NBD) (Fig. 2-5a). NBD is highly fluorescent in hydrophobic environments and has been used for imaging biological components.¹⁴ Recently, Xu et al. reported that NBD could serve as an efficient reporter for intracellular fiber formation of peptide-based small molecules based on its bright fluorescence upon intracellular fiber formation.¹⁵ The co-assembly of Mito-FF-NBD with Mito-FF into fiber assembly in PBS was confirmed by TEM analysis (Fig. 2-5b) and molecular simulation (CGMD) (Fig. 2-5c-f). The TEM showed that the co-assembled fibers have a decreased diameter (7.1 ± 0.8 nm averaged over 100 nanofibers in triplicate) compared with Mito-FF nanofiber alone or Mito-FF-NBD alone, suggesting that they assemble each other. We co-incubated HeLa cells with Mito-FF-NBD (10 μM) and Mito-FF (10 μM) and analyzed fluorescence emission using confocal microscopy as represented in the schematic diagram (Fig. 2-5g). Only Mito-FF-NBD showed diminished fluorescence. In contrast, co-incubation of Mito-FF-NBD and Mito-FF resulted in intense green emission of NBD and good co-localization with Mito Tracker (Fig. 2-5h). Mito-FF-NBD could form fiber (Fig. 2-5j) above their CAC of 1.5 mM (Fig. 2-5i).

400 450 500 550

0 100 200 300

400 0.125 

0.25 

0.5 

1 

Intensity (a.u)

Wavelength (nm)

400 450 500 550

0 20 40 60 80 100

10 

5 

Intensity (a.u)

Wavelength (nm) 0.0 0.2 0.4 0.6 0.8 1.0

100 200 300 400

Intensity at 377 nm

Concentration ()

y = 355.68x + 24.504

b c a

4 6 8 10 12

0 4 8 12

Mitochondrial Accumulation (mM)

Incubation Concentration (M) 3.14±1.01

11.23±2.85

d e BP 380-420 BP 420-480

Page 27 of 106

r

(a) (b)

(c) (d)

5.6 nm

20 nm

Water

z x

y

Pyrene Backbone

Phenyl ring

P⁺

TPP NBD

Backbone Phenyl ring

P⁺

TPP

Phosphorus atom Nitrogen atom

Carbon atom Oxygen atom Hydrogen atom

Pyrene P⁺‒C2

Backbone Phosphorus atom Nitrogen atom

Carbon atom Oxygen atom Hydrogen atom

Phosphorus atom Nitrogen atom

Carbon atom Oxygen atom Hydrogen atom

Phosphorus atom Nitrogen atom

Carbon atom Oxygen atom Hydrogen atom

NBD

Backbone P⁺‒C3

Benzene

Fibril formation and bright fluorescence Co incubation

With Mito-FF Mito-FF-NBD

Below CAC, no fibril formation, no fluorescence

= Mito-FF

= Mito-FF-NBD a

b

Mito-FF-NBD Mito-tracker Merge

With Mito-FFWithout Mito-FF

c d

e f

g h

1000 0

250 500 750

Intensity at 653 nm (a.u)

Log [C](M)

CAC = 1.5 mM

200 nm

i j

600 650 700

0 100 200 300

2 mM 1.81 mM 1.67 mM 1.54 mM 1.43 mM 1.33 mM 1.25 mM 1.18 mM 1.11 mM 1.05 mM 0.8 mM 0.6 mM 0.4 mM 0.2 mM

Intensity (a.u)

Wavelength (nm)

k

0.0 0.4 0.8

Intensity at 540 nm

Concentration ()

y = 1856.9x + 3.1489 R² = 0.9989

520 560 600

10 dosage 100 dosage

Intensity(a.u)

Wavelength (nm)

0 1000 2000 3000

100

IMC (M)

Concentration (M) 10

m l

c n

Page 28 of 106 We observed that Mito-FF-NBD showed a concentration dependent fluorescence inside the cell.

No fluorescence observed for 10 μM dosage concentration, negligible fluorescence for 20 μM and bright green fluorescence starts appearing above 40 μM, which well-overlaps with red fluorescence of Mito Tracker as indicated by the 2D (Fig. 2-6a) and 3D (Fig. 2-6b). This observation indicates that much higher concentration is needed for Mito-FF-NBD to rise intra-mitochondrial concentration above the CAC compared with Mito-FF. When we have analyzed the IMC of Mito-FF-NBD in the HeLa cells by using the same procedure as described for Mito-FF, we found very low cellular uptake for 10 μM while the IMC increased to 3.2±0.7 mM at high dosage concentration (100 µM) (Fig. 2-5k). These results suggest that Mito-FF-NBD could co-assemble well with Mito-FF and NBD located inside the hydrophobic fiber to emit bright green fluorescence. However, Mito-FF-NBD without Mito-FF could not self-assemble into the fiber because it could not reach to the high CAC of 1.5 mM in the mitochondria with an external concentration of 10 μM.

Figure 2-5. (a) Molecular structure of Mito-FF-NBD. (b) TEM image showing Mito-FF co-assembly with Mito-FF-NBD to form fibers of narrow diameter. CGMD simulation model for the co-assembly of Mito-FF-NBD with Mito-FF. Coarse-grained (CG) model of (c) Mito-FF-NBD and (d) Mito-FF.

NBD, phenyl ring, TPP and pyrene groups consist of six, ten and eight CG beads, respectively. NBD group contains O‒N‒O, N‒O‒N, N‒C3‒O and benzene ring. Backbone of Mito-FF-NBD consists of five CG beads, which are 3 amides and 2 amines, and Mito-FF consists of seven CG beads, which are 1 ketone, 3 amides, 2 amines, and 1 alkyl groups. (e) Snapshot of the simulation result of cylindrically self-assembled 174 Mito-FF-NBDs and 174 Mito-FFs in the box (i.e. 25×25×30 nm³) filled with water after performing CGMD for 3.6 μs. The cylinder shows effective radius and length of the Mito-FF- NBD and Mito-FF fibril. (f) Radial number density of four constituent molecules in the fibril from the principal axis of fibril to its surface. The numbers of groups in the same volume of radial shell were counted six times within the final 50 ns of the MD simulation and averaged with a 10 ns interval.

Hydrophilic TPP and hydrophobic phenyl groups showed high density compared to others since they were contained in both Mito-FF-NBD and Mito-FF. High density of phenyl group at the center of fibril indicated the hydrophobic characteristics inside of the fibril. NBD groups were well distributed in that hydrophobic environment within the fibril. (g) Schematic diagram showing co-assembly of Mito-FF- NBD with Mito-FF. (h) Co-assembly inside mitochondria indicated by the bright green fluorescence of Mito-FF-NBD in presence of Mito-FF (lower panel); however, such emission was not observed with Mito-FF-NBD alone (upper panel) (scale bar, 2μm). (i) CAC determination of Mito-FF-NBD using nile red encapsulation method showing CAC value of 1.5 mM. (j) The TEM images of Mito-FF-NBD showing fibrous structure. (k) Mitochondrial accumulation of Mito-FF-NBD.

Page 29 of 106 Figure 2-6. (a) Dosage dependent confocal microscopic analysis for Mito-FF-NBD. Blue: Nuclei stain, Red: Mito Tracker, Green: Mito-FF-NBD. (b) 3D localization for Mito-FF-NBD (middle: zoom view, right: orthogonal view). Inset: co-localization between Mito Tracker and Mito-FF-NBD. Blue: Nuclei stain, Red: Mito Tracker Red FM, Green: Mito-FF-NBD (left) Zoom image (middle) Orthogonal view (right).

Mito tracker Mito-FF-NBD Hoechst Merge

10 μM20 μM100 μM

5 μm 5 μm 5 μm 5 μm

5 μm 5 μm 5 μm 5 μm

5 μm 5 μm 5 μm 5 μm

3 D view Zoom Orthogonal view

5 μm

b a

Page 30 of 106 To visualize intra-mitochondrial self-assembly, TEM images were taken after application of Mito-FF to mitochondria isolated from a mouse brain (C3H female).¹⁶ The TEM images showed fiber structures with a diameter of 9.0 ± 1.5 nm averaged over 100 nanofibers (triplicate) within the mitochondria (Fig. 2-7a-b). For clear demonstration of the nanofibers within the mitochondria, TEM tomography (TEMT) was performed. TEMT is a powerful method to study the internal architecture of the cellular structure or macromolecular complexes.¹⁷ In here, depending upon the three dimensional (3D) orientation of the fibrils within the mitochondria and internal membrane at different tilt angles, we could differentaite the fibrils which are cylindrial in shape from the internal mitochondrial membranes which have a lamellar structure. Using TEMT we figure out the fibrils within the mitochondria with respect to tilt angles from -68° to +68°, where at -60° focused fibril has observed in its surface view and at +20° the same fibril has observed from the top as shown in the Fig. 2-7e (-60° (left), +20° (right)).

The mitochondrial cristae enclose a dense protein-rich matrix and are surrounded by an energy- transducing inner membrane and an outer membrane. As shown in Fig. 2-7e, the fibril formation altered the topology of the mitochondrial membrane compared with the untreated control (Fig. 2-7c). As a consequence, a considerable adverse effect on cell function may be expected, as the mitochondria play a vital role in controlling cellular metabolism.

Figure 2-7. (a) TEM images for Mito-FF fibrils inside mitochondria isolated from a C3H female nude mouse brain. (b) Diameter distribution of Mito-FF nanofibrils within the isolated mitochondria. (c) TEM image for isolated mitochondria (control). (d) 3D volume image of Mito-FF fibrils inside mitochondria isolated from a C3H female nude mouse brain. (e) Construction images showing the side and cross-sectional views of fibrils at different tilt-angles of −60° and +20° (from left to right) (scale bar, 100 nm).

Control Mitochondria FF fibril formation inside the mitochondria

c d e

7 8 9 10 11 12

0 10 20 30 40

Populations

Diameter (nm)

a b

Page 31 of 106 2.3.4. Mito-FF fibrils induce mitochondrial dysfunction. The mitochondria showed severe morphological damage with fragmentation after 1 h incubation of Mito-FF in HeLa cells (Fig. 2-8a).

To further investigate the mitochondrial dysfunction induced by Mito-FF fibrils, the membrane potential depolarization of the mitochondria was analyzed by using tetramethyl rhodamine dye (TMRM), which shows bright fluorescence under normal conditions that vanishes following membrane depolarization.¹⁸ Confocal analysis of TMRM-labeled HeLa cells after treatment with Mito-FF showed robust red fluorescence after 1 h incubation, which began to diminish within 3 h and had completely vanished after 6 h (Fig. 2-8b), suggesting that the Mito-FF fibrils in the mitochondria adversely affected their function. This could imbalance mitochondrial reactive oxygen species (ROS) production; excess production of ROS contributes to mitochondrial damage in several pathologies and causes mitochondrial DNA damage, membrane disruption, and protein damage. The excess mitochondrial ROS production induced by Mito-FF fibrils was monitored with Mito SOX Red, which showed red fluorescence after 6 h of incubation with Mito-FF (Fig. 2-8d). Furthermore, staining with dihydroethidium dye (DHE), which intercalates into nuclear DNA and oxidizes to the ethidium ion to show red fluorescence in the presence of ROS, showed strong red emission in HeLa cells after treatment with Mito-FF, suggesting that the Mito-FF fibrils markedly induced cell damage¹⁹ (Fig. 2-8c).

Figure 2-8. (a) Confocal analysis showing mitochondrial shrinkage after Mito-FF treatment on HeLa cells (right) compared with control (left) (scale bar, 1 μm). (b) Membrane depolarization of mitochondria over time after treatment with Mito-FF was measured by TMRM (scale bar, 2 μm). (c) ROS generation measured by DHE (d) Mito SOX Red (scale bar, 5 μm).

a

b

Control Mito-FF treated c

TMRM – 1h TMRM – 6h

ROS - DHE

1 μm 1 μm

2 μm 2 μm

5 μm

5 μm

d ROS – Mito SOX

Page 32 of 106 Figure 2-9. (a-c) Molecular design of the control peptides. (d) TEM image of Mito-VV (scale bar, 500 nm). (e) Membrane depolarization induced by Mito-VV after 1 or 6 h (scale bar, 2 μm). (f) Intracellular ROS generation (scale bar, 5 μm) by DHE and (g) MitoSOX. (h) TEM image of Mito-FxFx (scale bar, 200 nm). (i) Membrane depolarization induced by Mito-FxFx after 1 or 6 h (scale bar, 2 μm). (j) Intracellular ROS generation (scale bar, 5 μm) by DHE and by (k) MitoSOX. (l) TEM image of Mito- GG (scale bar, 200 nm). (m) Membrane depolarization induced by Mito-VV after 1 or 6 h (scale bar, 5 μm), and (n) intracellular ROS generation by DHE and (o) MitoSOX. (p) TEM images of mitochondria within the HeLa cell showing the morphological damage induced by Mito-FF. Fibers formed inside the mitochondria and extruded out of the membrane (scale bar, from left to right; 1 μm, 200 nm, 50 nm).

(q) Mito-GG-treated mitochondria HeLa cell showing no damage of mitochondria. (r) Control HeLa cell mitochondria (scale bar, 500 nm).

d

h

l

e

i

m

f

j

n

TMRM – 1h TMRM – 6h ROS - DHE TEM

2 μm 2 μm

2 μm 2 μm

2 μm 2 μm

5 μm

5 μm

5 μm 500 nm

200 nm

200 nm

5 μm

5 μm

5 μm

a b c

g

k

o

ROS - MitoSOX

q Mito-GG treated r Control Intra-mitochondrial fibril formation of Mito-FF

500 nm 500 nm

50 nm

1.0 μm 200 nm

p

Page 33 of 106 Diphenylalanine is a crucial building block for accelerating the amyloid assembly process in Alzheimer’s disease and other neurodegenerative diseases, and it ultimately induces toxicity by the formation of toxic fibril aggregates.²⁰ We speculate that the mitochondrial damage arose specifically from the fibril formation of diphenylalanine rather than other morphologies. To investigate this further, our molecular design was extended to Mito-VV and Mito-FxFx, where phenylalanine was replaced by valine and cyclohexylalanine, respectively (Fig. 2-9a-b). Both peptides assembled into fibrous structures (Fig. 2-9d, h) with a CAC of 70 µM and 30 µM, respectively (Experimental; Fig. 2.5-6). The membrane depolarization ability in HeLa cells was evaluated, both peptides could efficiently depolarize the mitochondrial membrane within 6 h, which was followed by ROS generation as evidenced with DHE (Fig. 2-9f, j) and Mito SOX Red (Fig. 2-9g, k). Notably, Mito-GG, a designer peptide forming a micelle of ~50 nm diameter (Fig. 2-9c) above the CAC of 114 µM) (Experimental; Fig. 2.5-7) showed no membrane depolarization by TMRM (Fig. 2-9n). Additionally Mito-GG induced considerably less ROS production as shown in Fig. 2-9o. From these results, we considered that mitochondrial stress induced by the small micelles might be significantly lower than that induced the fibril analogs, resulting in a reduced extent of dysfunction caused by Mito-GG.

A TEM experiment for HeLa cells treated with Mito-FF (20 µM for 3 h) was conducted to investigate the structural change of mitochondria induced by intra-mitochondrial fibril formation.²¹ As shown in (Fig. 2-9p), the mitochondria were found to be severely damaged with distorted membrane after Mito-FF treatment, and there were devoid of normal mitochondria morphology within HeLa cells.

It was observed that the fibrils were centered within the destroyed mitochondria. It might be expected that fibril formation inside the mitochondria induces damage to the mitochondria with membrane disruption. However, the mitochondria in HeLa cells treated with Mito-GG remained unaffected (Fig.

2-9q) under similar conditions and appeared identical to the control (Fig. 2-9r). These results indicate that fibril assembly plays an important role in mitochondria dysfunction by disruption of the mitochondrial membrane, whereas spherical assembly does not.

Figure 2-10. Surface charge analysis of Mito peptide fibers. (a) Mito-FF, (b) Mito-GG.

Page 34 of 106 2.3.5. Proposed mechanism for mitochondrial dysfunction. We speculate that the mitochondrial dysfunction induced by the supramolecular assembly resulted from a loss of mitochondrial membrane integrity and consequent leakage of mitochondrial content. Our analyses above suggest that a fibrous assembly, rather than a spherical assembly, was primarily responsible for the loss of mitochondrial membrane integrity. The high positive surface charge of +43±1.3 mV (triplicate measurement) (Fig. 2- 10), and hydrophobicity of Mito-FF fibrils favors strong interactions with the mitochondrial membrane, which could destroy the negatively charged mitochondrial membrane. ^{22, 23}

To determine whether the fibrous assembly could induce membrane disruption, we conducted a dye-leakage assay using a model liposome encapsulated with a self-quenching dye (calcein). The addition of Mito-FF fibrils (500 μM) rapidly caused a loss of membrane integrity in the liposomes, as indicated by the leakage of the encapsulated calcein dye. Dye leakage increased over time and reached a maximum leakage of 50% within 45 min. However, the addition of Mito-FF in the molecular state (i.e., below the CAC of 10 μM) did not induce liposome leakage and Mito-GG (500 μM), which formed a spherical assembly as described above, showed no liposome leakage (Fig. 2-11a).

Figure 2-11. (a) Membrane disruption ability of Mito-FF fibrils monitored using calcein encapsulated in a model liposome; a schematic diagram is provided. Data represent mean ± s.d. from three independent experiments. (b) Leakage of mitochondrial protein to the cytosol monitored using APEX labeling (scale bar, 2 μm).

Mito-GGMito-FFControl

Anti-V5/anti-mouse

488 MitoTracker Merge

0 10 20 30 40

0 20 40 60

Mito-FF_500 M Mito-GG_500 M Mito-FF_10 M Mito-GG_10 M

Dye leakage (%)

Time (m)

Mito-FF fibril Interacting with liposome

Membrane disruption calcine release

No fluorescence due to self quenching of calcine

Fluorescence “ turn on “ due to dye leakage

a b