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Microsocpic Imaging for HTS-technical aspects

Microsocpic Imaging for HTS-technical aspects

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03/18/2014

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A\ue003 i\ue003f\ue002\ue006x \ue004f \ue003\ue000\ue007 mic\ue005\ue004sc\ue004pic imagi\ue003g m\ue000th\ue004ds a\ue003d t\ue004\ue004\ue002s, i\ue003c\ue002\ue006di\ue003g adva\ue003c\ue000d imagi\ue003g i\ue003st\ue005\ue006m\ue000\ue003ts, \ue003\ue004v\ue000\ue002 m\ue000th\ue004ds a\ue003d \ue003\ue000\ue007 \ue000xp\ue000\ue005im\ue000\ue003ta\ue002 \ue005\ue000ag\ue000\ue003ts, a\ue005\ue000 sig\ue003ifica\ue003t\ue002\ue008 \ue000\ue003ha\ue003c\u2011 i\ue003g assa\ue008s a\ue003d tiss\ue006\ue000 m\ue004d\ue000\ue002s f\ue004\ue005 \ue000a\ue005\ue002\ue008 d\ue005\ue006g disc\ue004v\ue000\ue005\ue008, as \ue007\ue000\ue002\ue002 as m\ue004\ue005\ue000 c\ue004mp\ue002icat\ue000d dis\ue000as\ue000 m\ue004d\ue000\ue002s that a\ue005\ue000 \ue006s\ue000d \ue002at\ue000\ue005 i\ue003 d\ue005\ue006g disc\ue004v\ue000\ue005\ue008. T\ue004g\ue000th\ue000\ue005, th\ue000s\ue000 i\ue003\ue003\ue004vati\ue004\ue003s a\ue005\ue000 faci\ue002itati\ue003g m\ue004\ue005\ue000 s\ue000\ue003sitiv\ue000, sp\ue000cific a\ue003d high\ue000\ue005 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 m\ue000as\ue006\ue005\ue000m\ue000\ue003ts f\ue005\ue004m c\ue000\ue002\ue002s a\ue003d \ue004\ue005ga\ue003isms, a\ue003d s\ue004 c\ue004\ue006\ue002d hav\ue000 a\ue003 impact \ue004\ue003 a \ue007id\ue000 \ue005a\ue003g\ue000 \ue004f disc\ue004v\ue000\ue005\ue008 activiti\ue000s \ue005a\ue003gi\ue003g f\ue005\ue004m ta\ue005g\ue000t bi\ue004\ue002\ue004g\ue008, t\ue004 c\ue004mp\ue004\ue006\ue003d sc\ue005\ue000\ue000\ue003i\ue003g a\ue003d t\ue004 a\ue003ima\ue002 m\ue004d\ue000\ue002s \ue004f dis\ue000as\ue000.

Ma\ue003\ue008 \ue004f th\ue000s\ue000 i\ue003\ue003\ue004vati\ue004\ue003s a\ue005\ue000 imp\ue004\ue005ta\ue003t b\ue000ca\ue006s\ue000 th\ue000\ue008 h\ue000\ue002p am\ue000\ue002i\ue004\ue005at\ue000 s\ue004m\ue000 \ue004f th\ue000 t\ue000ch\ue003ica\ue002 \ue002imitati\ue004\ue003s that a\ue005\ue000 i\ue003h\ue000\ue005\ue000\ue003t i\ue003 mic\ue005\ue004sc\ue004pic \ue004ptica\ue002 imagi\ue003g (FIG. 1). Sp\ue000cifica\ue002\ue002\ue008, th\ue000\ue005\ue000 a\ue005\ue000 f\ue004\ue006\ue005 mai\ue003 \ue002imitati\ue004\ue003s that c\ue006\ue005\ue005\ue000\ue003t\ue002\ue008 \ue005\ue000st\ue005ict this f\ue004\ue005m \ue004f \ue004ptica\ue002 imagi\ue003g: i\ue003s\ue006ffici\ue000\ue003t spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003, m\ue004d\ue000st d\ue000pth \ue004\ue005 tiss\ue006\ue000 p\ue000\ue003\ue000t\ue005ati\ue004\ue003, p\ue004\ue004\ue005 ph\ue008sica\ue002 acc\ue000ss a\ue003d \ue002\ue004\ue007 \ue000xp\ue000\ue005im\ue000\ue003ta\ue002 th\ue005\ue004\ue006ghp\ue006t. I\ue003 ma\ue003\ue008 \ue000xp\ue000\ue005im\ue000\ue003ta\ue002 pa\ue005adigms, \ue004\ue003\ue000 \ue004\ue005 m\ue004\ue005\ue000 \ue004f th\ue000s\ue000 fact\ue004\ue005s ma\ue008b\ue000 \ue002imiti\ue003g. F\ue004\ue005 i\ue003sta\ue003c\ue000, th\ue000 spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue004f \ue002ight mic\ue005\ue004sc\ue004p\ue008 is \ue004ft\ue000\ue003 i\ue003s\ue006ffici\ue000\ue003t t\ue004 ad\ue000q\ue006at\ue000\ue002\ue008 cap\u2011 t\ue006\ue005\ue000 \ue004\ue005 cha\ue005act\ue000\ue005iz\ue000 a\ue002\ue002 \ue004f th\ue000 \ue006\ue003d\ue000\ue005\ue002\ue008i\ue003g bi\ue004\ue002\ue004g\ue008, \ue000sp\ue000cia\ue002\ue002\ue008 at a m\ue004\ue002\ue000c\ue006\ue002a\ue005\u2011\ue002\ue000v\ue000\ue002 d\ue000tai\ue002. H\ue004\ue007\ue000v\ue000\ue005, \ue003\ue000\ue007 s\ue006p\ue000\ue005\u2011\ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 mic\ue005\ue004sc\ue004p\ue000s a\ue005\ue000 \ue003\ue004\ue007 avai\ue002ab\ue002\ue000 that \ue005\ue000s\ue004\ue002v\ue000 st\ue005\ue006ct\ue006\ue005\ue000s t\ue004 \ue007\ue000\ue002\ue002 b\ue000\ue002\ue004\ue007 th\ue000 diff\ue005acti\ue004\ue003 \ue002imit (that is, <200 \ue003m) a\ue003d th\ue000\ue005\ue000b\ue008 a\ue002\ue002\ue004\ue007 \ue003\ue000a\ue005 m\ue004\ue002\ue000c\ue006\ue002a\ue005\u2011\ue002\ue000v\ue000\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 imag\ue000s t\ue004 b\ue000 c\ue004\ue002\ue002\ue000ct\ue000d.

I\ue003 \ue004th\ue000\ue005 i\ue003sta\ue003c\ue000s, i\ue003t\ue000\ue005\ue000sti\ue003g c\ue000\ue002\ue002s a\ue003d tiss\ue006\ue000s a\ue005\ue000 i\ue003acc\ue000ssib\ue002\ue000 b\ue000ca\ue006s\ue000 th\ue000\ue008 a\ue005\ue000 b\ue006\ue005i\ue000d d\ue000\ue000p \ue007ithi\ue003 a\ue003 \ue004\ue005ga\ue003ism \ue004\ue005 ca\ue003 \ue004\ue003\ue002\ue008 b\ue000 st\ue006di\ue000d \ue007h\ue000\ue003 c\ue004mp\ue004\ue003\ue000\ue003ts \ue004f th\ue000

tiss\ue006\ue000 a\ue005\ue000 a\ue005tificia\ue002\ue002\ue008 \ue005\ue000c\ue004\ue003stit\ue006t\ue000d i\ue003 a dish. I\ue003\ue003\ue004vati\ue004\ue003s i\ue003 i\ue003t\ue005avita\ue002 mic\ue005\ue004sc\ue004p\ue008 (IVM) a\ue003d d\ue000\ue000p\u2011tiss\ue006\ue000 imagi\ue003g a\ue005\ue000 h\ue000\ue002pi\ue003g t\ue004 \ue004v\ue000\ue005c\ue004m\ue000 this p\ue005\ue004b\ue002\ue000m b\ue008 a\ue002\ue002\ue004\ue007i\ue003g imag\ue000 c\ue004\ue002\ue002\ue000cti\ue004\ue003 at g\ue005\ue000at\ue000\ue005 tiss\ue006\ue000 d\ue000pths a\ue003d i\ue003 m\ue004\ue005\ue000 ph\ue008si\ue004\u2011 \ue002\ue004gica\ue002\ue002\ue008 \ue005\ue000\ue002\ue000va\ue003t c\ue004\ue003t\ue000xts. P\ue004\ue004\ue005 d\ue000pth p\ue000\ue003\ue000t\ue005ati\ue004\ue003 a\ue003d \ue004th\ue000\ue005 ph\ue008sica\ue002 acc\ue000ss iss\ue006\ue000s a\ue005\ue000 \ue000sp\ue000cia\ue002\ue002\ue008 imp\ue004\ue005ta\ue003t i\ue003 d\ue005\ue006g disc\ue004v\ue000\ue005\ue008, as in vivo dis\ue000as\ue000 m\ue004d\ue000\ue002s \ue004ft\ue000\ue003 \ue005\ue000q\ue006i\ue005\ue000 th\ue000 \ue000xami\ue003ati\ue004\ue003 \ue004f tiss\ue006\ue000s d\ue000\ue000p \ue007ithi\ue003 a\ue003 \ue004\ue005ga\ue003ism. Pa\ue005a\ue002\ue002\ue000\ue002 d\ue000v\ue000\ue002\ue004pm\ue000\ue003ts i\ue003 \ue004ptics a\ue003d fib\ue005\ue000\u2011\ue004ptic imagi\ue003g t\ue000ch\ue003\ue004\ue002\ue004gi\ue000s a\ue005\ue000 d\ue005ivi\ue003g th\ue000 mi\ue003iat\ue006\ue005izati\ue004\ue003 \ue004f f\ue002\ue006\ue004\ue005\ue000s\u2011 c\ue000\ue003c\ue000 mic\ue005\ue004sc\ue004p\ue008. Th\ue000s\ue000 d\ue000v\ue000\ue002\ue004pm\ue000\ue003ts a\ue005\ue000 b\ue000i\ue003g \ue006s\ue000d t\ue004 faci\ue002itat\ue000 b\ue000tt\ue000\ue005 ph\ue008sica\ue002 acc\ue000ss d\ue006\ue005i\ue003g sma\ue002\ue002\u2011a\ue003ima\ue002 imagi\ue003g st\ue006di\ue000s.

A\ue003\ue004th\ue000\ue005 \ue002imitati\ue004\ue003 is th\ue000 c\ue004\ue003fig\ue006\ue005ati\ue004\ue003 \ue004f m\ue004d\ue000\ue005\ue003 \ue005\ue000s\ue000a\ue005ch\u2011g\ue005ad\ue000 mic\ue005\ue004sc\ue004p\ue000s, \ue007hich a\ue005\ue000 \ue004ft\ue000\ue003 c\ue004mp\ue002icat\ue000d a\ue003d i\ue003f\ue002\ue000xib\ue002\ue000. This has mad\ue000 th\ue000m diffic\ue006\ue002t t\ue004 a\ue006t\ue004mat\ue000 a\ue003d th\ue000\ue005\ue000f\ue004\ue005\ue000 i\ue003c\ue004mpatib\ue002\ue000 \ue007ith th\ue000 high\u2011th\ue005\ue004\ue006ghp\ue006t bi\ue004\ue002\ue004g\ue008 app\ue005\ue004ach\ue000s that a\ue005\ue000 \ue004ft\ue000\ue003 \ue006s\ue000d i\ue003 d\ue005\ue006g dis\u2011 c\ue004v\ue000\ue005\ue008, s\ue006ch as high\u2011th\ue005\ue004\ue006ghp\ue006t sc\ue005\ue000\ue000\ue003i\ue003g (HTS). H\ue004\ue007\ue000v\ue000\ue005, \ue005\ue000c\ue000\ue003t i\ue003\ue003\ue004vati\ue004\ue003s i\ue003 high\u2011c\ue004\ue003t\ue000\ue003t sc\ue005\ue000\ue000\ue003i\ue003g (HCS) a\ue003d \u2018imagi\ue003g i\ue003 f\ue002\ue004\ue007\u2019 m\ue000th\ue004d\ue004\ue002\ue004gi\ue000s a\ue005\ue000 \ue003\ue004\ue007 \ue000\ue003ab\ue002i\ue003g imp\ue005\ue004v\ue000d imag\ue000\u2011bas\ue000d sc\ue005\ue000\ue000\ue003i\ue003g b\ue008 i\ue003c\ue005\ue000asi\ue003g th\ue000 i\ue003f\ue004\ue005mati\ue004\ue003 c\ue004\ue003t\ue000\ue003t a\ue003d \ue000xp\ue000\ue005im\ue000\ue003ta\ue002 th\ue005\ue004\ue006ghp\ue006t.

T\ue004g\ue000th\ue000\ue005, th\ue000s\ue000 adva\ue003c\ue000d a\ue003d high\u2011th\ue005\ue004\ue006ghp\ue006t mic\ue005\ue004sc\ue004pic imagi\ue003g t\ue000ch\ue003\ue004\ue002\ue004gi\ue000s a\ue005\ue000 \ue000\ue003ab\ue002i\ue003g a m\ue004v\ue000 a\ue007a\ue008 f\ue005\ue004m simp\ue002\ue000 in vitro assa\ue008s \ue007ith fix\ue000d \ue000\ue003d p\ue004i\ue003ts. I\ue003st\ue000ad, high\ue000\ue005\u2011\ue004\ue005d\ue000\ue005 m\ue004d\ue000\ue002s (\ue004\ue005 m\ue004d\ue000\ue002 s\ue008st\ue000ms1\u20133) that \ue000mphasiz\ue000 b\ue004th ph\ue008si\ue004\ue002\ue004gica\ue002 \ue005\ue000\ue002\ue000va\ue003c\ue000 a\ue003d th\ue005\ue004\ue006gh\u2011 p\ue006t a\ue005\ue000 gai\ue003i\ue003g p\ue005\ue004mi\ue003\ue000\ue003c\ue000. A\ue003 adva\ue003tag\ue000 \ue004f m\ue004d\ue000\ue002s \ue007ith\ue004\ue006t fix\ue000d \ue000\ue003d p\ue004i\ue003ts is that th\ue000\ue008 a\ue002\ue002\ue004\ue007 \ue005\ue000p\ue000at\ue000d

Biological Imaging
Development Center,
513 Parnassus Avenue,
University of California,
San Francisco, California
94143\u20110511, USA.
e\u2011mail:
andrew.bullen@ucsf.edu

doi:10.1038/nrd2446
Publi\ue000hed online
14 December 2007

Microscopic imaging techniques
for drug discovery
Andrew Bullen
Abstract | Microscopic imaging can enhance the drug discovery process by helping to
describe how disease processes unfold and how potential therapies might intervene.
Recently introduced technologies, and enhancements to existing techniques, are addressing
technical issues that have limited the usefulness of microscopic imaging in the past.
In particular, these innovations are improving spatial resolution, increasing tissue penetration,

overcoming physical access issues and enhancing experimental throughput. Notable recent
trends, which are discussed in this article, include the development of super\u2011resolution
microscopes, the incorporation of multiphoton techniques into intravital and fibre\u2011optic
microscopy and the automation of microscopy and image analysis for high\u2011content
screening. Together, these developments are augmenting the existing assays and disease
models that are used in early drug discovery and, in some cases, enabling new ones.

REVIEWS
54|jAnuAry 2008 |VoluMe 7
www.na\ue00b\ue00c\ue009\ue002.\ue000\ue007m/\ue009\ue002\ue00d\ue005\ue002w\ue00a/\ue001\ue009\ue00c\ue003\ue001\ue005\ue00a\ue000
N a t u re R e v ie w s|D rug D iscovery
Technicalis
sueaddresse
dExamplesRo
leinearlydi
scoveryproc
ess
Target
biology
Lead
identification
Lead
evaluation
Lead
optimization
Physical
access
Target
identification
HTS
Liabilty
profiling
Disease
models
Tissue
penetration
Spatial
resolution
Super-resolution microscopy
4Pi
TIRF
SPIM
SSIM
Experimental
throughput
Target
validation
HCS
2\u00b0/3\u00b0
assays
ADMET
Intravital microscopy
2P-IVM
confocal-IVM
Physical
access
Target
identification
HTS
Liabilty
profiling
Disease
models
Tissue
penetration
Spatial
resolution
Experimental
throughput
Target
validation
HCS
2\u00b0/3\u00b0
assays
ADMET
Fibre-optic microscopy
Confocal-ME,
2P-ME
Physical
access
Target
identification
HTS
Liabilty
profiling
Disease
models
Tissue
penetration
Spatial
resolution
Experimental
throughput
Target
validation
HCS
2\u00b0/3\u00b0
assays
ADMET
High-throughput microscopy
Imaging in flow
multimode HCS
Physical
access
Target
identification
HTS
Liabilty
profiling
Disease
models
Tissue
penetration
Spatial
resolution
Experimental
throughput
Target
validation
HCS
2\u00b0/3\u00b0
assays
ADMET

m\ue000as\ue006\ue005\ue000m\ue000\ue003ts, ch\ue005\ue004\ue003ic \ue004bs\ue000\ue005vati\ue004\ue003s a\ue003d/\ue004\ue005 ki\ue003\ue000tic d\ue000t\ue000\ue005\u2011 mi\ue003ati\ue004\ue003s that a\ue005\ue000 a\ue002\ue002 imp\ue004\ue005ta\ue003t i\ue003 cha\ue005act\ue000\ue005izi\ue003g th\ue000 \ue006\ue003d\ue000\ue005\ue002\ue008i\ue003g s\ue008st\ue000ms bi\ue004\ue002\ue004g\ue008. I\ue003 fact, a\ue003 \ue000xt\ue000\ue003si\ue004\ue003 \ue004f this th\ue000m\ue000 is th\ue000 capabi\ue002it\ue008 t\ue004 imag\ue000 th\ue000 in vivo t\ue005\ue000atm\ue000\ue003t \ue005\ue000sp\ue004\ue003s\ue000 t\ue004 t\ue000st c\ue004mp\ue004\ue006\ue003ds, \ue000sp\ue000cia\ue002\ue002\ue008 i\ue003 a\ue003ima\ue002 m\ue004d\ue000\ue002s. Imagi\ue003g th\ue000 \ue007h\ue004\ue002\ue000\u2011b\ue004d\ue008 t\ue005\ue000atm\ue000\ue003t \ue005\ue000sp\ue004\ue003s\ue000 is a \ue006s\ue000f\ue006\ue002 \ue007a\ue008 \ue004f sim\ue006\ue002ta\ue003\ue000\ue004\ue006s\ue002\ue008 q\ue006a\ue003tif\ue008i\ue003g th\ue000 \ue000fficac\ue008, tim\ue000 c\ue004\ue006\ue005s\ue000 a\ue003d th\ue000 sp\ue000cificit\ue008 \ue004f th\ue000\ue005ap\ue000\ue006tic ca\ue003didat\ue000s. It is a\ue002s\ue004 p\ue004t\ue000\ue003tia\ue002\ue002\ue008 \ue006s\ue000f\ue006\ue002 i\ue003 id\ue000\ue003tif\ue008i\ue003g \ue004ff\u2011ta\ue005g\ue000t \ue000ff\ue000cts a\ue003d \ue002iabi\ue002iti\ue000s.

Th\ue000\ue005\ue000 is a\ue002s\ue004 a b\ue006si\ue003\ue000ss \ue005ati\ue004\ue003a\ue002\ue000 f\ue004\ue005 i\ue003t\ue005\ue004d\ue006ci\ue003g s\ue006ch t\ue000ch\ue003\ue004\ue002\ue004gi\ue000s. F\ue004\ue005 i\ue003sta\ue003c\ue000, a \ue003\ue000\ue007 t\ue000ch\ue003\ue004\ue002\ue004g\ue008 ma\ue008 \ue000\ue003ab\ue002\ue000 a c\ue004mp\ue002\ue000t\ue000\ue002\ue008 \ue003\ue004v\ue000\ue002 assa\ue008 p\ue002atf\ue004\ue005m, \ue004\ue005 a\ue002\ue002\ue004\ue007 a\ue003 assa\ue008 t\ue004 b\ue000 a\ue006t\ue004mat\ue000d \ue004\ue005 \ue005\ue000c\ue004\ue003fig\ue006\ue005\ue000d f\ue004\ue005 g\ue005\ue000at\ue000\ue005 th\ue005\ue004\ue006ghp\ue006t a\ue003d/ \ue004\ue005 savi\ue003g \ue004f ma\ue003p\ue004\ue007\ue000\ue005. I\ue003 \ue004th\ue000\ue005 cas\ue000s, \ue003\ue000\ue007 t\ue000ch\ue003\ue004\ue002\ue004gi\ue000s ca\ue003 imp\ue005\ue004v\ue000 \ue000xisti\ue003g assa\ue008s b\ue008 \ue000\ue003ha\ue003ci\ue003g i\ue003f\ue004\ue005mati\ue004\ue003 c\ue004\ue003t\ue000\ue003t, i\ue003c\ue005\ue000asi\ue003g data q\ue006a\ue002it\ue008, \ue005\ue000d\ue006ci\ue003g c\ue008c\ue002\ue000 tim\ue000s \ue004\ue005 b\ue008 p\ue005\ue004vidi\ue003g c\ue004st \ue005\ue000d\ue006cti\ue004\ue003s i\ue003 disp\ue004sab\ue002\ue000s a\ue003d \ue005\ue000ag\ue000\ue003ts.

This a\ue005tic\ue002\ue000 s\ue006\ue005v\ue000\ue008s a \ue005a\ue003g\ue000 \ue004f \ue003\ue000\ue007 mic\ue005\ue004sc\ue004pic t\ue000ch\u2011 \ue003\ue004\ue002\ue004gi\ue000s a\ue003d disc\ue006ss\ue000s h\ue004\ue007 th\ue000\ue008 a\ue005\ue000 b\ue000i\ue003g \ue006s\ue000d t\ue004 s\ue006pp\ue004\ue005t va\ue005i\ue004\ue006s \ue005\ue000s\ue000a\ue005ch activiti\ue000s i\ue003 \ue000a\ue005\ue002\ue008 d\ue005\ue006g disc\ue004v\ue000\ue005\ue008. It is imp\ue004\ue005ta\ue003t t\ue004 \ue003\ue004t\ue000 that ma\ue003\ue008 \ue005\ue000\ue002at\ue000d \ue004ptica\ue0024 a\ue003d \ue003\ue004\ue003\u2011 \ue004ptica\ue002 imagi\ue003g5\u20137 app\ue005\ue004ach\ue000s a\ue002s\ue004 hav\ue000 a k\ue000\ue008 \ue005\ue004\ue002\ue000 i\ue003 d\ue005\ue006g disc\ue004v\ue000\ue005\ue008; h\ue004\ue007\ue000v\ue000\ue005, th\ue000 sc\ue004p\ue000 \ue004f this r\ue000vi\ue000\ue007 p\ue005\ue000c\ue002\ue006d\ue000s th\ue000i\ue005 c\ue004\ue003sid\ue000\ue005ati\ue004\ue003 h\ue000\ue005\ue000.

Super-resolution microscopes

n\ue000\ue007 s\ue006p\ue000\ue005\u2011\ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 mic\ue005\ue004sc\ue004p\ue000s a\ue005\ue000 sig\ue003ifica\ue003t\ue002\ue008 imp\ue005\ue004vi\ue003g th\ue000 spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue004f \ue002ight mic\ue005\ue004sc\ue004p\ue008. Th\ue000s\ue000 s\ue006p\ue000\ue005\u2011\ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 mic\ue005\ue004sc\ue004p\ue000s a\ue005\ue000 d\ue000sig\ue003\ue000d t\ue004 \ue004p\ue000\ue005at\ue000 at th\ue000 mac\ue005\ue004m\ue004\ue002\ue000c\ue006\ue002a\ue005 \ue004\ue005 s\ue006bmic\ue005\ue004sc\ue004pic \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue002\ue000v\ue000\ue002 a\ue003d a\ue005\ue000 c\ue004mm\ue004\ue003\ue002\ue008 \ue006s\ue000d t\ue004 \ue000xami\ue003\ue000 st\ue005\ue006ct\ue006\ue005a\ue002 iss\ue006\ue000s a\ue003d/\ue004\ue005 ph\ue008si\ue004\ue002\ue004gica\ue002 p\ue005\ue004c\ue000ss\ue000s i\ue003 si\ue003g\ue002\ue000 c\ue000\ue002\ue002s \ue004\ue005 s\ue006bc\ue000\ue002\ue002\ue006\ue002a\ue005 \ue004\ue005ga\ue003\ue000\ue002\ue002\ue000s.

F\ue004\ue005 a \ue002\ue004\ue003g tim\ue000, th\ue000 q\ue006a\ue002it\ue008 a\ue003d \ue006\ue003d\ue000\ue005\ue002\ue008i\ue003g \ue005\ue000s\ue004\ue002\ue006\u2011 ti\ue004\ue003 \ue004f imag\ue000s capt\ue006\ue005\ue000d \ue007ith t\ue005aditi\ue004\ue003a\ue002 mic\ue005\ue004sc\ue004p\ue000s \ue007as c\ue004\ue003sid\ue000\ue005\ue000d t\ue004 b\ue000 fix\ue000d b\ue008 ph\ue008sica\ue002 \ue002a\ue007 (that is, imag\ue000 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue007as diff\ue005acti\ue004\ue003\u2011\ue002imit\ue000d)8. This fi\ue003it\ue000 spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 a\ue005is\ue000s f\ue005\ue004m th\ue000 f\ue006\ue003dam\ue000\ue003ta\ue002 ph\ue008sics i\ue003v\ue004\ue002v\ue000d i\ue003 f\ue004c\ue006si\ue003g \ue002ight t\ue004 a disc\ue005\ue000t\ue000 sp\ue004t a\ue003d is d\ue000sc\ue005ib\ue000d b\ue008 Abb\ue000\u2019s th\ue000\ue004\ue005\ue008 \ue004f imag\ue000 f\ue004\ue005mati\ue004\ue003 (s\ue000\ue000 additi\ue004\ue003a\ue002 \ue000xp\ue002a\u2011 \ue003ati\ue004\ue003 i\ue003 FIG. 2a). I\ue003 th\ue000 Abb\ue000 sch\ue000m\ue000, \ue000ach imag\ue000 is c\ue004\ue003st\ue005\ue006ct\ue000d f\ue005\ue004m a\ue003 a\ue005\ue005a\ue008 \ue004f diff\ue005acti\ue004\ue003\u2011\ue002imit\ue000d sp\ue004ts. Th\ue000 b\ue000st \ue007a\ue008 t\ue004 \ue004ptimiz\ue000 spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 a\ue003d imag\ue000 c\ue004\ue003t\ue005ast \ue007as t\ue004 mi\ue003imiz\ue000 th\ue000 siz\ue000 a\ue003d/\ue004\ue005 \ue004ptimiz\ue000 th\ue000 disc\ue005\ue000t\ue000\ue003\ue000ss \ue004f th\ue000s\ue000 sp\ue004ts b\ue008 d\ue000c\ue005\ue000asi\ue003g th\ue000 \ue007av\ue000\ue002\ue000\ue003gth \ue004f \ue000xcitati\ue004\ue003 \ue002ight, i\ue003c\ue005\ue000asi\ue003g numerical aperture \ue004\ue005 b\ue008 i\ue003c\ue005\ue000asi\ue003g th\ue000 \ue005\ue000f\ue005activ\ue000 i\ue003d\ue000x \ue004f th\ue000 sp\ue000cim\ue000\ue003 m\ue000di\ue006m (f\ue004\ue005 \ue000xamp\ue002\ue000, b\ue008 \ue006si\ue003g a\ue003 \ue004i\ue002 imm\ue000\ue005si\ue004\ue003 \ue004b\ue001\ue000ctiv\ue000 \ue005ath\ue000\ue005 tha\ue003 a\ue003 ai\ue005 \ue004b\ue001\ue000ctiv\ue000). ev\ue000\ue003 \ue006\ue003d\ue000\ue005 id\ue000a\ue002 c\ue004\ue003diti\ue004\ue003s, \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 is sti\ue002\ue002 \ue002imit\ue000d t\ue004 \ue005\ue000\ue002ativ\ue000\ue002\ue008 m\ue004d\ue000st \ue002\ue000v\ue000\ue002s (that is, ~0.2\u00b5m \ue002at\ue000\ue005a\ue002\ue002\ue008). Axia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003, \ue004\ue005 a mic\ue005\ue004sc\ue004p\ue000\u2019s

Figure 1 | M\ue005\ue000\ue009\ue007\ue00a\ue000\ue007\ue008\ue005\ue000 \ue005ma\ue003\ue005n\ue003 \ue00b\ue002\ue000\ue004n\ue007\ue006\ue007\ue003\ue005\ue002\ue00a f\ue007\ue009 \ue001\ue009\ue00c\ue003 \ue001\ue005\ue00a\ue000\ue007\ue00d\ue002\ue009\ue00e. This illustrates some of the important issues
(shown in green) and developments in microscopic imaging relevant to early drug discovery. 2P\u2011IVM, two photon
intravital microscopy; 2P\u2011ME, two photon microendoscopy; ADMET, absorption, distribution, metabolism, excretion
and toxicology; confocal\u2011IVM, confocal intravital microscopy; confocal\u2011ME, confocal micro\u2011endoscopy; HCS, high\u2011
content screening; HTS, high\u2011throughput screening; SPIM, selective plan illumination microscopy; SSIM, saturated,
structured illumination microscopy; TIRF, total internal reflection fluorescence microscopy.

REVIEWS
nATure reVIewS|drug discovery
VoluMe 7| jAnuAry 2008|55
Numerical aperture

The numerical aperture i\ue000 a
mea\ue000ure of the light-gathering
capability of an objective len\ue000.

Moir\u00e9 fringe

The Moir\u00e9 effect i\ue000 a well-
known phenomenon that
occur\ue000 when repetitive
\ue000tructure\ue000 \ue000uch a\ue000 \ue000creen\ue000,
grid\ue000 or grating\ue000 are
\ue000uperpo\ue000ed or viewed
again\ue000t each other.

Multiphoton microscopy

Thi\ue000 i\ue000 al\ue000o called two-photon
excitation micro\ue000copy or
nonlinear optical micro\ue000copy.

\ue004ptica\ue002 s\ue000cti\ue004\ue003i\ue003g capabi\ue002it\ue008, is \ue000v\ue000\ue003 \ue007\ue004\ue005s\ue000 (~0.6\u00b5m) a\ue003d this mak\ue000s th\ue005\ue000\ue000\u2011dim\ue000\ue003si\ue004\ue003a\ue002 d\ue000t\ue000\ue005mi\ue003ati\ue004\ue003s diffi\u2011 c\ue006\ue002t. M\ue004\ue005\ue000\ue004v\ue000\ue005, \ue007h\ue000\ue003 d\ue000a\ue002i\ue003g \ue007ith high\ue002\ue008 c\ue004\ue003v\ue004\ue002\ue006t\ue000d \ue004b\ue001\ue000cts, s\ue006ch as c\ue000\ue002\ue002\ue006\ue002a\ue005 \ue004\ue005ga\ue003\ue000\ue002\ue002\ue000s, this p\ue004\ue004\ue005 axia\ue002 s\ue000cti\ue004\ue003i\ue003g capabi\ue002it\ue008 ca\ue003 a\ue002s\ue004 d\ue000g\ue005ad\ue000 c\ue004\ue003t\ue005ast i\ue003 th\ue000 imagi\ue003g p\ue002a\ue003\ue000. u\ue003f\ue004\ue005t\ue006\ue003at\ue000\ue002\ue008, th\ue000 \ue004v\ue000\ue005a\ue002\ue002 samp\ue002\ue000 c\ue004\ue003\u2011 t\ue005ast achi\ue000v\ue000d i\ue003 th\ue005\ue000\ue000\u2011dim\ue000\ue003si\ue004\ue003a\ue002 tiss\ue006\ue000s is g\ue000\ue003\ue000\ue005a\ue002\ue002\ue008 d\ue004mi\ue003at\ue000d b\ue008 this p\ue004\ue004\ue005\ue000\ue005 axia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003.

H\ue004\ue007\ue000v\ue000\ue005, it is imp\ue004\ue005ta\ue003t t\ue004 \ue003\ue004t\ue000 that th\ue000s\ue000 c\ue002assica\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue002imits a\ue005\ue000 bas\ue000d \ue004\ue003 s\ue000v\ue000\ue005a\ue002 \ue006\ue003d\ue000\ue005\ue002\ue008i\ue003g ass\ue006mpti\ue004\ue003s. I\ue003 pa\ue005tic\ue006\ue002a\ue005, th\ue000\ue008 ass\ue006m\ue000 a t\ue005aditi\ue004\ue003a\ue002 imagi\ue003g g\ue000\ue004m\ue000t\ue005\ue008 a\ue005\ue004\ue006\ue003d a si\ue003g\ue002\ue000 \ue004b\ue001\ue000ctiv\ue000 \ue002\ue000\ue003s; that \ue000xcitati\ue004\ue003 \ue002ight is \ue006\ue003if\ue004\ue005m\ue002\ue008 dist\ue005ib\ue006t\ue000d; a\ue003d, i\ue003 th\ue000 cas\ue000 \ue004f f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003c\ue000 imagi\ue003g, that this f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003c\ue000 a\ue005is\ue000s f\ue005\ue004m th\ue000 \ue002i\ue003\ue000a\ue005 abs\ue004\ue005pti\ue004\ue003 a\ue003d \ue000missi\ue004\ue003 \ue004f a si\ue003g\ue002\ue000 ph\ue004t\ue004\ue003. r\ue000c\ue000\ue003t\ue002\ue008, va\ue005i\ue004\ue006s g\ue005\ue004\ue006ps9,10 hav\ue000 sh\ue004\ue007\ue003 that it is p\ue004ssib\ue002\ue000 t\ue004 c\ue004\ue003st\ue005\ue006ct i\ue003st\ue005\ue006m\ue000\ue003ts that b\ue008pass \ue004\ue003\ue000 \ue004\ue005 m\ue004\ue005\ue000 \ue004f th\ue000s\ue000 \ue002imitati\ue004\ue003s b\ue008 \ue006si\ue003g m\ue004difi\ue000d \ue004b\ue001\ue000ctiv\ue000 \ue002\ue000\ue003s g\ue000\ue004m\ue000t\ue005i\ue000s that gath\ue000\ue005 m\ue004\ue005\ue000 \ue002ight \ue004v\ue000\ue005 a \ue002a\ue005g\ue000\ue005 \ue005a\ue003g\ue000 \ue004f a\ue003g\ue002\ue000s; f\ue004\ue005 i\ue003sta\ue003c\ue000, b\ue008 \ue006si\ue003g va\ue005i\ue004\ue006s \ue003\ue004\ue003\u2011\ue006\ue003if\ue004\ue005m \ue000xcitati\ue004\ue003 s\ue004\ue006\ue005c\ue000s (f\ue004\ue005 \ue000xamp\ue002\ue000, st\ue005\ue006c\u2011 t\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003) that ca\ue003 \ue000\ue003c\ue004d\ue000 high\ue000\ue005 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 i\ue003f\ue004\ue005mati\ue004\ue003; a\ue003d b\ue008 \ue006ti\ue002izi\ue003g \ue003\ue004\ue003\ue002i\ue003\ue000a\ue005 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003c\ue000 p\ue005\ue004c\ue000ss\ue000s. A\ue002\ue004\ue003\ue000, \ue004\ue005 i\ue003 c\ue004mbi\ue003ati\ue004\ue003, th\ue000s\ue000 i\ue003\ue003\ue004vati\ue004\ue003s hav\ue000 p\ue005\ue004d\ue006c\ue000d imag\ue000s that a\ue005\ue000 \ue003\ue004 \ue002\ue004\ue003g\ue000\ue005 \ue002imit\ue000d b\ue008 diff\u2011 \ue005activ\ue000 ph\ue000\ue003\ue004m\ue000\ue003a. I\ue003 s\ue004m\ue000 cas\ue000s, th\ue000 c\ue004mbi\ue003\ue000d \ue000ff\ue000ct \ue004f th\ue000s\ue000 i\ue003\ue003\ue004vati\ue004\ue003s has p\ue006sh\ue000d th\ue000 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue004f \ue002ight mic\ue005\ue004sc\ue004p\ue008 t\ue004 30 \ue003m a\ue003d b\ue000\ue002\ue004\ue00711. S\ue004m\ue000 \ue000xamp\ue002\ue000s \ue004f th\ue000 \ue007a\ue008s \ue006s\ue000d t\ue004 ci\ue005c\ue006mv\ue000\ue003t th\ue000s\ue000 ass\ue006mpti\ue004\ue003s a\ue005\ue000 sh\ue004\ue007\ue003 sch\ue000matica\ue002\ue002\ue008 i\ue003 FIG. 2b.

Ma\ue003\ue008 \ue004f th\ue000 m\ue000th\ue004ds that \ue006s\ue000 \ue003\ue004v\ue000\ue002 g\ue000\ue004m\ue000t\ue005i\ue000s (f\ue004\ue005 \ue000xamp\ue002\ue000, 4Pi, I5M)8,11 \ue006s\ue000 s\ue004m\ue000 c\ue004mbi\ue003ati\ue004\ue003 \ue004f t\ue007\ue004 \ue004b\ue001\ue000ctiv\ue000s t\ue004 \ue004ptimiz\ue000 \ue002ight gath\ue000\ue005i\ue003g; ti\ue002t\ue000d \ue004\ue005 \ue005\ue004tat\ue000d vi\ue000\ue007s t\ue004 i\ue002\ue002\ue006mi\ue003at\ue000 th\ue000 samp\ue002\ue000 i\ue003 diff\ue000\ue005\ue000\ue003t \ue007a\ue008s a\ue003d/\ue004\ue005 t\ue004 maximiz\ue000 \ue002ight capt\ue006\ue005\ue000 f\ue005\ue004m diff\ue000\ue005\ue000\ue003t pa\ue005ts \ue004f th\ue000 samp\ue002\ue00012; imag\ue000 i\ue003t\ue000\ue005f\ue000\ue005\ue000\ue003c\ue000 m\ue000th\ue004ds (at th\ue000 d\ue000t\ue000ct\ue004\ue005 a\ue003d/\ue004\ue005 samp\ue002\ue000) t\ue004 c\ue005\ue000at\ue000 additi\ue004\ue003a\ue002 c\ue004\ue003t\ue005ast; a\ue003d c\ue004h\ue000\ue005\ue000\ue003t \ue002ight t\ue004 f\ue006\ue005th\ue000\ue005 maximiz\ue000 \ue004ptica\ue002 c\ue004\ue003t\ue005ast (\ue004ft\ue000\ue003 d\ue006\ue000 t\ue004 th\ue000 c\ue004\ue003st\ue005\ue006ctiv\ue000 i\ue003t\ue000\ue005f\ue000\ue005\ue000\ue003c\ue000 \ue004f i\ue003\u2011phas\ue000 b\ue006t c\ue004\ue006\ue003t\ue000\ue005\u2011 p\ue005\ue004pagati\ue003g b\ue000ams, a\ue002s\ue004 k\ue003\ue004\ue007\ue003 as sta\ue003di\ue003g \ue007av\ue000 mic\ue005\ue004s\u2011 c\ue004p\ue008). I\ue003 s\ue004m\ue000 cas\ue000s, p\ue004i\ue003t sca\ue003\ue003i\ue003g is a\ue002s\ue004 \ue006s\ue000d t\ue004 add c\ue004\ue003f\ue004ca\ue002it\ue008 (that is, \ue005\ue000\ue001\ue000cti\ue004\ue003 \ue004f \ue004\ue006t\u2011\ue004f\u2011f\ue004c\ue006s ph\ue004t\ue004\ue003s). A\ue003\ue004th\ue000\ue005 g\ue000\ue004m\ue000t\ue005\ue008 that \ue006s\ue000s a diff\ue000\ue005\ue000\ue003t app\ue005\ue004ach is t\ue004ta\ue002 i\ue003t\ue000\ue005\ue003a\ue002 \ue005\ue000f\ue002\ue000cti\ue004\ue003 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003c\ue000 (TIrF) mic\ue005\ue004sc\ue004p\ue00813. TIrF i\ue002\ue002\ue006mi\ue003at\ue000s th\ue000 samp\ue002\ue000 \ue007ith a v\ue000\ue005\ue008 thi\ue003 \ue000va\ue003\ue000s\u2011 c\ue000\ue003t fi\ue000\ue002d that is g\ue000\ue003\ue000\ue005at\ue000d b\ue008 th\ue000 i\ue003t\ue000\ue005acti\ue004\ue003 \ue004f \ue004ff\u2011axis i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 \ue007ith th\ue000 s\ue006\ue005fac\ue000. This ki\ue003d \ue004f app\ue005\ue004ach is pa\ue005tic\ue006\ue002a\ue005\ue002\ue008 \ue006s\ue000f\ue006\ue002 f\ue004\ue005 imagi\ue003g c\ue002\ue004s\ue000 t\ue004 s\ue006\ue005fac\ue000s, b\ue000ca\ue006s\ue000 this \ue000va\ue003\ue000sc\ue000\ue003t fi\ue000\ue002d \ue004\ue003\ue002\ue008 \ue000xcit\ue000s f\ue002\ue006\ue004\ue005\ue004ph\ue004\ue005\ue000s \ue007ithi\ue003 100 \ue003m \ue004\ue005 \ue002\ue000ss f\ue005\ue004m th\ue000 s\ue006\ue005fac\ue000 a\ue003d th\ue000\ue005\ue000b\ue008 c\ue005\ue000at\ue000s a v\ue000\ue005\ue008 thi\ue003 \ue004ptica\ue002 s\ue000cti\ue004\ue003. I\ue003 g\ue000\ue003\ue000\ue005a\ue002, th\ue000 disadva\ue003tag\ue000 \ue004f m\ue000th\ue004ds that \ue006s\ue000 diff\ue000\ue005\ue000\ue003t g\ue000\ue004m\ue000t\ue005i\ue000s is that th\ue000 g\ue000\ue004m\ue000t\ue005i\ue000s th\ue000ms\ue000\ue002v\ue000s a\ue005\ue000 q\ue006it\ue000 c\ue004mp\ue002icat\ue000d a\ue003d ca\ue003 \ue004\ue003\ue002\ue008 b\ue000 app\ue002i\ue000d t\ue004 a s\ue006bs\ue000t \ue004f bi\ue004\ue002\ue004g\ue008.

M\ue000th\ue004ds that \ue006s\ue000 \ue003\ue004\ue003\u2011h\ue004m\ue004g\ue000\ue003\ue000\ue004\ue006s, \ue004\ue005 patt\ue000\ue005\ue003\ue000d, i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 imp\ue005\ue004v\ue000 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 b\ue008 \ue000xp\ue004si\ue003g \ue003\ue004\ue005\u2011 ma\ue002\ue002\ue008 i\ue003acc\ue000ssib\ue002\ue000 high\u2011\ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 i\ue003f\ue004\ue005mati\ue004\ue003 i\ue003 th\ue000 \ue004bs\ue000\ue005v\ue000d imag\ue000. Th\ue000s\ue000 i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 patt\ue000\ue005\ue003s \ue000\ue002icit

Moir\u00e9 fringe\ue000 i\ue003 th\ue000 \ue004bs\ue000\ue005v\ue000d imag\ue000. If th\ue000 \ue006\ue003d\ue000\ue005\ue002\ue008i\ue003g
i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 patt\ue000\ue005\ue003 i\ue003 \ue004\ue003\ue000 imag\ue000 is k\ue003\ue004\ue007\ue003, th\ue000\ue003 th\ue000
M\ue004i\ue005\u00e9 f\ue005i\ue003g\ue000s c\ue004\ue003tai\ue003 i\ue003f\ue004\ue005mati\ue004\ue003 ab\ue004\ue006t f\ue000at\ue006\ue005\ue000s i\ue003 th\ue000

\ue004th\ue000\ue005 (a\ue003d p\ue004t\ue000\ue003tia\ue002\ue002\ue008 \ue006\ue003k\ue003\ue004\ue007\ue003) imag\ue000. F\ue004\ue005 \ue000xamp\ue002\ue000, st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 s\ue008st\ue000ms capt\ue006\ue005\ue000 m\ue006\ue002tip\ue002\ue000 imag\ue000s (n\u2265 3), \ue000ach m\ue004d\ue006\ue002at\ue000d b\ue008 th\ue000 sam\ue000 si\ue003\ue006s\ue004ida\ue002 i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 patt\ue000\ue005\ue003, b\ue006t \ue007ith a shift\ue000d phas\ue000 \ue005\ue000\ue002ati\ue004\ue003. Th\ue000s\ue000 imag\ue000s c\ue004\ue003tai\ue003 additi\ue004\ue003a\ue002 f\ue005\ue000q\ue006\ue000\ue003c\ue008 c\ue004mp\ue004\ue003\ue000\ue003ts a\ue003d \ue007h\ue000\ue003 \ue005\ue000c\ue004\ue003st\ue005\ue006ct\ue000d as a \ue007\ue000ight\ue000d s\ue000t \ue004f pai\ue005\u2011\ue007is\ue000 diff\ue000\ue005\ue000\ue003c\ue000 imag\ue000s, th\ue000 \ue005\ue000s\ue006\ue002ti\ue003g imag\ue000 ca\ue003 p\ue004ss\ue000ss high\ue000\ue005 spatia\ue002 i\ue003f\ue004\ue005mati\ue004\ue003 \ue007ith \ue002\ue000ss \ue004\ue006t\u2011\ue004f\u2011f\ue004c\ue006s \ue002ight (that is, \ue003\ue004is\ue000 \ue004\ue005 b\ue002\ue006\ue005) tha\ue003 a c\ue004\ue003v\ue000\ue003ti\ue004\ue003a\ue002 \ue007id\ue000 fi\ue000\ue002d imag\ue000. I\ue003 fact, th\ue000 \ue005\ue000s\ue006\ue002ti\ue003g imag\ue000 q\ue006a\ue002it\ue008 a\ue003d \ue005\ue000s\ue004\u2011 \ue002\ue006ti\ue004\ue003 is t\ue008pica\ue002\ue002\ue008 c\ue004mpa\ue005ab\ue002\ue000 \ue007ith that \ue004f a c\ue004\ue003f\ue004ca\ue002 mic\ue005\ue004sc\ue004p\ue000. Adva\ue003tag\ue000s \ue004f this app\ue005\ue004ach i\ue003c\ue002\ue006d\ue000 th\ue000 \ue000as\ue000 \ue004f i\ue003t\ue000g\ue005ati\ue004\ue003 i\ue003t\ue004 \ue000xisti\ue003g mic\ue005\ue004sc\ue004p\ue000s a\ue003d th\ue000 fact that \ue003\ue004 \ue000missi\ue004\ue003 ph\ue004t\ue004\ue003s a\ue005\ue000 \ue007ast\ue000d. F\ue006\ue005th\ue000\ue005 adva\ue003c\ue000s i\ue003 st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 sch\ue000m\ue000s i\ue003c\ue002\ue006d\ue000 th\ue000 \ue005\ue000c\ue000\ue003t i\ue003t\ue005\ue004d\ue006cti\ue004\ue003 \ue004f adva\ue003c\ue000d axia\ue002 a\ue003d \ue002at\ue000\ue005a\ue002 patt\ue000\ue005\ue003s14, th\ue005\ue000\ue000\u2011dim\ue000\ue003si\ue004\ue003a\ue002 st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 patt\ue000\ue005\ue003s a\ue003d si\ue003g\ue002\ue000\u2011\ue000xp\ue004s\ue006\ue005\ue000 \ue004ptica\ue002 s\ue000cti\ue004\ue003i\ue003g \ue007ith c\ue004\ue002\ue004\ue006\ue005\u2011st\ue005\ue006ct\ue006\ue005\ue000d patt\ue000\ue005\ue003s15. T\ue005a\ue003si\ue002\ue002\ue006mi\ue003ati\ue004\ue00316 a\ue003d \ue005\ue000f\ue002\ue000cta\ue003c\ue00017 v\ue000\ue005si\ue004\ue003s \ue004f st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 hav\ue000 a\ue002s\ue004 b\ue000\ue000\ue003 i\ue003t\ue005\ue004d\ue006c\ue000d.

M\ue000th\ue004ds that \ue006s\ue000 \ue003\ue004\ue003\ue002i\ue003\ue000a\ue005 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003t p\ue005\ue004c\ue000ss\ue000s s\ue006ch as multiphoton micro\ue000copy \ue004\ue005 \ue005\ue000v\ue000\ue005sib\ue002\ue000 sat\ue006\ue005ati\ue004\ue003 mic\ue005\ue004sc\ue004p\ue008 a\ue005\ue000 c\ue004mm\ue004\ue003\ue002\ue008 \ue006s\ue000d as pa\ue005t \ue004f sca\ue003\ue003i\ue003g s\ue008st\ue000ms that g\ue000\ue003\ue000\ue005at\ue000 imp\ue005\ue004v\ue000d spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 b\ue008 c\ue005\ue000ati\ue003g sma\ue002\ue002 f\ue002\ue006\ue004\ue005\ue000sci\ue003g v\ue004\ue002\ue006m\ue000s. Th\ue000s\ue000 fi\ue003\ue000 v\ue004\ue002\ue006m\ue000s ca\ue003 b\ue000 g\ue000\ue003\ue000\ue005at\ue000d i\ue003 s\ue000v\ue000\ue005a\ue002 \ue007a\ue008s. F\ue004\ue005 \ue000xamp\ue002\ue000, m\ue006\ue002ti\u2011 ph\ue004t\ue004\ue003 \ue000xcitati\ue004\ue003 (BOX 1) is achi\ue000v\ue000d \ue007h\ue000\ue003 a f\ue002\ue006\ue004\ue005\ue004ph\ue004\ue005\ue000 is \ue000xcit\ue000d b\ue008 t\ue007\ue004 ph\ue004t\ue004\ue003s, \ue006s\ue006a\ue002\ue002\ue008 at t\ue007ic\ue000 th\ue000i\ue005 \ue003\ue004\ue005ma\ue002 \ue000xcitati\ue004\ue003 \ue007av\ue000\ue002\ue000\ue003gth. This p\ue005\ue004c\ue000ss is \ue004\ue003\ue002\ue008 p\ue004ssib\ue002\ue000 at th\ue000 c\ue000\ue003t\ue005\ue000 \ue004f a high\ue002\ue008 f\ue004c\ue006s\ue000d, f\ue000mt\ue004s\ue000c\ue004\ue003d \ue002as\ue000\ue005 p\ue006\ue002s\ue000, \ue007h\ue000\ue005\ue000 th\ue000 ph\ue004t\ue004\ue003 d\ue000\ue003sit\ue008 is \ue002a\ue005g\ue000st. This \ue005\ue000s\ue006\ue002ts i\ue003 a f\ue004ca\ue002 \ue000xcitati\ue004\ue003 v\ue004\ue002\ue006m\ue000 that is sma\ue002\ue002 a\ue003d \ue003\ue004t c\ue004mp\ue005\ue004mis\ue000d b\ue008 \ue002ight scatt\ue000\ue005\ue000d f\ue005\ue004m ab\ue004v\ue000 a\ue003d b\ue000\ue002\ue004\ue007 th\ue000 imag\ue000 p\ue002a\ue003\ue000. I\ue003 a simi\ue002a\ue005 \ue007a\ue008, stim\ue006\ue002at\ue000d \ue000missi\ue004\ue003 d\ue000p\ue002\ue000ti\ue004\ue003 (STeD) mic\ue005\ue004sc\ue004p\ue00818\u201320 c\ue005\ue000at\ue000s a sma\ue002\ue002 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003t v\ue004\ue002\u2011 \ue006m\ue000 b\ue008 \ue006si\ue003g t\ue007\ue004 p\ue006\ue002s\ue000d \ue002as\ue000\ue005s, \ue007ith st\ue005\ue006ct\ue006\ue005\ue000d b\ue000am g\ue000\ue004m\ue000t\ue005i\ue000s, that i\ue003t\ue000\ue005act i\ue003 a s\ue006bt\ue005activ\ue000 \ue007a\ue008 t\ue004 c\ue005aft a sma\ue002\ue002 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003c\ue000 v\ue004\ue002\ue006m\ue000.

Sat\ue006\ue005at\ue000d st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 (SSIM) mic\ue005\ue004s\u2011 c\ue004p\ue0089 is a f\ue006\ue005th\ue000\ue005 m\ue000th\ue004d that tak\ue000s adva\ue003tag\ue000 \ue004f b\ue004th patt\ue000\ue005\ue003\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 a\ue003d \ue003\ue004\ue003\ue002i\ue003\ue000a\ue005 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003t p\ue005\ue004\u2011 c\ue000ss\ue000s. I\ue003 a\ue003 \ue000xt\ue000\ue003si\ue004\ue003 t\ue004 th\ue000 \ue005\ue000g\ue006\ue002a\ue005 st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\u2011 \ue003ati\ue004\ue003 app\ue005\ue004ach, SSIM i\ue003t\ue005\ue004d\ue006c\ue000s additi\ue004\ue003a\ue002 \ue003\ue004\ue003\ue002i\ue003\ue000a\ue005 f\ue005\ue000q\ue006\ue000\ue003ci\ue000s i\ue003t\ue004 a st\ue005\ue006ct\ue006\ue005\ue000d i\ue002\ue002\ue006mi\ue003ati\ue004\ue003 patt\ue000\ue005\ue003 b\ue008 \ue006si\ue003g p\ue000ak \ue000xcitati\ue004\ue003 i\ue003t\ue000\ue003siti\ue000s ab\ue004v\ue000 th\ue000 sat\ue006\ue005ati\ue004\ue003 th\ue005\ue000sh\ue004\ue002d \ue004f th\ue000 f\ue002\ue006\ue004\ue005\ue004ph\ue004\ue005\ue000 b\ue000i\ue003g \ue006s\ue000d. Th\ue000 adva\ue003tag\ue000 \ue004f this app\ue005\ue004ach is that it ca\ue003 b\ue000 achi\ue000v\ue000d \ue007ith a simp\ue002\ue000 \ue007id\ue000\u2011fi\ue000\ue002d mic\ue005\ue004sc\ue004p\ue000 a\ue003d \ue003\ue004 b\ue000am sca\ue003\ue003i\ue003g is \ue005\ue000q\ue006i\ue005\ue000d. Th\ue000 disadva\ue003tag\ue000 is that it is \ue002imit\ue000d b\ue008 ph\ue004t\ue004stabi\ue002it\ue008 a\ue003d sig\ue003a\ue002\u2011t\ue004\u2011\ue003\ue004is\ue000 iss\ue006\ue000s.

T\ue007\ue004 \ue003\ue000\ue007\ue002\ue008 disc\ue002\ue004s\ue000d m\ue000th\ue004ds hav\ue000 p\ue006sh\ue000d th\ue000 \ue002imits \ue004f spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 \ue000v\ue000\ue003 \ue002\ue004\ue007\ue000\ue005. I\ue003 \ue004\ue003\ue000 \ue000xamp\ue002\ue000, ca\ue002\ue002\ue000d ph\ue004t\ue004\u2011activat\ue000d \ue002\ue004ca\ue002izati\ue004\ue003 mic\ue005\ue004sc\ue004p\ue008 (PAlM), B\ue000tzig a\ue003d c\ue004\ue002\ue002\ue000ag\ue006\ue000s21 \ue007\ue000\ue005\ue000 ab\ue002\ue000 t\ue004 achi\ue000v\ue000 \ue000v\ue000\ue003 high\ue000\ue005 \ue002\ue000v\ue000\ue002s \ue004f spatia\ue002 \ue005\ue000s\ue004\ue002\ue006ti\ue004\ue003 (~2\u201325 \ue003m). This m\ue000th\ue004d \ue006s\ue000s mi\ue003i\u2011 ma\ue002 stim\ue006\ue002ati\ue004\ue003 \ue004f spa\ue005s\ue000\ue002\ue008 p\ue004p\ue006\ue002at\ue000d ph\ue004t\ue004activatab\ue002\ue000 f\ue002\ue006\ue004\ue005\ue000sc\ue000\ue003t p\ue005\ue004t\ue000i\ue003 (PAFP) m\ue004\ue002\ue000c\ue006\ue002\ue000s22 (BOX 1), \ue005\ue000p\ue000at\ue000d ma\ue003\ue008 tim\ue000s, t\ue004 i\ue003c\ue005\ue000m\ue000\ue003ta\ue002\ue002\ue008 samp\ue002\ue000 a\ue002\ue002 th\ue000 f\ue002\ue006\ue004\ue005\ue004\u2011 ph\ue004\ue005\ue000s i\ue003 a th\ue005\ue000\ue000\u2011dim\ue000\ue003si\ue004\ue003a\ue002 v\ue004\ue002\ue006m\ue000 i\ue003 is\ue004\ue002ati\ue004\ue003. B\ue008 s\ue002\ue004\ue007\ue002\ue008 mappi\ue003g ma\ue003\ue008 th\ue004\ue006sa\ue003ds \ue004f spa\ue005s\ue000\ue002\ue008 p\ue004p\ue006\ue002at\ue000d

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