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by Meng-Chih Su, Wesley Wei-Wen Hsiao, Huan-Cheng Chang
Fluorescent Nanodiamonds
Cover
Preface
Part I: Basics
1 Introduction to Nanotechnology
1.1 Nanotechnology: From Large to Small
1.2 Nanocarbons: Now and Then
References
2 Nanodiamonds
2.1 Ah, Diamonds, Eternal Beautiful
2.2 Diamonds: From Structure to Classification
2.3 Diamond Synthesis
2.4 Nanodiamonds: A Scientist’s Best Friend
References
3 Color Centers in Diamond
3.1 Nitrogen Impurities
3.2 Crystal Defects
3.3 Vacancy‐Related Color Centers
3.4 The NV Center
References
4 Surface Chemistry of Nanodiamonds
4.1 Functionalization
4.2 Bioconjugation
4.3 Encapsulation
References
5 Biocompatibility of Nanodiamonds
5.1 Biocompatibility Testing
5.2 In Vitro Studies
5.3 Ex Vivo Studies
5.4 In Vivo Studies
References
6 Producing Fluorescent Nanodiamonds
6.1 Production
6.2 Characterization
References
Part II: Specific Topics
7 Single Particle Detection and Tracking
7.1 Single Particle Detection
7.2 Single Particle Tracking
References
8 Cell Labeling and Fluorescence Imaging
8.1 Cell Labeling
8.2 Fluorescence Imaging
References
9 Cell Tracking and Deep Tissue Imaging
9.1 Cellular Uptake
9.2 Cell Tracking
9.3 Deep Tissue Imaging
References
10 Nanoscopic Imaging
10.1 Diffraction Barrier
10.2 Superresolution Fluorescence Imaging
10.3 Cathodoluminescence Imaging
10.4 Correlative Light‐Electron Microscopy
References
11 Nanoscale Quantum Sensing
11.1 The Spin Hamiltonian
11.2 Temperature Sensing
11.3 Magnetic Sensing
References
12 Hybrid Fluorescent Nanodiamonds
12.1 Silica/Diamond Nanohybrids
12.2 Gold/Diamond Nanohybrids
12.3 Silver/Diamond Nanohybrids
12.4 Iron Oxide/Diamond Nanohybrids
References
13 Nanodiamond‐Enabled Medicine
13.1 NDs as Therapeutic Carriers
13.2 Drug Delivery
13.3 Gene Therapy
13.4 Animal Experiments
References
14 Diamonds in the Sky
14.1 Unidentified Infrared Emission
14.2 Extended Red Emission
14.3 Cosmic Events at Home on Earth
References
Further Reading
Review Articles
General References
Index
End User License Agreement
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Title Page
Table of Contents
Cover
Preface
Part I: Basics
1 Introduction to Nanotechnology
1.1 Nanotechnology: From Large to Small
1.2 Nanocarbons: Now and Then
References
2 Nanodiamonds
2.1 Ah, Diamonds, Eternal Beautiful
2.2 Diamonds: From Structure to Classification
2.3 Diamond Synthesis
2.4 Nanodiamonds: A Scientist’s Best Friend
References
3 Color Centers in Diamond
3.1 Nitrogen Impurities
3.2 Crystal Defects
3.3 Vacancy‐Related Color Centers
3.4 The NV Center
References
4 Surface Chemistry of Nanodiamonds
4.1 Functionalization
4.2 Bioconjugation
4.3 Encapsulation
References
5 Biocompatibility of Nanodiamonds
5.1 Biocompatibility Testing
5.2 In Vitro Studies
5.3 Ex Vivo Studies
5.4 In Vivo Studies
References
6 Producing Fluorescent Nanodiamonds
6.1 Production
6.2 Characterization
References
Part II: Specific Topics
7 Single Particle Detection and Tracking
7.1 Single Particle Detection
7.2 Single Particle Tracking
References
8 Cell Labeling and Fluorescence Imaging
8.1 Cell Labeling
8.2 Fluorescence Imaging
References
9 Cell Tracking and Deep Tissue Imaging
9.1 Cellular Uptake
9.2 Cell Tracking
9.3 Deep Tissue Imaging
References
10 Nanoscopic Imaging
10.1 Diffraction Barrier
10.2 Superresolution Fluorescence Imaging
10.3 Cathodoluminescence Imaging
10.4 Correlative Light‐Electron Microscopy
References
11 Nanoscale Quantum Sensing
11.1 The Spin Hamiltonian
11.2 Temperature Sensing
11.3 Magnetic Sensing
References
12 Hybrid Fluorescent Nanodiamonds
12.1 Silica/Diamond Nanohybrids
12.2 Gold/Diamond Nanohybrids
12.3 Silver/Diamond Nanohybrids
12.4 Iron Oxide/Diamond Nanohybrids
References
13 Nanodiamond‐Enabled Medicine
13.1 NDs as Therapeutic Carriers
13.2 Drug Delivery
13.3 Gene Therapy
13.4 Animal Experiments
References
14 Diamonds in the Sky
14.1 Unidentified Infrared Emission
14.2 Extended Red Emission
14.3 Cosmic Events at Home on Earth
References
Further Reading
Review Articles
General References
Index
End User License Agreement
List of Tables
Chapter 02
Table 2.1 Typical physical properties of diamond.
Table 2.2 Classification of diamonds.
Chapter 03
Table 3.1 Nomenclature for zero‐phonon lines of color centers in diamond.
Table 3.2 Spectroscopic properties of vacancy‐related color centers in bulk diamonds.
Table 3.3 Calibrations of the optical absorption in zero‐phonon lines of some vacancy‐related color centers in bulk diamonds.
Table 3.4 Character table of the
C
3v
point group.
Chapter 06
Table 6.1 Methods of creating vacancies in nanodiamonds by radiation damage.
Chapter 08
Table 8.1 Cells successfully labeled with FNDs by endocytosis [7–32].
Chapter 09
Table 9.1 Biochemical and physiological actions of some representative endocytosis inhibitors.
Table 9.2 Characteristics of an ideal imaging technology for stem cell tracking during clinical trial.
Chapter 14
Table 14.1 Types, abundances, and sizes of stardust in meteorites.
Table 14.2 Characteristics of extended red emission and its carrier.
List of Illustrations
Chapter 01
Figure 1.1 A molecular model of C
60
.
Figure 1.2 (a) Formation of SWCNT from a carbon sheet showing the unit vectors (
a
1
and
a
2
), the chiral vector (
C
h
), the chiral indices (
n
,
m
), and the chiral angle (
θ
). The wrapping angle is
ϕ
= 30 °−
θ
. In this particular example, by rolling up the sheet along
C
h
such that the origin (0,0) coincides with point C, a nanotube indicated by indices (11,7) is formed. (b) Three representative SWCNT structures in similar diameter: zigzag, armchair, and chiral.
Figure 1.3 A molecular model of graphene. The almost perfect web is only one atom thick.
Chapter 02
Figure 2.1 Unit cell of the diamond cubic crystal structure with a lattice constant of 3.57 Å. Carbon atoms are presented in gray spheres.
Figure 2.2 Optical transmission spectra of natural and synthetic diamonds of various types. Note the change in scale at 1 μm.
Figure 2.3 Diamond crystallites synthesized by the HPHT method. The particles, diameter approximately 400 μm, contain typically 100 ppm nitrogen.
Figure 2.4 Phase and reaction diagram of carbon. The regions where the three synthesis methods, HPHT, CVD, and detonation (or shock wave), discussed in the text are indicated in the figure.
Figure 2.5 Ultrahigh pressure belt apparatus used by General Electric for the HPHT diamond synthesis.
Figure 2.6 Schematic diagram of the CVD diamond growth mechanism. Methane is used as the carbon source in this example.
Figure 2.7 (a) High‐resolution TEM image of DNDs. The particles are surrounded by graphitic and soot‐like materials. (b) Structure model of the DND agglomerates.
Figure 2.8 Structures of adamantane (red), diamantane (green), and triamantane (blue) as segments of the diamond lattice.
Figure 2.9 Diamond nanoparticles (white powders in vial) extracted from a piece of the carbonaceous Murchison meteorite.
Chapter 03
Figure 3.1 (a) IR absorption spectrum of a synthetic diamond crystal with [N
0
] = 109 ppm. The ratio of the height of the hump at 1130 cm
−1
versus the depth of the dip at 2120 cm
−1
was measured to determine the nitrogen concentration. (b) IR absorption spectrum of a natural diamond crystal with a nitrogen concentration of [N
A
] = 900 ppm. The ratio of the height of the peak at 1282 cm
−1
versus the depth of the dip at 2120 cm
−1
was measured to determine the nitrogen concentration.
Figure 3.2 An irradiation‐treated green diamond.
Figure 3.3 Structures of vacancy‐related color centers in diamond: (a) V
0
, (b) NV, (c) H3, and (d) N3. The carbon atoms, nitrogen atoms, and vacancies are denoted by black spheres, dark red spheres, and blue dashed circles, respectively.
Figure 3.4 UV‐Vis absorption spectra of natural and synthetic diamonds after irradiation at room temperature with 2‐MeV electrons: (a) type Ia, (b) type IIa, (c) type Ib with additional annealing, and (d) type Ia with additional annealing. All the spectra were recorded at liquid‐nitrogen temperature.
Figure 3.5 Luminescence spectra of NV
0
, NV
−
, and V
0
in diamond at 77 K. The characteristic ZPLs are located at 575, 637, and 741 nm, respectively.
Figure 3.6 Photographs of fluorescent diamonds containing high‐density ensembles of (a) NV and (b) H3 centers excited by green and blue light, respectively.
Figure 3.7 Schematic illustration of the photoinduced ionization and recombination of NV
−
and NV
0
. Processes 1 and 2 are associated with ionization and processes 3 and 4 are associated with recombination.
Figure 3.8 Electronic energy diagram of the NV
−
center in diamond.
Figure 3.9 ODMR spectra of a single NV
−
center in bulk diamond in the presence of an increasing magnetic field.
Chapter 04
Figure 4.1 An idealized cubic ND with a perfect crystal structure and a length of 2.85 nm (or eight unit cells in one dimension).
Figure 4.2 IR transmission spectra of DNDs subjected to (a) LiAlH
4
reduction, (b) borane reduction, (c) no treatment, (d) oxidation with HClO
4
, (e) oxidation with HNO
3
and H
2
SO
4
(1 : 1, v/v), and (f) reaction with ozone under ultraviolet irradiation.
Figure 4.3 Overview of the commonly used methods for chemical modification and functionalization of ND surfaces.
Figure 4.4 (a) Synthesis of hydrophobic HPHT‐NDs by reduction and silanization of acid‐treated NDs. (b) Photograph of 40‐nm HPHT‐NDs suspended in toluene/water before and after the reduction and silanization treatments.
Figure 4.5 (a) Zeta potentials of oxidative‐acid‐treated HPHT‐NDs (nominal size ∼100 nm) as a function of solution pH. (b) Conductometric titration of oxidative‐acid‐treated 100‐nm HPHT‐NDs. In this titration, an excessive amount of NaOH was first added into the ND suspension and then neutralized with 0.1 N HCl.
Figure 4.6 (a) UV‐Vis absorption spectra of horse cytochrome
c
solutions before (solid curves) and after (dash dot curves) exposure to HPHT‐NDs at two different protein concentrations. (b) Adsorption isotherms of horse heart cytochrome
c
(HCC), horse heart myoglobin (Mb), and bovine serum albumin (BSA) on 100‐nm HPHT‐NDs at pH 10.5, 6.9, and 4.7, respectively.
Figure 4.7 Scope of typical applications of the SPEED platform to proteome analysis. ESI, electrospray ionization; LC, liquid chromatography; MALDI, matrix‐assisted laser desorption/ionization; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; and SDS, sodium dodecyl sulfate.
Figure 4.8 (a) Size distributions of HPHT‐NDs before and after noncovalent conjugation with BSA and α‐LA in DDW and PBS, measured by DLS. The mean diameters of the particles with size distributions from left to right are 30.0, 35.1, 41.9, and 752.7 nm, respectively. (b) Stability tests of the colloidal suspensions of BSA‐ and α‐LA‐conjugated HPHT‐NDs in PBS at room temperature for nine days. The mean diameters of the particles with size distributions from left to right are 35.0, 37.0, 41.9, and 43.2 nm, respectively.
Figure 4.9 Encapsulation of NDs in liposome by rehydration of lipid thin films containing cholesterol and biotinylated lipids in concentrated ND solution. The molecule in red is epirubicin, a chemotherapy drug.
Figure 4.10 TEM images of (a) as‐received and (b) silica‐coated HPHT‐ND particles.
Figure 4.11 Synthesis of silica‐coated NDs by liposome‐based encapsulation. MLVs, multilamellar vesicles; SUVs, unilamellar vesicles; TEOS, tetraethyl orthosilicate; and SDS, sodium dodecyl sulfate.
Chapter 05
Figure 5.1 Molecular structures of (a) trypan blue, (b) propidium iodide, and (c) ethidium homodimer.
Figure 5.2 The MTT assay involving the reduction of 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (left) to 1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenylformazan (right) by mitochondrial reductase.
Figure 5.3 Confocal fluorescence images of a single 293T human kidney cell after FND uptake. The cross‐sectional image in each three‐dimensional scan (as indicated by the yellow dashed square) has a vertical thickness of 0.25 μm and an area of 42 × 42 μm
2
. The bright red spots correspond to FNDs. Inset: Cytotoxicity tests with the 293T cells and the MTT reduction assay.
Figure 5.4 (a) Cell proliferation and (b) comet assays of human fibroblasts after treatments with X‐ray, TiO
2
, and FNDs. The X‐ray and TiO
2
treatments served as positive controls.
Figure 5.5 Cytotoxicity measurements after 24‐h incubation of various nanocarbons in (a) neuroblastoma cells and (b) macrophages. GdO nanoparticles served as the positive control.
Figure 5.6 Hemolysis studies of GOs and oxidized NDs of different size with human RBCs. (a) Photographs of human RBCs treated with GOs and NDs of four different sizes at the concentration range of 25−400 μg ml
−1
. (b) Hemolysis percentages measured at the concentration range of 25−400 μg ml
−1
for GOs and four different ND samples (35, 100, 250 and 500 nm in diameter) incubated with RBCs at 25 °C for two hours. GOs served as the positive control.
Figure 5.7 Histopathological examination of the tissue sections of FND‐injected (treated) and saline‐injected (control) rats with and without recovery. The results show no specific pathological changes in both FND‐treated and control groups (magnification 200×).
Figure 5.8 Biodistribution of
125
I‐labeled HPHT‐NDs in mice after intravenous injection with a dose of 20 mg kg
−1
body weight for 30 minutes.
Chapter 06
Figure 6.1 (a) Numbers of vacancies created as a function of depth by 1, 2, and 5 MeV electrons through diamond. (b) Maximum penetration depths of diamond damage as a function of electron energy.
Figure 6.2 (a) Numbers of vacancies created as a function of depth by H
+
and He
+
ions through diamond. (b) Maximum penetration depths of diamond damage as a function of the energy of H
+
and He
+
ions. A displacement energy of 37.5 eV was used in the simulations.
Figure 6.3 Spatial distribution of vacancies produced in diamond as a function of target depth predicted by SRIM Monte Carlo simulations. The proton beams used for the irradiation are (a) 3 MeV and (b) 40 keV, respectively. The numbers of damage events used in both simulations are 9999. Note that the vacancies are more uniformly distributed along the target depth in (b).
Figure 6.4 Photographs of HPHT microdiamonds before (a) and after (b) electron irradiation and thermal annealing. A bright‐field transmission image of the corresponding particles (~400 μm in diameter) is shown on the right of each panel.
Figure 6.5 Experimental setup used to create vacancies in NDs with a medium‐energy ion beam.
Figure 6.6 Procedures of FND production. The method is applicable for all color centers, not limited to NV as illustrated herein.
Figure 6.7 Experimental setup for the characterization of air‐oxidized NDs. (a) An artistic view of the confocal beam incident from the bottom and through the glass coverslip combined with the AFM tip probing the sample from above. The inset is a photograph of the sample from directly above. (b, c) A confocal fluorescence intensity map (b) and an AFM height map (c) of the sample. The scan area is 50 × 50 μm.
Figure 6.8 Optical layout of the experimental setup for fluorescence measurements of red FNDs suspended in water. A round electromagnet supplies a time‐varying magnetic field of 0–50 mT, as controlled by a power amplifier. By changing the laser and optics, the setup can be applied to detect FNDs containing other color centers such as H3.
Figure 6.9 Fluorescence spectra of H3 and NV centers in green and red FNDs, respectively, suspended in water. The sizes of both particles are approximately 100 nm in diameter and the FND concentration is 1 mg ml
−1
each.
Figure 6.10 (a) Bright field and (b) epifluorescence images of red FNDs. Both images were obtained with a 40× objective. (c) Photostability tests of FND (red) and fluorescent polystyrene beads (blue) excited under the same conditions. The excitation was made with light of 510–560 nm in wavelength.
Figure 6.11 (a) Comparison of the fluorescence lifetimes of FNDs with different sizes (100 and 30 nm in diameter). The concentration of both FND suspensions is 1 mg ml
−1
. (b) Variation of the fluorescence lifetimes of 100‐nm FNDs in water, biological buffer, and aqueous solutions with different pHs. The amplitude‐weighted mean lifetime (
τ
m
) and the intensity‐weighted mean lifetime (
τ
i
) are calculated from
τ
1
and
τ
2
according to Eqs. (6.2) and (6.3) in text.
Figure 6.12 Ultrasensitive quantification of FNDs by magnetically modulated fluorescence. (a) Normalized fluorescence spectrum of FNDs in water (1 mg ml
−1
), excited with a 532 nm laser. (b) Time trace of the peak fluorescence intensity at 687 nm under magnetic modulation at
f
= 2 Hz. The inset shows the corresponding frequency spectrum after FFT. (c) Normalized fluorescence spectrum of FNDs (0.1 μg ml
−1
) in water (black) and its FFT spectrum (red) after demodulation at
f
= 2 Hz. The asterisk denotes the Raman peaks of water. (d) Dependence of fluorescence modulation depth on the magnetic field strength at
f
= 2 and 10 Hz for FNDs in water (0.1 mg ml
−1
). (e) Demodulated fluorescence intensity of FNDs as a function of the particle concentration at
f
= 10 Hz and
B
= 40 mT. Solid line is a linear fit of the experimental data. Note that the magnetic modulation depth is independent of the FND concentration even when the concentration is as low as 60 ng ml
−1
.
Chapter 07
Figure 7.1 Fluorescence images and spectra of single 35‐nm FNDs. Each pixel corresponds to 200 nm. (a) Confocal scanning image of 35‐nm FNDs dispersed on a coverglass slide. (b) Fluorescence spectra of three different 35‐nm FND particles.
Figure 7.2 Spectroscopic characterization of 35‐ and 100‐nm FNDs. (a) Typical time traces of the fluorescence from a single 100‐nm FND, a single 35‐nm FND, and a single Alexa Fluor 546 dye molecule attached to a single double‐stranded DNA molecule. (b) Time traces of the fluorescence from a single 100‐nm FND and a single 35‐nm FND acquired with a time resolution of 1 ms, showing no photoblinking behavior. (c) Plot of the fluorescence intensity as a function of the laser power density over the range of 1 × 10
2
–1 × 10
6
W cm
−2
for 35‐ and 100‐nm FNDs. (d) Fluorescence lifetime measurements of 100‐nm FNDs and Alexa Fluor 546 dye molecules. Fitting the time traces with two exponential decays for the FND reveals a fast component of 1.7 ns (4%) and a slow component of 17 ns (96%). The latter is approximately 4 times longer than that (~4 ns) of Alexa Fluor 546.
Figure 7.3 Observation of a single poly‐
L
‐lysine‐coated FND particle bound with a single T4 DNA molecule on an amine‐terminated glass substrate. (a) Dual‐view fluorescence images of a single DNA/FND complex. An overlay (right) of the images from the shorter (545–605 nm) and longer (675–685 nm) wavelength channels shows that the T4 DNA molecule is wrapped around the 100‐nm FND particle and stretched to a V‐shape configuration. (b) Fluorescence decays of the 100‐nm FND particle and the TOTO‐1 dyes intercalated in the T4 DNA molecule.
Figure 7.4 Measuring the numbers of color centers in FNDs. (a) Sketch of the experimental setup combining an atomic force microscope (AFM) with an inverted confocal microscope: NDs, nanodiamonds; O, oil immersion microscope objective; DM, dichroic beamsplitter; F, long‐pass filter; PH, pinhole; FM, flip mirror directing the collected photoluminescence either to an imaging spectrograph equipped with a back‐illuminated cooled charge‐coupled‐device array, or to a Hanbury–Brown–Twiss interferometer consisting of two silicon avalanche photodiodes (APDs) placed on the output ports of a 50/50 beamsplitter. (b, c) Topography image (b) and photoluminescence raster scan (c) of the same sample. Red dotted circles indicate NDs hosting NV
−
defects. (d) Typical second‐order autocorrelation function
g
(2)
(
τ
) recorded for a single NV
−
defect.
Figure 7.5 Three‐dimensional tracking of a single FND in solution by a wide‐field fluorescence microscope. (a) Schematic diagram of the experimental setup: OBJ, objective; Ex, excitation filter; DM, dichroic mirror; Em, emission filter; TL, tube lens; EMCCD, electron‐multiplying charge‐coupled device; PZT, piezoelectric translational stage. (b) Trajectory of a single FND moving freely in 80% glycerol–water solution. Time traces are shown in pseudo‐color. (c) Mean square displacement (MSD) of the tracked FND. Black line is the best fit of the experimental data to Eq. (7.6) in text.
Figure 7.6 Three‐dimensional tracking of a single FND in a living cell. (a) Merged bright‐field and epifluorescence (red) image of the cell after FND uptake. (b) Location (left) and 3D trajectory (right) of the single FND surrounded by a yellow box in (a) over a time span of 200 seconds.
Figure 7.7 Orientation tracking of a single FND in a living cell. (a) Changes in orientation of the NV crystallographic axis (also the NV
−
spin orientation) relative to an external magnetic field. (b) Measurement of the orientation of the internalized FND as a function of time.
Figure 7.8 Tracking of a single EGFP‐conjugated FND particle along the connecting membrane nanotube of two living cells. (a) Forward and (b) backward movement of two different particles. White arrows indicate the directions of the particle movement and orange arrows denote the particles of interest in the frames. Scale bar: 5 μm.
Figure 7.9 Intraneuronal transport monitoring by FND tracking. (a) Experimental pipeline from hippocampal neuron culture dissociated from E18.5 mouse embryo to endosome trajectory acquisition using pseudo‐TIRF microscopy. DIV, day
in vitro
; TIRF, total internal reflection fluorescence. (b) Transmission white‐light illumination image of the neuronal branches merged with the fluorescence channel showing four FNDs moving within dendrites (yellow arrows). (c) Superimposition onto a white light image of the positions of these two FNDs (1 in yellow; 2 in green), determined by particle tracking, with a persistence of 10 seconds, at different time points. Scale bars: 5 μm in (b) and 1 μm in (c).
Figure 7.10 Two‐photon fluorescence correlation spectroscopy of bare and lipid‐encapsulated 40‐nm FNDs in live HeLa cells. The solid line is the simulated result using Eq. (7.9) in text.
Figure 7.11 Tracking of single FNDs in a
Drosophila
embryo. (a) Confocal fluorescence image of FNDs in the blastoderm cells during stage 5 of development. (b) Schematic drawing of the structure of an early stage 5 embryo, showing the cellularization furrows introgressing between nuclei, which invade the yolk‐free periplasm during the later syncytial divisions indicated as arrows.
Figure 7.12 Tracking the intercellular transport of single FNDs in
C. elegans
. (a) A time‐course localization analysis of GFP::YLC‐FNDs in specific tissues and organs of the worms over 55 minutes post‐injection. (b) A merged bright‐field and time‐gated fluorescence image of a representative worm at 30 minutes after injection of GFP::YLC‐FNDs into the intestinal cell. The blue arrow indicates the injection site. (c) A cartoon illustrating the excretion of GFP::YLC‐FNDs from the intestine (in) to the body cavity (bc) and after passing through the sheath cells (sc), the excreted GFP::YLC‐FNDs enter oocytes (oo). (d) A merged bright‐field and time‐gated fluorescence image of a representative worm at 55 minutes after injection of GFP::YLC‐FNDs into the intestinal cell. (e, f) An enlarged image (e) and a cartoon (f) of the area in the red box in (d), showing the presence of GFP::YLC‐FNDs in both intestine (with a boundary marked by the yellow dotted line) and oocytes (with boundaries marked by thin white lines and nuclei labeled with blue stars). Scale bars: 50 μm.
Chapter 08
Figure 8.1 Flow cytometric analysis of 100‐nm FNDs internalized by HeLa cells as a function of particle concentration after three hours of incubation with (black) or without (blue) 10% FBS in cell medium. Inset: Kinetics of the uptake of FNDs (without FBS in medium) at the particle concentration of 25 μg ml
−1
.
Figure 8.2 Flow cytometric analysis of the 1 : 1 mixture of FND‐labeled (magenta) and unlabeled (green) 489‐2.1 cells in the (a) far‐red fluorescence and (b) side scattering channels.
Figure 8.3 Flow cytometric analysis of FND‐labeled and unlabeled 489‐2.1 cells utilizing the light scattering property of FNDs at various cell number ratios of 1 : 10, 1 : 100, and 1 : 1000. Green data points represent the major live cell population and the blue data points represent the gated FND‐positive cells.
Figure 8.4 Surface functionalization of FNDs with proteins. (a) Grafting of neoglycoproteins on FND. The proteins are attached to the surface of acid‐washed FND by physical adsorption. (b) Grafting of streptavidin on FND. The grafting starts with activation of the surface carboxyl groups on FND with EDC and NHS, forming amine‐reactive terminus. The FND is then conjugated with carboxyl PEG amines via carboxyl‐to‐amine cross‐linking. Further activation leads to covalent coupling between of the carboxyl groups of PEG‐FND and the primary amine groups (–NH
2
) of streptavidin through amide bond formation. Finally, the SA‐conjugated PEG‐FND is noncovalently covered with BSA to prevent particle aggregation in PBS.
Figure 8.5 Flow cytometric analysis of the uptakes of protein‐conjugated FNDs by HepG2 cells. Mean fluorescence intensities as a function of the concentration (0, 10, 25, and 50 μg ml
−1
) of the labeling agent, as annotated in the figure, reflect different levels of the uptake of FNDs conjugated with BSA, neoglycoprotein, or glycoprotein.
Figure 8.6 Competition assays for the uptake of Lac‐BSA‐FND by HepG2 cells measured by flow cytometry. The cellular uptake of Lac‐BSA‐FND (50 μg ml
−1
) is significantly suppressed due to the presence of lactose (0.3 M), showing the competition for binding with ASGPRs by free lactose.
Figure 8.7 Vertical cross‐section scans (0–8 μm) of the wide‐field epifluorescence image of a HeLa cell after FND uptake by endocytosis. As evidenced by the images taken at 3–5 μm, most of the FND particles are distributed in the cytoplasm and do not enter the nucleus of the cell.
Figure 8.8 (a) Colocalization study of FNDs with early endosomes labeled with EEA1‐FITC fluorescent conjugates. (b) Colocalization of FNDs with lysosomes labeled with green LysoTracker. FNDs colocalized with endosomes or lysosomes appear in yellow in the merged fluorescence scans. Images were acquired by confocal fluorescence raster scans of HeLa cells incubated with FNDs (10 μg ml
−1
) in normal (control) conditions, then fixed, followed by labeling with endosomal or lysosomal markers. From left to middle: raster scan in the green channel (500–530 nm) showing the endocytic compartments and in the red channel (600–750 nm) showing the FNDs. Images on the right represent the merged green and red scans.
Figure 8.9 Bright‐field and confocal fluorescence images of live ASB145‐1R cells labeled with DyLight488‐SA or SA‐PEG‐FND before and after endocytosis. The cells were first stained with biotinylated anti‐CD44 antibody and then incubated with DyLight488‐SA (a−d) or SA‐PEG‐FND (e–h) at 4 °C. Panels (a) and (e) are bright‐field images, (b), (d), (f), and (h) are Z‐stacked confocal fluorescence images, and (c) and (g) are merged bright‐field and Z‐stacked confocal fluorescence images of the cells before endocytosis. The contrast in photostability between these two types of fluorophores is evident in (d) and (h), where the organic dye photobleached during the second scan of the specimen. FND‐labeled CD44 on the cell surface was then internalized through endocytosis at 37 °C (i−k). The orange spots in the merged fluorescence image (k) evidence that the FND (red, i) are colocalized with endosome/lysosomes, which were stained by the endo‐lysosomal marker, Rab7 (green, j). Scale bar: 10 μm.
Figure 8.10 One‐photon and two‐photon excited fluorescence images of 140‐nm FNDs in a fixed HeLa cell. (a) Typical OPE and TPE confocal fluorescence images of the same cell. (b) Three‐dimensional TPE image of the cell and the internalized FNDs. (c) Lateral and axial cross‐sections of the FND labeled with a red box in (a). Note that the resolution of TPE for this particular particle was approximately 300 nm in the lateral direction and approximately 800 nm in the axial direction, both of which are close to their theoretical diffraction limits.
Figure 8.11 Time‐resolved confocal fluorescence images of a fixed cell containing FNDs. (a) Raster‐scan image obtained by detecting all photons, displaying NV
−
fluorescence together with cell autofluorescence. (b) Time‐gated raster scan constructed from photons detected between 15 and 53 ns after pulsed laser excitation. Scan area: 25 × 25 μm
2
. (c) Fluorescence time decay from one of the FNDs shown in (a).
Figure 8.12 (a, b) Wide‐field fluorescence images of FND‐labeled HeLa cells attached to a coverglass slide and immersed in human blood without (a) or with (b) time gating. The exposure times used for the fluorescence imaging with a 100 × oil‐immersion objective lens in (a) and (b) are 0.1 and 0.3 seconds, respectively. (c) Intensity profiles along the black and red color lines denoted in (a) and (b), respectively.
Chapter 09
Figure 9.1 Flow cytometric analysis of the mechanism of FND uptakes by (a) human HeLa cells and (b) murine 3T3‐L1 pre‐adipocytes. Cells were preincubated with various inhibitors for specified time, followed by incubation with FNDs for three hours before cell harvest for the analysis. PAO, phenylarsine oxide; CytD, cytochalasin D; Noco, nocodazole. The first two columns in each figure represent control experiments.
Figure 9.2 Schematic illustration of a proposed model on how Ub‐coated FNDs bind with autophagy receptors when entering the selective autophagy pathway, leading to entrapment in the lysosome.
Figure 9.3 Detection and quantification of FNDs in cells and tissues. (a) Dose‐dependent uptakes of HSA‐coated FNDs by A549 cells and HeLa cells in culture, analyzed by MMF. (b) Comparison of the uptakes of HSA‐coated FNDs by A549 cells, analyzed by both MMF and flow cytometry. (c) Normalized fluorescence spectrum of FNDs (5 μg ml
−1
) in an acid digest of pig liver tissue (black) and its FFT spectrum (red) after demodulation at
f
= 2 Hz. The asterisk denotes the Raman peaks of water. FFT, fast Fourier transform.
Figure 9.4 (a) Time‐lapse images of a FND‐labeled HeLa cell undergoing division, acquired by differential interference contrast and epifluorescence microscopy. (b) Long‐term tracking of FND‐labeled HeLa cells over eight days by flow cytometry. The fluorescence intensity of each cell decreases exponentially with time due to cell proliferation.
Figure 9.5 Comparison of the long‐term tracking capability of EdU, CFSE, and FND. The assays were conducted for the mammospheres generated from AS‐B145‐1R cells labeled separately with EdU, CFSE, and FND and then dissociated for flow cytometric analysis with a 4‐day period for 20 days.
Figure 9.6 Tracking the engraftment and regenerative capability of transplanted lung stem cells using FNDs in a lung‐injured mouse model. Lung tissue sections were examined on day 1 and 7 after intravenous injection of the FND‐labeled lung stem cells. Arrows indicate the identified cells.
Figure 9.7 Comparison of the fluorescence spectrum (red curve) of FNDs with the near‐infrared window of biological tissues. The black and gray curves are the absorption spectra of H
2
O, oxygen‐bound hemoglobin (HbO
2
), and hemoglobin (Hb), respectively.
Figure 9.8 (a–d) Bright‐field images of two FMD particles covered with chicken breast tissues with thickness of 0, 1.5, 3, and 5 mm, respectively. (e–h) Corresponding fluorescence images of FMDs in (a–d) illuminated by a continuous‐wave laser operating at 637 nm.
Figure 9.9
In vivo
imaging of FNDs in rats after (a) intraperitoneal or (b) subcutaneous injection. The times indicated in both panels are the time points post‐injection. White and blue arrows indicate the sites of injection.
Figure 9.10
In vivo
and
ex vivo
lymph node imaging of a nude mouse after intradermal injection. (a) Image showing the accumulation of BSA‐FND particles in the right axillary lymph node (indicated by the blue arrow) on day 8. Note that most of the injected BSA‐FND particles remain at the injection site. (b)
Ex vivo
fluorescence image of four extracted lymph nodes, where ALN1 and ALN2 are the lymph nodes located at the right and left axilla, respectively, and BLN1 and BLN2 are the lymph nodes located at the right and left brachial region, respectively.
Figure 9.11 NV‐based deep tissue imaging. (a) FND phantoms made of double‐sticky tape and FNDs. (b) Chicken breast illuminated by a LED. The gray stripe on the piece of chicken breast represents the edge of the phantom, which is placed inside the chicken breast, 5 mm back from the front surface, facing the LED operating at 621 nm. Fluorescence is collected off to the side. (c) FND phantoms imaged outside of the chicken breast.
Figure 9.12 Tracking of single FND‐labeled cells in a mouse ear. (a) Bright‐field image of a mouse ear tissue. The green arrow indicates the position of an FND‐labeled mouse lung cancer cell in the blood vessel of approximately 50 μm in diameter. (b) Enlarged view of the fluorescence image of the square green region in (a). The bright spot corresponds to the FND‐labeled cell. (c) Enlarged view of the fluorescence image of the rectangular green region in (b), showing the trajectory of the FND‐labeled lung cancer cell moving in the vessel. Scale bars: 100 nm in (a), 50 nm in (b), and 10 μm in (c).
Chapter 10
Figure 10.1 An Airy disk resulting from the diffraction of light on a circular aperture.
Figure 10.2 Principle of STED microscopy. A blue excitation (EXC) beam is focused to a diffraction‐limited excitation spot, shown in the adjacent panel in blue, while the orange STED beam de‐excites molecules. The STED beam is phase‐modulated to form the focal doughnut shown in the top right panel. Superimposition of the two focal spots confines the area in which emission is only possible in the doughnut center, yielding the effective fluorescent spot of subdiffraction size shown in green in the lower panel.
Figure 10.3 (a) STED and (b) SEM images of a single FND particle.
Figure 10.4 Confocal and STED imaging of HeLa cells labeled with BSA‐conjugated FNDs by endocytosis. (a) Confocal image acquired by raster scanning of an FND‐labeled cell. The fluorescence image of the entire cell is shown in the white box and demonstrates fairly uniform cell labeling by BSA‐conjugated FNDs. (b) STED image of single BSA‐FND particles enclosed within the green box in (a). (c) Confocal and STED fluorescence intensity profiles of the particle are indicated in (b) with a blue line. Solid curves are best fits to one‐dimensional Gaussian (confocal) or Lorentzian (STED) functions. The corresponding full widths at half‐maximum are given in parentheses.
Figure 10.5 Fluorescence intensity profiles on the lateral (
x
) and axial (
z
) axes, calculated for different demodulation frequencies in saturated excitation fluorescence microscopy. The excitation intensities used to obtain the demodulated intensity profiles at
ω
, 2
ω
, 4
ω
, and 8
ω
are 1.5, 15, 400, and 700 kW cm
−2
, respectively.
Figure 10.6 (a) Energy level diagram of NV
−
, showing how preferential shelving of the
m
s
= ± excited states (
3
E
) into the dark metastable state (
1
A
) gives rise to a typical electron spin resonance spectrum. (b) Fluorescence of two NV
−
centers in the presence of a static magnetic field as a function of applied microwave frequency. The splitting of the two dips (Δ
ω
A
and Δ
ω
B
) is given by the projection of the incident magnetic field on the magnetic moment of the NV
−
. The contrast of each dip has an inverted Lorentzian shape. (c) Illustration of NV
−
centers in a static magnetic field, each having field splitting frequencies corresponding to their uniquely oriented magnetic moments relative to the magnetic field. (d) Schematic diagram of the method for resolving a switchable emitter by taking the difference between two images where a uniquely addressed emitter is dimmed and not dimmed by resonant microwave excitation at
I
(Ω
ZFS
± Δ
ω
) and
I
(Ω
o
), respectively. ZPL, zero‐phonon line; ZFS, zero‐field splitting.
Figure 10.7 (a) AFM image, (b, c) fluorescence images, and their corresponding line profiles of a 30‐nm FND particle (b) with and (c) without a gold tip close to it. The resolution of the fluorescence image is significantly improved by the tip enhancement effect from (c) 290 nm to (b) 40 nm. The gray scale in (a) is in unit of nm. Black solid curves are best Gaussian fits to the experimental data. Scale bar: 400 nm.
Figure 10.8 (a) STEM image of a FND mixture. (b) Color‐coded intensity map of NV
0
(red) and H3 (green) centers in the FNDs. (c, d) Typical CL spectra of FNDs containing NV
0
and H3 centers in (c) and (d), respectively.
Figure 10.9 Images of both green and red FNDs internalized in HeLa cells, acquired with (a) phase contrast microscopy and (b) direct electron beam excitation‐assisted (D‐EXA) fluorescence microscopy. The magnified D‐EXA image of the area marked with a red square in (c) shows a spatial resolution of 155 nm in (d).
Figure 10.10 (a) SEM images of a HeLa cell labeled with anti‐CD44 antibody, neutravidin, and bL‐FND. (b–g) Enlarged views of (b, c) SEM images, (d, e) fluorescence images, and (f, g) CLEM images of the white boxes in (a). The tilt angles to achieve complete overlaps of the FNDs are 39° and 1° for (b, d) and (c, e), respectively. (h) Intensity profiles of the white dashed line drawn in panel (i), which is an enlarged view of the white box in (g). Scale bars: 10 μm in (a) and 2 μm in (b, c).
Figure 10.11 (a) TEM image of suspended HeLa cells without labeling. (b–d) TEM images of suspended HeLa cells labeled with biotin‐anti‐CD44 antibody, neutravidin, and then biotinylated lipid‐coated FND. (e, f) Fluorescence and CLEM images of the same sample in (d). The electron energy used to obtain the TEM images is 120 kV. Scale bars: 5 μm in (a, d) and 1 μm in (c).
Chapter 11
Figure 11.1 Magneto‐optical nanothermometry. (a) Frequency scan of a single FND containing approximately 500 NV
−
centers. The four red points indicate the measurement frequencies used to extract the temperature. (b) Two‐dimensional confocal scan of FNDs (circles) and gold nanoparticles (cross) spin‐coated onto a glass coverslip. (c) Temperature rise of a single FND as a function of laser power for two different laser‐focus locations with (red) or without (blue) laser illumination on a nearby gold nanoparticle. (d) Temperature changes measured at six FND locations, indicated by circles in (b), as a function of the distance from the illuminated gold nanoparticle indicated by the cross. The blue curve represents the best‐fitting of the temperature profile to a steady‐state solution of the heat conduction equation.
Figure 11.2 Comparison of sensor sizes and temperature accuracies for the NV‐based quantum thermometer and other reported techniques. Biocompatible methods are labeled in red. The open red circle indicates the ultimate expected accuracy for FNDs. SthM, scanning thermal microscopy.
Figure 11.3 Pictorial presentation of the three‐point method based on an ODMR spectrum consisting of only one peak. The peaks before (blue) and after (red) temperature change are both Lorentzian and have the same width, although their heights may vary. Overlaid on the spectra are three frequencies (
f
1
,
f
2
,
f
3
) chosen for the intensity measurement.
Figure 11.4 Time‐resolved nanothermometry. (a) Experimental scheme for the time‐resolved temperature measurement with a 100‐nm FND particle submerged in aqueous solution containing 10‐nm × 41‐nm GNRs (black rods) heated by an 808 nm laser (red hyperboloid). (b) ODMR spectra of the GNR solution heated by the near‐infrared laser with its power varying from 0 to 10 mW. (c) Time sequences of the laser, microwave, and detection pulses (all in μs) used in the time‐resolved nanothermometry with a three‐point method. (d) Time evolution of the heat dissipation of the GNR solution at the radial positions of
r
= 1.0 and 1.5 μm, as indicated in (a).
Figure 11.5 All‐optical nanothermometry. (a) Area‐normalized temperature‐dependent fluorescence spectra of 100‐nm FNDs illuminated by a 594 nm laser in solution. Inset: Enlarged view of the temperature‐induced shift of the ZPLs for spectra acquired at 28–75 °C. (b) Typical fluorescence spectrum of a single 100‐nm FND spin‐coated on a glass coverslip at room temperature over 610–660 nm. The exposure time of the sample to the 594 nm laser with a power of 30 μW is 0.1 second. Inset: Changes of
λ
0
with time over six seconds, with a mean value of 638.519 ± 0.013 nm for a 95% confidence interval.
Figure 11.6 Thermal imaging of a laser‐heated gold nanoparticle. (a) Schematic diagram of the experimental design. Both the AFM tip and the gold nanoparticle are immersed in water to ensure thermal equilibrium between the probe and the sample. (b, c) Fluorescence image (b) and temperature map (c) obtained simultaneously by scanning a 40‐nm gold nanoparticle relative to the FND probe and its excitation laser. (d) ODMR spectra corresponding to three different pixels of the scan, located as indicated by the crosses in (b) with matching colors. (e) Line cuts extracted from (b) and (c) taken along the dashed lines shown in (b) with matching colors. The black solid curve is a fit of the experimental data to a theoretical model.
Figure 11.7 Scanning probe magnetometry. (a) Diagram of the magnetic field imaging experiment. A magnetic nanoparticle (red) is imaged with a single NV
−
in FND (green dot within the blue nanocrystal) fixed at the scanning probe tip (black). (b) Optical image of a FND attached to an AFM tip. The scattered light image of the tip is overlapped with the fluorescence image of the nanocrystal. The bright red spot (arrowed) represents fluorescence of the single NV
−
. (c) Field reconstruction using the scanning probe single spin magnetometer, showing an AFM image of a nickel magnetic nanostructure (top left), a magneto‐optical image of the same structure (bottom left), and the fluorescence signal when resonant microwaves at 2.750 GHz are applied to the NV
−
center.
Figure 11.8 ODMR spectra (left) and camera images (right) of a magnetic nanoparticle (red square) and a FND (green square) separated by a distance of (a) 7.25 μm, (b) 3.62 μm, and (c) 1.50 μm. Red curves in the spectra are Lorentzian fits to the measured data and dashed lines in purple and orange are the center frequencies of the Lorentzian fits in (a) and (c), respectively. PL, photoluminescence. Scale bar: 3 μm.
Figure 11.9 Schematic of the nanoscopic detection of spin labels in an artificial cell membrane using a single‐spin FND sensor. (a) A supported lipid bilayer (SLB) formed around a FND immobilized on a glass substrate. (b) A FND hosting a single NV
−
center acting as a single‐spin sensor by virtue of the magnetic levels in the ground state. (c) A Gd
3+
ion binding with a lipid molecule in the SLB. (d) Gd spin labels causing magnetic field fluctuations that affect the quantum states of the NV
−
centers in FNDs, measured through its spin–lattice relaxation time,
T
1
. (e) Electronic energy structure of the NV
−
center, showing the fluorescence cycle and optical spin readout of the spin states,
m
s
= 0 and ±1, and the protocol for the
T
1
measurement. (f) Schematic illustration of the
T
1
measurement. The relaxation of the NV spin in the target environment is compared with that in the reference environment. Measurement at a single time point in the evolution allows faster detection.
Chapter 12
Figure 12.1 (a) Schematic diagram of the biosensing experiment with silica‐coated FNDs. A quartz slide passivated with biotinylated PEG was first saturated with streptavidin. The biotinylated FNDs were then flowed into a microfluidic cell and specific attachment to streptavidin–biotin‐PEG was probed by comparing the number of bound particles before and after washing to remove nonspecifically bound particles. FNDs were excited by the evanescent field (green) in a prism‐type TIRFM. (b, c) Images showing that (b) 60% of the biotinylated silica‐coated FNDs and (c) 4% of the nonbiotinylated silica‐coated FNDs remained attached to the surface after acid–base washes.
Figure 12.2 TEM images of FNDs and MSN‐FNDs. (a) Pure FND cores as imaged by TEM and (b) individual MSN‐FNDs imaged by high‐resolution TEM. (c, d) Corresponding (c) bright field and (d) dark field images revealing the presence of a crystalline diamond core.
Figure 12.3 Experimental realization and numerical simulation of a GNP‐FND hybrid. (a) AFM images of a single diamond nanocrystal (left), to which one (middle) or two (right) GNPs (or Au NPs) are coupled. (b) Corresponding numerical simulations of the intensity enhancement of the excitation light linearly polarized along the
x
axis. Upper row: schematic representation of the particle configuration. Middle row:
x–y
cross section. Lower row:
x–z
cross section. The field intensity is normalized to the value at the center of the bare FND.
Figure 12.4 Comparison of resolution and penetration depth between different imaging modalities, including confocal microscopy, multiphoton microscopy (MPM), optical coherence tomography (OCT), photoacoustic tomography (PAT), ultrasonography (US), and magnetic resonance imaging (MRI).
Figure 12.5 Characterization of GNP‐FND nanohybrids. (a) TEM image of GNP‐FNDs. The inset depicts the size distribution of the particles with a mean diameter of 33 nm. (b) High‐resolution TEM image of the GNP‐FND on graphene as a supporting film. The lattice fringes of FND (0.206 nm) and GNP (0.235 nm) are clearly resolved. The separation width between these two nanoparticles is approximately 1 nm. Scale bars: 200 nm in (a) and 5 nm in (b).
Figure 12.6 (a) Illustration of plasmon oscillation for a metal nanosphere. (b) Measured absorbance spectrum of a GNR solution. The insets show the schematic of the transverse and longitudinal SPR modes responsible for the two absorption bands observed in the spectrum. (c) TEM images of as‐synthesized GNRs with different longitudinal SPR wavelengths as noted. Unlabeled scale bars: 20 nm. (d) Measured absorbance spectra of gold nanospheres and GNRs, whose TEM images are shown in (c).
Figure 12.7 (a) Comparison between the absorption spectrum of 10‐nm × 16‐nm GNRs and the emission spectrum of 100‐nm GNR‐FNDs. The thick green bar denotes the excitation wavelength at 594 nm. Inset: TEM image of a GNR‐decorated FND having more than 30 GNRs on the observable hemisphere or a surface coverage of greater than 50%. (b) Merged bright‐field/fluorescence images of an HEK293T cell cluster with GNR‐FNDs attached to the plasma membrane after exposure to the 594 nm laser in the presence of PI at different time points. Intracellular PI was observed almost immediately after laser irradiation of the particle indicated by the white arrow. Many blebs appeared on the cells not being irradiated at five minute after the hyperlocalized hyperthermia treatment. Scale bar: 10 μm.
Figure 12.8 Nanohyperthermia with GNR‐FNDs in membrane nanotubes. (a) Scanning electron microscopy (SEM) image of a membrane nanotube formed between two HEK293T cells. Elemental analysis by energy‐dispersive X‐ray spectroscopy (EDS) shown to the right reveals the presence of GNR‐FNDs (denoted by black and red crosses) in the TNT. Scale bar: 10 μm. (b) Pictorial presentation of the experiment using a tightly focused 594 nm laser beam for both heating and temperature sensing of the GNR‐FNDs trapped in the endosomes of a membrane nanotube. (c, d) Fluorescence (c) and merged bright‐field/fluorescence (d) images of HEK293T cells transduced with actin‐GFP fusion proteins (green) and labeled with GNR‐FNDs (red). (e) Empirical cumulative distribution plot of the membrane temperatures at which TNTs are ruptured by local heating and global heating of GFP‐transduced HEK293T cells. Data of two independent global heating experiments (blue dots) are shown in the plot. (f, g) Fluorescence (f) and merged bright‐field/fluorescence (g) images of GNR‐FND‐labeled, GFP‐transduced HEK293T cells after exposure to the 594 nm laser with a power of 330 μW for six seconds. TNT breaking and retraction could be clearly observed after laser irradiation. Insets of (d) and (g): Enlarged views of the regions with the particle irradiated by the 594 nm laser in the yellow boxes. White arrows indicate the particles being irradiated. GFP, green fluorescent protein.
Figure 12.9 (a) Schematic synthetic paradigm illustrating different growth stages (S1‐S6). (b) A typical large‐scale TEM image showing excellent dispersion and uniformity of hybrid Ag‐FND nanostructures made by following synthetic scheme in (a). Scale bar: 200 nm.
Figure 12.10 Characterization of USPIOs, FNDs, and USPIO‐FNDs. (a) Size distributions, (b) TEM images, (c) magnetizations, and (d) transverse relaxivities (
r
2
) of USPIOs before and after conjugation with FND. Scale bars in (b): 10 nm (USPIO) and 20 nm (USPIO‐FND). The values of the transverse relaxivity (
r
2
) are given by the slopes of the linear fitting to the experimental data in (d).
Figure 12.11 MRI for the growth of glioblastoma tumors in a mouse model. (a)
T
2
mappings of three coronal slices on day 11, 18, 25, and 32. The unlabeled glioblastoma tumors (right thigh) were circled by thin red lines and the USPIO‐FND‐labeled glioblastoma tumors were circled by thin black lines (left thigh). Given shorter
T
2
times, the contrast‐enhanced regions within the labeled glioblastoma tumors (left thigh) are in dark blue due to the presence of USPIO‐FNDs. (b) Three‐dimensional images of the unlabeled glioblastoma tumor (right thigh, red area) and the USPIO‐FND‐labeled glioblastoma tumor (left thigh, green area) on day 11, 18, 25, and 32.
Chapter 13
Figure 13.1 Chemical structures of some commonly used drugs loaded on NDs. (a) Doxorubicin, (b) 10‐hydroxycamptothecin, (c) purvalanol A, (d) 4‐hydroxytamoxifen, and (e) Paclitaxel.
Figure 13.2 (a) Schematic drawing and (b) photograph of NaCl‐mediated loading and release of doxorubicin. Blue spheres denote DNDs and red dots denote DOX.
Figure 13.3 Viability of HeLa cells treated with DNDs, HCPT‐DNDs, and HCPT alone at different concentrations and time points.
Figure 13.4 Dispersion of Purvalanol A and 4‐hydroxytamoxifen (4‐OHT) in water before and after complexation with monodisperse DNDs. Vials were prepared against background, and the reduction in turbidity mediated by the DNDs was confirmed under the following conditions: (a) 1 mg ml
−1
DND in 5% DMSO; (b) 1 mg ml
−1
DND, 0.1 mg ml
−1
Purvalanol A in 5% DMSO; (c) 0.1 mg ml
−1
Purvalanol A in 5% DMSO; (d) 1 mg ml
−1
DND in 25% DMSO; (e) 1 mg ml
−1
DND, 0.1 mg ml
−1
4‐OHT in 25% DMSO; (f) 0.1 mg ml
−1
4‐OHT in 25% DMSO. (g, h) TEM images of pristine DNDs (g) and 4‐OHT–DND complexes (h).
Figure 13.5 (a) Amino acid sequence of human insulin. (b) Five‐day insulin desorption test of ND–insulin complexes treated with NaOH (pH 10.5) and water, showing insulin release in an alkaline pH environment.
Figure 13.6 (a, b) Confocal fluorescence microscopy and (c) flow cytometric analysis of GFP knockdown in M4A4 cells transfected with GFP‐expressing plasmids. Images shown in (a, b) are that of the negative control (a) and the treatment group with ND‐PEI + siRNA (b). Lipo stands for lipofectamine, a commonly used transfection reagent.
Figure 13.7 Colocalization studies between (a) PEI‐FND vectors and (b) siRNA labeled by FITC in NIH/3T3 EF cells. (c) Quantitative estimate of the siRNA release time, using the photoluminescence intensities (PL) of FITC over the whole cell, normalized to that of FND.
Figure 13.8 Imaging DNA release from the FND–PEI–DNA complexes in IC‐21 macrophages. (a) Confocal fluorescence images of the cells incubated with FND–PEI–DNA for 30 minutes, where DNA was labeled with fluorescein amidite (FAM) before forming complexes with FND–PEI. (b) Fluorescence spectra of NV centers measured by 532 nm laser excitation, measured for FND–PEI–DNA in water, medium, and cells after incubation for 30, 60, and 120 minutes. TDI, transmission/bright field image.
Figure 13.9 Improving glioblastoma therapy efficiency via convection‐enhanced delivery of DND‐DOX. (Top) Representative images of H&E staining from each treatment group, showing tumor size regression at the indicated day after tumor inoculation in glioma‐bearing rats. CON: 0.9% saline; NDs: 5 mg ml
−1
DNDs, DOX: 1 mg ml
−1
DOX; DND‐Dox: 5 mg ml
−1
DND and 1 mg ml
−1
DOX. (Middle) 200 × magnification of boxed areas. (Bottom) Apoptotic cell death, measured by TUNEL staining of the boxed areas. Nuclei were counterstained with DAPI (blue). H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; DAPI, 4’,6‐diamidino‐2‐phenylindole.
Chapter 14
Figure 14.1 Comparison of the 3.43‐ and 3.53‐μm emission bands of
HD 97048
to a number of other astronomical features.
Figure 14.2 (a) Structure of a hydrogenated diamond C(111) surface. Gray spheres denote carbon atoms and white spheres denote hydrogen atoms. (b) Scanning electron microscopy image of gravel‐like diamond nanocrystals used in the laboratory studies. (c) Laboratory absorption spectra of hydrogenated ND films prepared in different thickness (1 and 8 μm) and treated at different microwave plasma temperatures (1000 and 1300 K). The spectra were acquired at 300 K.
Figure 14.3 Temperature‐dependent shifts of the absorption bands of the CH stretching vibrations on {100} and {111} facets of hydrogenated NDs.
Figure 14.4 Comparison between the infrared emission spectrum of
Elias 1
(green) and the laboratory absorption spectrum (red) of hydrogenated NDs measured at 300 K. Note that the absorption spectrum is redshifted by 5 cm
−1
in comparison.
Figure 14.5 The proto‐planetary nebula
HD 44179
(also called the “
Red Rectangle
”) in the constellation of
Monoceros
. This picture was obtained with the Hubble Space Telescope (courtesy of Hans Van Winckel, Martin Cohen, NASA, and ESA).
Figure 14.6 (a) The planetary nebula
NGC 7027
in the constellation of
Cygnus
. This picture was obtained with the Hubble Space Telescope (Courtesy of William B. Latter, NASA, and ESA). (b) Comparison between the observed ERE spectrum of
NGC 7027
and the laboratory fluorescence spectrum of FND excited with 510–560 nm light.
Figure 14.7 Emission spectra of FNDs excited with 170‐ and 532‐nm light at 300 K. The spectra were obtained for the same sample. Note the occurrence of photoionization, which converts NV
−
to NV
0
under far‐UV excitation.
Figure 14.8 (a) The reflection nebula
NGC 7023
in the constellation of
Cepheus
. This picture was obtained with the Hubble Space Telescope (Courtesy of NASA and ESA). (b) Comparison of a laboratory photoluminescence spectrum and the ERE band from
NGC 7023
. The ERE band was obtained on dividing the nebular spectrum by the spectrum of the illuminating star. The sharp features denoted with asterisks result from incomplete cancellation of night‐sky and nebular emission bands. The laboratory spectra were synthesized by combining the photoluminescence signals of NV
0
and NV
−
with (green) or without (red) corrections for the interstellar reddening effect.
Figure 14.9 Comparison between the observed ERE spectra of the
Red Rectangle
at 10″ south and 6″ south of
HD 44179
and the laboratory fluorescence spectra of FNDs with different contents of NV
0
and NV
−
. The fluorescence intensity ratios of NV
0
: NV
−
in samples FND1 and FND2 are roughly 3 : 2 and 1 : 0, respectively.
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