Emerging Tools for Single-Cell Analysis

Index

Abbé equation, for resolution, 245

Acoustic devices, for cell transport and manipulation, 108

Acoustic pollution, 38–39

Acoustic signal, in droplet cell sorters, 34–39, 38f

Acoustic-optical modulator (AOM), 154–155 Active pixel sensor (APS) camera, 334–335 Acute myelogenous leukemia, CD34+

hematopoietic stem cells for, 77 ADC. See Analog-to-digital converter Adequate sampling, 301

Air-cooled lasers, 313, 317 Air-handling units, standards for, 83 Airy disk size

in charge-coupled device (CCD) cameras, 303–304

in digital microscopy, 300–302 Alexandrite lasers, 320

Aliasing

in camera systems, 304, 336 temporal, in confocal microscopy, 264

Altra cell sorter, 8, 8f, 85

Aluminum gallium indium phosphide (AlGaInP) lasers, 319

Amplitude modulation, in measuring fluorescence lifetimes, 179–181

Analog-to-digital converter (ADC), 255, 276, 324

Anticoincidence systems, for rare-cell sorting, 69f–70f, 69–71

Antigen presentation, monitoring of, flow cytometry for, 204–215

Antigen processing, 197–199, 198f

MHC II-peptide complexes in, 215–217, 216f protease inhibitors and, 207, 208f study/monitoring of

comparison of methods, 207–208, 215 flow cytometry for, 204–215 fluorescence intensity measurements for,

204–208, 205f, 207f–209f fluorescence lifetime measurements for,

213–215, 214t

fluorescence polarization measurements for, 209–213, 210f–212f

fluorescence-derivatized bovine serum albumin for, 200–201, 201f

two-photon fluorescence microscopy for, 202–204, 203f

APS camera. See Active pixel sensor Arc lamps, 309–310, 310f

arc wander with, 313–314 costs of, 314

operating lifetimes of, 314 power supplies of, 314

Arc wander, 313–314 Areance, radiant, 308 Argon-ion lasers, 313, 317

Note: Page numbers followed by letters f and t indicate figures and tables, respectively.

339

340

Artifacts

in confocal microscopy, 247

in high-speed cell sorting, 29–31 in rare-cell sorting, 29

Autoclaving, 85 Autofluorescence, tissue, 225

Autoimmune disease, CD34+ hematopoietic stem cells for, 78

Automation

in cell collection, 45

in digital microscopy, 291–292 Avalanche photodiode (APD)

cost of, 130

count rates of, 130 dead time of, 118, 130

correction of, 127, 128f

for DNA fragment analysis, 115, 118–119, 123–127, 130, 135–136

use of two, 135–136

Averaging mode, of digital microscopy, 299–300

Background rejection, in fluorescent lifetime imaging microscopy, 168–169

Backscattered light, 247 Beckman-Coulter cell sorters

droplet, 8, 8f

for research samples, 80 sterilization of, 85

Becton Dickinson cell sorters fluid-switching, 16f–17f, 17 for research samples, 80 sterilization of, 85

Binning, definition of, 324

Bio-Rad MRC-600, 255–257, 257f, 261 Bio-Rad MRC-1000, 256–257, 257f Biosafety

of fluid-switching cell sorters, 3, 17, 55 standards for, 83

Blank field test, for confocal microscopy, 240–241

Blind deconvolution algorithm, for deep-tissue two-photon fluorescence microscopy, 229–231, 230f

Blooming effect, 304–305, 332 Bone marrow cells

CD34+, 73–93

high-speed sorting of, 22, 73–93

tagged data for classifier systems of, 58f–59f Bovine serum albumin, fluorescence-derivatized

as exogenous antigen, 200–201, 201f in study of antigen processing, 200–217

Break-off point, of droplet cell sorters, 34–36, 35f, 40

Index

piezosignal amplitude and, 37f stability of, 43–44, 55

Breast cancer, CD34+ hematopoietic stem cells for, 76, 84, 88t, 88–89

Breast cancer cells

ROC analysis of, 57, 60f

tagged data for classifier systems of, 58f–59f Brightness, in white-light scanning digital

microscopy, 297–298, 302–303

Broadcast scanning conventions, 325–326, 326f Brownian motion, in microfluidics, 96, 98 Burst, in DNA fragment detection, 116, 118,

119f, 132–134, 133f

Camera technologies accuracy of, 323

active pixel sensor (APS), 334–335 aliasing in, 304, 336

charge injection device (CID), 332–334 construction of, 332, 333f

operation of, 333–334 performance of, 334

charge-coupled device (CCD), 329–332 blooming effect in, 304–305, 332 charge transfer efficiency of, 331 construction of, 329–330

dark current of, 331 dead pixels in, 331 dynamic range of, 332 false data with, 304 fill factor of, 331–332

in fluorescence lifetime imaging microscopy, 141, 154f, 154–156

frame transfer, 330, 330f full-frame, 330, 330f

interline transfer, 330f, 330–331 operation of, 330f, 330–331 performance of, 331–332, 337 readout noise in, 331, 336 resolution of, 303–305

scan rate of, 331 selection of, 336–337

signal-to-noise ratio of, 332 silicon light sensors in, 329 spectral sensitivity of, 332

in video rate two-photon fluorescence microscopy, 231

white-light scanning digital microscopy versus, 303–305

for cytometry applications, 323–338 definitions in, 324

image digitization in, 335–336 image size of, 327

Index	341
interpolation in, 305, 336	Cell fusion, 104, 106f
light-sensing, 324–325	Cell separation, cell sorting versus, 2
linearity of, 325, 328	Cell sorter(s)
magnification in, 327	for clinical-scale tissue, 80
noise in, 328, 331	cycle time of, 3, 13–14, 14f, 23, 29
photon, 328, 336	dead time of, 13, 23
quantization, 335–336	droplet. See Droplet cell sorters
sources of, 335–336	duty cycle of
performance of, 326–328, 331–332, 334, 337	50 percent, 23
photometric, 325	full, 23
pixel size and spacing in, 327, 336	high, 23, 46
resolution of, 303–305, 327	effective rate of, 22–23
optical, 327	fluid-switching, 2–3, 16f–17f, 17–18
spatial, 336	high-speed. See High-speed cell sorting
scanning conventions for, 325–326	parallel, 47
broadcast, 325–326, 326f	peak rate of, 22–23
video, 325	performance of, defining, 14–15
selection of, 323–324	photodamage, 3
task requirements of, 328	processing rates of, 22–23
tube-type, 328–329	quality assurance for, 82–86
types of, 328–335	recovery of, 14–15, 23
vidicon, 328–329	for research samples, 80
Cathepsins, in antigen processing, 197–199, 207	robustness of, 23, 46
Cathode ray tube, in digital microscopy,	sterilization of, 85–86
293–294, 305	types of, 2–3
CCD. See Charge-coupled device	well-tempered, 23
CD34 antigen, 74–76, 197–199	yield of, 14–15, 23
CD34+ hematopoietic stem cells	Cell sorting
clinical applications of	versus cell separation, 2
potential, 76–80	emerging tool in, 18
results with, 88t–89t, 88–90	high-speed. See High-speed cell sorting
engraftment data, 89, 89t	indexed, 53
FDA regulations for, 74, 80, 82	push-button approach to, 16f–17f, 17
high-speed sorting of, 73–93	rare. See Rare-cell sorting
immunomagnetic separation in, 79, 81–82	as real-time data analysis, 55, 57
in-process and final product testing in,	technology
86–88, 87f	lack of understanding of, 50
pre-enrichment for, 79–81	review of, 1–19
SyStemix process for, 76, 79–90, 84f,	smart, 50
88t–89t	utilization of, 50
technical challenges in, 79	Cell therapy
manufacturing practices for, 80, 82	current Good Manufacturing Practices for
purified hematopoietic stem cells versus,	(cGMP), 80, 82
77–78	FDA regulations for, 74, 80, 82
quality assurance for, 80, 82–86	quality assurance for, 80, 82–86
sources of, 75	Cell viability
subfractionation of, 75	in confocal microscopy, 243–244, 244f
Thy-1+ subset of, 76–77, 80–82, 84	in high-speed sorting, 46, 80
Cell assays, in microfabricated devices,	in rare-cell sorting, 66
107–108, 108f–109f	Centrifugal elutriation, 79, 81
Cell biology, scanning near-field optical	Ceprate-LC, 79
imaging in, 271–290	cGMP. See Good Manufacturing Practices,
Cell diameter, and transit time, 26–27, 28f	current

342

Charge, in cell sorters, 6–7, 11–12, 12f adjusting synchronization of, 41–43 and drop deflection, 44–45

stages of, 40 timing of, 39–40

Charge injection device (CID), 332–334 construction of, 332, 333f

operation of, 333–334 performance of, 334

Charge transfer efficiency, of CCD cameras, 331 Charge-coupled device (CCD) cameras,

329–332 aliasing in, 304

blooming effect in, 304–305, 332 charge transfer efficiency of, 331 construction of, 329–330

dark current of, 331 dead pixels in, 331 dynamic range of, 332 false data with, 304 fill factor of, 331–332

in fluorescence lifetime imaging microscopy, 141, 154f, 154–156

frame transfer, 330, 330f full-frame, 330, 330f

interline transfer, 330f, 330–331 interpolation in, 305

linearity of, 325, 328 noise in, 331

readout, 331, 336 operation of, 330f, 330–331

performance of, 331–332, 337 resolution of, 303–305

scan rate of, 331 selection of, 336–337

signal-to-noise ratio of, 332 silicon light sensors in, 329 spectral sensitivity of, 332

in video rate two-photon fluorescence microscopy, 231

white-light scanning digital microscopy versus, 303–305

Charge-coupled device (CCD) chips, 329–330, 336–337

Charged-coupled devices (CCD), cooled in confocal microscopy, 254–255, 265 quantum efficiency of, 254f, 254–255

Charge-to-mass ratio, in droplet cell sorters, 44–45

Chinese hamster ovary cells. See CHO cells Chloride concentrations, fluorescent lifetime imaging microscopy of, 163f–165f,

163–166

Index

CHO cells

fluorescence lifetime flow cytometry of, 185–186, 188f

green fluorescent protein in, scanning nearfield optical microscopy of, 277–278, 278f

Chromosome(s)

fluorescence lifetime flow cytometry of, 189f, 190

sorting eight-way, 45

high-speed, 22, 45 photodamage technique for, 3

Chronic myelogenous leukemia, CD34+ hematopoietic stem cells for, 77

CID. See Charge injection device Class II vesicle (CIIV), 199 Clinical trials

cell sorting for, 74, 83–86 regulations for, 74, 80, 82

Coaxial flow system, of droplet cell sorters, 3–5, 5f, 9–10

Coincidence

in cell sorters, 12–13, 23, 32

in rare-cell sorting, 62–63, 63f–64f

minimal cell-cell in excitation source, while maintaining sufficient overall sensitivity, 66–67

Color scanning microscope (COSMIC) averaging mode of, 299–300

brightness and color calibration in, 302–303 brightness and focus change in, 297–298 communications of, 300

comparison with camera systems, 303–305 contrast enhancement and suppression in,

298–299, 298f–299f detection system in, 294 future applications of, 306 image size for, 295

operational considerations with, 305–306 oversampling in, 297, 300–302, 301f resolution of, super, 297, 300–302, 302f response function of, 299

scan rate of, 294 scanning spot in, 293–294 schematic layout of, 293f

signal processing in, 294–296, 295f spot modulation in, 298f, 298–299 summing mode of, 299–300

zoom magnification in, 296–297, 296f–297f, 302

Complementary metal-oxide-semiconductor APS camera, 334–335

Computers, for DNA data collection, 124–125

Index	343
Confocal microscopy	temporal, 246, 264
basic concept of, 222	Rose criterion for, 263
biological reliability versus physical accuracy	sampling theory for, 261–267
in, 243–244	sectioning ability of, 239–240
blank field test for, 240–241	signal-to-noise ratio in, 263
contrast transfer function of, 262, 262f	spatial frequencies of, 262
and resolution, 263	specimen response to dye in, 243–244
cooled CCD in, 254f, 254–255, 265	stage drift in, 266
counting statistics in, 240–243	task of, 240
deep-tissue, 222–225	temporal aliasing in, 264
detection and measurement losses in, 249–261	versus two-photon fluorescence, 222–225,
detection geometry of, 223, 224f	224f
detector in	visibility in, 263
quantum efficiency of, 249–255, 250f–251f,	wagon-wheel effect in, 264
253f–254f	zoom settings for, 245–246, 261, 264
testing, 249–255	Continuous wave (CW) lasers, 320
digitization in, 255, 256f, 264–265	Continuous-wave two-photon imaging, with
fluorescence saturation in, 240, 243–244	scanning near-field optical microscopy,
singlet-state, 243	282f, 283, 283t
interaction problems in, 243–246, 244f	Contrast enhancement and suppression, in
lifetime imaging with, 141	white-light scanning digital microscopy,
limits of, 239–269, 267t	298–299, 298f–299f
imposed by spatial and temporal	Contrast transfer function, of confocal mi-
quantization, 261	croscopy, 262f, 262–263
parameters related to, 240, 241f	Cooled CCD
mechanical stability of, 265–267	in confocal microscopy, 254f, 254–255, 265
mirrors in, 247–248, 258, 266	quantum efficiency of, 254f, 254–255
noise in, 240–243	Cooling, for lasers
extrinsic, 240	air, 313, 317
fixed-pattern, 243	water, 317
intrinsic, 240–243	Cornea
level of, 250	anatomy of, 225–226, 226f
Nyquist criterion for, 245–246, 261, 264–265	as deep-tissue imaging model, 225–226
objective transmission in, 246–247, 247t	two-photon fluorescence microscopy of,
optical system losses in, 246–249, 247t	225–226, 234f, 234–235
penetration depth of, 222	Correlation error, 29–30, 30f
photomultiplier tube in, 239, 250–252, 251f,	COSMIC. See Color scanning microscope
253f, 256, 258–261	Coulter particle counter device, microchip,
photon counting in, 255–258, 256f–257f	99–100, 100f
photon efficiency of, 246	Coulter volume instrument, 3–4
measuring, 258–261, 260f	Critical illumination, 314
practical tests of, 246–261	Cuvette systems, 28
pinholes of, testing, 248	CW. See Continuous wave
pixels in	Cycle time
shape, mismatch with probe, 264–265	definition of, 23
size of, 245–246, 264	determination of, 29
position and motion in, determination of, 266	of droplet cell sorters, 13–14, 14f
pulse heights in, 252, 253f	of fluid-switching cell sorters, 3
repeatability of scanning system in, 265–267	Cytomation sorters
resolution of, 245–246, 261–267	droplet cell, 8, 8f
practical considerations in relating to	high-speed, 22, 80
distortion, 265–267	Cytometry
spatial, 222, 246	flow. See Flow cytometry

344

Cytometry (continued) lasers for, 314–317

light sources for, 314–317 requirements for, 307

microfabricated, 98–100, 99f microfluidics and, 96–100

Dark current, 331 Dead pixels, 331

Dead time, in equipment, 13, 23 calculation of, 64–65

in DNA fragment detection, 118, 130 correction of, 127, 128f

functional, 64

in rare-cell sorting, 63–65, 65f minimal, 67

Decompression, and cell viability, 46 Deconvolution, blind algorithm, for deep-tissue

two-photon fluorescence microscopy, 229–231, 230f

Deep-tissue imaging

confocal microscopy for, 222–225 of mouse ear, 228–229, 229f

skin and cornea as models of, 225–226 two-photon fluorescence microscopy for,

221–237

blind deconvolution algorithm for, 229–231, 230f

video rate, 231–233

two-photon fluorescence spectroscopy for, 233–235

biomedical applications of information, 234–235

importance of information, 233–234 Delay time, of droplet cell sorters, 41–43

accuracy of, 41–43

visual method for determining, 41, 42f Density gradient separation, 79

Detection time, of droplet cell sorters, 13–14, 14f

Dielectrophoresis, 103–104, 105f–106f negative, 103

positive, 103

Differential counting, 100, 100f Diffraction theory, 315–317 Diffusion, between sheath and core, 98 Digital microscopy, 291–293

development of, 291–292 important issues in, 291 remote-control, 292

in telepathology, 292, 305 white-light scanning, 291–306

Digital oversampling, 297, 300–302, 301f Digitization

Index

in camera systems, 335–336

in confocal microscopy, 255, 256f, 264–265 definition of, 324

Diode lasers, 319 Discriminant function analysis

assumptions of, 58–59

in rare-cell sorting, 57–59, 61f, 69 Distortion, definition of, 324

DNA fragment(s)

analysis and sizing, 115–137

avalanche photodiode for, 115, 118–119, 123–127, 130, 135–136

beam-shaping optics for, 121, 121f

burst data analysis in, 118, 119f, 132–134, 133f

capillary and pressurized sample delivery for, 122–123, 123f

versus conventional flow cytometry, 116 critical aspects of system, 128–131

data collection system for, 115, 124–125 data processing software for, 125–127, 126f detection volume in, 130–131

DNA samples for, 120

dye-DNA interactions and, 128–129, 129t, 134

flow cell and fluidics for, 121f, 121–122 future directions in, 135–136 instrument dead time in, 118, 127, 128f

instrument development for, 115, 117–118 instrumentation for, 120–127, 121f

of large fragments, 134–135, 135f laser for, 120–121, 121f

light collection path for, 123–124 materials and methods for, 120–127 MiniSizer for, 116–136

nucleic acid stain for, 120 results of, 131–136

sample delivery and flow rate in, 129–130 of small fragments, 131, 132f

physical length of calculation of, 134 extension in flow, 134–135 staining and, 128–129, 129t

single, detection by flow cytometry, 115–137 DNA injection, 101

DNA-binding dyes development of, 136

interactions with DNA, 128–129, 129t, 134 POPO-3, 117–118, 120, 128–129, 134 TOTO-1, 116

DNA-binding fluorochromes, 188f, 189 DNASizer, 124

Droplet cell sorters, 2–16 acoustic signal in, 34–39, 38f

Index

345

acoustically polluted, 38–39 automated cell collection in, 45 biohazards with, 2

break-off point of, 34–36, 35f, 40 piezosignal amplitude and, 37f stability of, 43–44, 55

brief history of, 3–4 charge of, 6–7, 11–12, 12f

adjusting synchronization of, 41–43 and drop deflection, 44–45

stages of, 40 timing of, 39–40

charge-to-mass ratio in, 44–45 coaxial flow system of, 3–5, 5f, 9–10 coincidence in, 12–13, 32 components of, 4f, 4–8 contaminating cells in, 12–13

cycle time of, 31

delay time of, 41–43, 42f droplet generation of, 5–6, 8–10

assemblies for, 8, 8f

frequency of, 8–10, 31–34, 33f–34f high-speed, 31–34

optimal, 32–34, 34f physics of, 32–34, 33f stability of, 2, 5, 8

efficiency of, 14–15, 23–26, 25f–26f eight-way configuration of, 45 electronic sorting system of

cycle time of, 13–14, 14f dead time of, 13

detection time of, 13–14, 14f

processing of cell measurement in, 13–14, 14f evaporation and, 45

exclusion zone of, 32, 43 field strength of, 44–45 flow cells of, 10–11, 11f

four-way sorting configuration of, 6f–7f, 7 frequency coupling of, 34–36, 35f

numerical example of, 36–37, 37f high-speed, 21–48

hydrodynamic properties of, 4–5, 9 kinetic energy of, 32–34

laser intersection of, location of, 10–11, 11f microdroplets of, 5–6

multiple sort directions in, 6f–7f, 7, 45 nozzle of, 10–11

assemblies of, 10–11, 11f, 37–39, 38f diameter of, 8–10

geometry of, 5 jet-in-air, 10–11, 11f, 28 quartz, 10–11, 11f

resonance frequency of, 37–38 performance of

defining, 14–15 predicting, 15–16 variables in, 14

Poisson statistics in assessment of, 23–26, 25f–26f

pressure of, 31–34 purity of, 14–15, 24, 26f queuing in, 12–13

for rare cells, 55 rates of, 2 recovery of, 14–15

sequence of operations in, 7–8 sheath pressure of, 8–10

for sorting into microwell plate, 6f–7f, 7 stream deflection in, variations of, 6f–7f, 7 stream undulations in, 5–9, 12–13, 34–36, 35f,

39–40

temperature stability of, 38–39

timing and drop deflection synchronization in, 39–40

typical, schematic of, 4f

velocity of, 32–34, 33f–34f, 39–40 parabolic profile of, 9–10

yield of, 14–15

Drosophila melanogaster Schneider cells, green fluorescent protein in, scanning near-field optical microscopy of, 277–278, 279f

Duty cycle

50 percent, 23 full, 23

of high-speed cell sorters, 23, 46 Dye lasers, 320

Dyes, DNA-binding, 116–118, 120, 128–129, 129t, 134, 136

Dynamic telepathology, 292

Ear, mouse, two-photon microscopy of, 228–229, 229f

E-beam radiation, for equipment sterilization, 85–86

Effective rate, definition of, 22–23 Electrical impedance, 104

Electrical transport, sorting and characterization, in microfabricated devices, 103–104, 105f–106f

Electrokinetic microchip, 107–108, 108f–109f Electronic(s)

in confocal microscopy, 255 defocusing, 151

for DNA analysis and sizing, 116 of droplet cell sorters, 13–14, 14f

in fluorescence lifetime flow cytometry, 182–183, 185f

of high-speed cell sorters, 29–31, 80

346

Electronic(s) (Continued) errors in, 29–31, 30f parameters for, 29 quality tests of, 30–31

of light sources, 311–314

in white-light scanning digital microscopy, 294–296, 295f

Electrophoretic separations continuous, 108, 109f

in microfabricated devices, 107–108, 108f–109f

Electrostatic field, strength of, in droplet cell sorters, 44–45

Embryo manipulation

microfabricated devices for, 102–103, 104f traditional methods of, 101

Empty magnification, 296–297, 301 Endocytic system, antigen processing in,

197–219

Epidermal growth factor receptor (EGFR), green fluorescent protein in, scanning nearfield optical microscopy of, 277–278, 278f

Epi-illumination system, 308 Error(s)

in charge synchronization, 41–43 correlation, 29–30, 30f

in high-speed cell sorting, 29–31, 30f Evaporation, and droplet cell sorters, 45 Event(s)

definition of, 22

in DNA fragment detection, 116 ghost, 29–30, 30f

interval distribution, 24–26, 25f, 27f Poisson statistics for, 23–26, 25f–26f processed per second, 22–23

rare, detection of, 49–72 Exclusion zone, in cell sorters, 32, 43 Exitance, of light sources, 307–309

Exponential delay, in fluorescent lifetime imaging microscopy, 170

Extrinsic noise, in confocal microscopy, 240

FACS Vantage, 85 FACSCalibur, 86–87, 87f Facsort, 16f–17f, 17

Fast Fourier transform

for fluorescence lifetime flow cytometry, 175 for fluorescent lifetime imaging microscopy,

153, 155

FDA. See Food and Drug Administration Fill factor, of CCD camera, 331–332

Fixed-pattern noise, in confocal microscopy, 243 FLIM. See Fluorescence lifetime imaging

microscopy

Index

Flow

laminar, 96–97 sheath, 97f, 97–98, 99f steady, 96–97 turbulent, 96–97 unsteady, 96–97

Flow cells

for DNA fragment analysis and sizing, 121f, 121–122

for droplet cell sorters, 10–11, 11f Flow cytometry

of CD34+ hematopoietic stem cells, 73–93 in clinical diagnostic medicine, 175 conventional, 95, 175

fluorescence lifetime. See Fluorescence lifetime flow cytometry

high-speed, 21–48. See also High-speed cell sorting

kinetics of, 207–208, 209f lasers for, 316–317

light sources for, requirements for, 307 microfabrication in, for single-cell handling

and analysis, 95–113

for monitoring antigen processing and presentation, 204–215

quality standards for, 82–86

of rare cells, 49–72. See also Rare-cell sorting for single DNA fragment detection, 115–137 technology, 1–19, 50

Fluid(s) classifications of, 96 flow

laminar, 96–97 sheath, 97f, 97–98, 99f steady, 96–97 turbulent, 96–97 unsteady, 96–97

Newtonian, 96 non-Newtonian, 96

Fluidic(s)

devices, microfabricated, for single-cell handling and analysis, 95–113

for DNA fragment analysis and sizing, 121f, 121–122

in high-speed cell sorting, 27–28, 80 microsystems

basics of, 96–98

and cytometry, 96–100 in rare-cell sorting, 55 sterilization of, 85

Fluid-switching cell sorters, 2–3, 16f–17f, 17–18 biosafety of, 3, 17, 55

cycle time of, 3 for rare cells, 55

Index

rates of, 3, 17–18 stability of, 3

Fluorescein isothiocyanate (FTIC) conjugated to bovine serum albumin

as exogenous antigen, 200–201, 201f in study of antigen processing, 200–217

in fluorescent lifetime imaging microscopy, 178

fluorescent properties of, 200–201 immunogenicity and antigenicity of, 200–201 pH dependence of, 200, 202f

spectral properties of, 200, 204 Fluorescence decay, 140, 176

Fluorescence decay time, methods of measuring, 140

Fluorescence emission signals, separation of fluorescence lifetimes for, 177–178 phase-resolved, 181–182

Fluorescence intensity

measurements, of antigen processing and presentation, 204–208, 205f, 207f–209f

in scanning near-field optical microscopy as function of excitation power, 283t,

283–284

as function of tip-sample displacement, 284–285, 286t

Fluorescence lifetime(s)

of fluorochromes used for labeling cellular complexes and cells, 176, 177t

measurement of, 179–181

amplitude modulation method of, 179–181 in fluorescence lifetime flow cytometry,

179–181, 184–187 phase-shift method of, 179–181 in steady state, 179–180 two-phase method of, 179

for separating overlapping emissions, 177–178 unique properties of, 141–142

Fluorescence lifetime flow cytometry, 175–196 of antigen processing, 213–215, 214t

cell preparation and staining in, 184–187 fluorescence lifetimes in

of markers for cellular complexes and cells, 176, 177t

measurement of, 178–181, 184–187 in frequency domain, 176–177, 182 instrumentation for, 183–184, 184f–185f materials and methods for, 178–187 multifrequency, 181

conceptual diagram of, 181f phase-resolved separation of emission signals

in, 181–182

results of, 187–192, 187f–192f signal processing in, 182–183, 185f

347

single-frequency, 180–181 conceptual diagram of, 180f

theory of operation, 178–183 in time domain, 176

Fluorescence lifetime imaging microscopy (FLIM), 139–173

background rejection and noise immunity in, 168–169

of chloride concentrations, 163f–165f, 163–166

comparison of techniques, 166–170 examples of, 162–166

frequency-domain heterodyning method of, 152–162, 153f

background of, 152–154

calculation of ac, dc, phase and modulation for, 153, 153f

instrumentation for, 154f, 154–159, 156f–158f

frequency-domain homodyning method of, 145–152, 147f–149f

background of, 145–150 instrument for, 150–152, 151f

modulation of excitation light source in, 150–151

modulation of microchannel plate image intensifier in, 151

signal detection in, 152 historical perspective on, 139–140

intensified camera-based, 154f, 154–156 modulation of laser in, 154–155

signal detection and data acquisition system in, 155–156, 156f

laser scanning versus wide-field illumination in, 167–168

lifetime resolution of, 166

microchannel plate-based, 154–156, 156f optical mixing methods of, 141, 159–162

background of, 159–160 instrument for, 160–162 photobleaching and, 169–170

photon and time efficiency of, 166–167 pump-probe method of, 159–162, 160f–161f

laser sources for, 161–162

optics, signal detection and data acquisition in, 162

recent developments in, overview of, 140–141 sequential laser scanning based, 156–159

excitation and optics in, 157f, 157–158 synchronization of instrument and data ac-

quisition in, 158f, 158–159

singleor multiple-exponential delay in, 170 stimulated emission technique of, 159–162 in time domain, 142–145, 143f

348

Fluorescence lifetime imaging microscopy (Continued)

background of, 142–144 instrument for, 144–145, 145f pulsed light source in, 144–145 time gating of detector in, 145

time-resolved, 141–142, 154f, 154–162, 157f, 160f–161f

two-photon. See Fluorescence microscopy, two-photon

Fluorescence microscopy, two-photon, 142–143, 156–157, 227f

advantages of, 223–225

of antigen processing, 202–204, 203f axial depth discrimination of, 223

biomedical applications of fluorescence spectroscopic information in, 234–235

blind deconvolution algorithm for, 229–231, 230f

challenges in clinical setting, 231 versus confocal, 222–225, 224f

of cornea, 225–226, 234f, 234–235 deep-tissue, 221–237

detection geometry of, 223, 224f excitation wavelengths of, 223–225 of mouse ear, 228–229, 229f penetration depth of, 223 photodamage with, 223

photon collection with, 223–225 setting up, 227f, 227–228

of skin, 225–226, 230f, 231, 232f, 233 three-dimensional imaging capabilities of,

202–203

tissue thermal damage with, 222–223 video rate, 231–233, 232f, 232–233

Fluorescence polarization measurements, of antigen processing, 209–213, 210f–212f

Fluorescence resonance energy transfer (FRET), 140

with scanning near-field optical microscopy, 273, 276–281

for photobleaching on cell surfaces, 278–279

Fluorescence resonance energy transfer, with scanning near-field optical microscopy, for donor photobleaching using labeled 3T3 cells in, 280f, 280–281

Fluorescence saturation

in confocal microscopy, 240, 243–244 singlet-state, 243

Fluorescence spectroscopy, two-photon, in deep tissue, 233–235

Fluorescence-derivatized bovine serum albumin

Index

as exogenous antigen, 200–201, 201f in study of antigen processing, 200–217

Food and Drug Administration (FDA), cell therapy regulations of, 74, 80, 82

Fourier transform

for fluorescence lifetime flow cytometry, 175 for fluorescent lifetime imaging microscopy,

146, 153, 155

Frame transfer CCD, 330, 330f Frequency coupling, 34–36, 35f

numerical example of, 36–37, 37f Frequency domain

fluorescence lifetime flow cytometry in, 176–177, 182

fluorescent lifetime imaging microscopy in, 145–162

background rejection and noise immunity in, 168–169

exponential delay in, 170 lifetime resolution of, 166 photobleaching and, 169–170

photon and time efficiency of, 166–167 for measuring fluorescence decay time, 140 spatial, confocal microscopy in, 262

Frequency-domain heterodyning method, of fluorescent lifetime imaging microscopy, 152–159, 153f

background of, 152–154

calculation of ac, dc, phase and modulation for, 153, 153f

instrumentation for, 154f, 154–159, 156f–158f pump-probe, 159–162, 160f–161f

Frequency-domain homodyning method, of fluorescent lifetime imaging microscopy, 145–152, 147f–149f

background of, 145–150 instrument for, 150–152, 151f

modulation of excitation light source in, 150–151

modulation of microchannel plate image intensifier in, 151

signal detection in, 152

FRET. See Fluorescence resonance energy transfer

FTIC. See Fluorescein isothiocyanate Full duty cycle, 23

Full-field illumination, 304 Full-frame CCD, 330, 330f Functional dead time, 64 Functional genomics, 95, 101 Fused-silica device, 98, 99f Fused-silica microcoils, 105 Fusion, cell, 104, 106f