Imaging modes of atomic force microscopy for application in molecular and cell biology

TL;DR: The basic principles, advantages and limitations of the most common AFM bioimaging modes are reviewed, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging.

Abstract: Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Summary

Introduction

• The emergence of atomic force microscopy (AFM) 30 years ago 1 in the then fledgling field of nanotechnology 2 has opened new avenues in physics, chemistry, biology, and medicine, and since then has continuously inspired researchers all over the world, as testified by more than 340,000 scientific articles in peer reviewed journals (web of science).
• The key invention was to contour nonconductive surfaces much below the diffraction limit of light by controlling a conglomerate of forces acting between a tiny probe and the object.
• This unique flexibility of AFM to image, probe and manipulate materials made it the most versatile toolkit in nanoscience and --technology, changed their perception of hard and soft matter and stimulated revolutionary discoveries and technologies 2 .
• Major advances in high--resolution imaging have also been achieved in complementary methods including super resolution microscopy and cryo--electron microscopy, which significantly enrich the imaging toolbox now available to molecular and cell biology (Table 1 ).

Imaging native biological systems in liquid

• The key breakthrough that led to biological AFM was the development of an optical detection system, followed by the design of a fluid chamber, enabling imaging in buffer solution and thus maintaining the native state of the biological system 4, 5 .
• In close proximity to the sample surface, the interactions between tip and sample change both the cantilever amplitude and resonance frequency allowing them to be used as feedback parameters for contouring fragile biological samples 34, 35, 36 .
• Applied to cellular systems contact and dynamic mode AFM reveal topographs below the resolution limit of conventional light microscopy.
• An elegant approach for imaging living cells and circumventing tip contamination problems is scanning ion conductance microscopy (SICM), which scans a nanopipette over the sample while measuring the ion current 47, 48, 49 .
• The force applied to the AFM tip can simply be adjusted for mechanical manipulation, and the tip can be functionalized with chemical groups to manipulate specific sample regions.

From force--distance curves to multiparametric imaging

• The question came up whether AFM can do more than just contouring a surface.
• Approach FD curves can quantify the height, surface forces, mechanical deformation of the sample, or derive its elastic modulus and energy dissipation.
• FD--based AFM thus opens the door to image complex biological systems and to simultaneously quantify and map their intrinsic physical properties, including elasticity and adhesion (Fig. 3d--e ).
• Y,z), it is often a challenge to determine the exact contact point between tip and sample (zero separation), particularly when long--range surface forces, surface roughness and deformation of the soft biological sample play roles.
• FD--based AFM also mapped the mechanical properties of heterogeneous lipid membranes 76 and correlated mechanical properties of human keratinocytes 77 and bacteria 78, 79 to their morphology and state.

Molecular recognition imaging

• This approach requires tip--sample interactions to be known, which is facilitated by functionalizing AFM tips with specific chemical groups or ligands 88, 89 .
• Using functionalized probes, FD--based AFM could detect and localize specific interactions of biological systems ranging from antibodies to living human cells 8, 63, 88, 89, 90, 93, 95 .
• Applied to live bacteria and yeast, the main components of microbial cell walls have been localized and force probed, including peptidoglycans 46 , teichoic acids 100 , and cell adhesion proteins 83, 101 .
• Therefore, before engaging functionalized tips, it is useful to characterize the sample with unmodified tips.
• This method was used to map the binding sites of cadherins on vascular endothelial cells 105 .

Multifrequency imaging

• Besides topographic imaging AFM can map mechanical and functional properties of the biological sample.
• Recently developed multifrequency AFM modes 37, 106 , which promise exciting possibilities to study biological systems are therefore discussed.
• Bimodal AFM has also been used to image ferritin while separating short--range mechanical (≈0.5 nm) from long--range magnetic (≈5-1,000 nm) forces.
• Initially, this AFM imaging mode has been applied to measure topography and viscoelastic properties of relatively large biological objects including viruses and cells (Fig. 4e ) 111, 112 .
• Torsional harmonics allow the topograph of the sample and the time--varying forces to be recorded by integrating the higher harmonics of the torsional movement.

High--speed imaging: imaging biological processes in real time

• Compared to fluorescence microscopy, AFM imaging is limited by its rather slow time resolution.
• Therefore, to achieve high--speed using amplitude modulation AFM, the cantilever's response time τ = Q/(πf0) has to be shortened, with Q being the quality factor and f0 the first resonance frequency of the cantilever in water (Fig. 5a ).
• This rotation is made possible by rotary propagation of three chemical states (empty, ATP--bound and ADP--bound states) and hence corresponding structural states over the β subunits.
• How nucleoporins form a selective barrier facilitating this transport has been unclear.
• The tip--scan HS--AFM developed very recently will thus significantly expand the applicability to study biological processes by AFM 133 .

Correlative imaging

• Living cells present a high level of structural and functional complexity.
• In such cases the full potential of AFM is achieved in combination with complementary microscopy techniques that can identify and correlate complex cellular structures of interest 9 .
• Exciting applications range from single animal cells, to tissues microbial cells, and to their assemblies.
• Such experiments allowed the furrow stiffening during cell division 65 to be observed, the adhesion of Dictyostelium discoideum to their substrate to be measured to molecular scale 138 , or to unravel whether cell adhesion or cortex tension determine cell sorting in the developing embryo 139 .
• Recent examples include the localization of receptors on CHO cells and endothelial cells 144 , and the visualization of the peptidoglycan insertion into the cell wall of L. lactis 46 while mapping the distribution of single peptidoglycan molecules on the outermost cell surface using the AFM.

Conclusions

• This year the authors are celebrating the 30 th birthday of AFM, which undoubtedly has revolutionized nanotechnology and now shows a considerable impact in life sciences.
• In the past years many new AFM--imaging modalities have been introduced, which in principle can be readily applied to biological systems and thus will further extend the variety of information that can be quantified and structurally mapped while imaging complex biological systems.
• It may be thus expected, that recently introduced ultrastable AFMs providing sub--pN force precision and high positional stability (< 0.03 Å) at extremely low lateral drift (≈ 5 pm min -1 ) 147, 148 , will guide the development of AFMs for new applications of biological significance.
• Biological systems are rather complex and require the acquisition of a wealth of information to be understood.

Full text

Delft University of Technology
Imaging modes of atomic force microscopy for application in molecular and cell biology
Dufrêne, Yves F.; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas;
Gerber, Christoph; Müller, Daniel J.
DOI
10.1038/nnano.2017.45
Publication date
2017
Document Version
Accepted author manuscript
Published in
Nature Nanotechnology
Citation (APA)
Dufrêne, Y. F., Ando, T., Garcia, R., Alsteens, D., Martinez-Martin, D., Engel, A., Gerber, C., & Müller, D. J.
(2017). Imaging modes of atomic force microscopy for application in molecular and cell biology.
Nature
Nanotechnology
,
12
(4), 295-307. https://doi.org/10.1038/nnano.2017.45
Important note
To cite this publication, please use the final published version (if applicable).
Please check the document version above.
Copyright
Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent
of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons.
Takedown policy
Please contact us and provide details if you believe this document breaches copyrights.
We will remove access to the work immediately and investigate your claim.
This work is downloaded from Delft University of Technology.
For technical reasons the number of authors shown on this cover page is limited to a maximum of 10.

! !
Review&article&for&Nature&Nanotechnology&
!
"#$%&'!($)'*!+&')$,'$-.!/%01&21!+$304&#&*,!
&2!+$4*'540)!023!6*44!7&$4$1.!
!
!"#$%&'%()*+,-#
.
/0%12$342%5-62
7
0%849:+62%;:+94:
<
0%(:"46%5=$>##-$
.
0%(:" 46%?:+>4-#@A?:+>4-
B
0%
5-6+#:$%C-D#=
E
0%F3+4$>2G3%;#+H#+
I
%:-6%(:-4#=%J'%?K==#+
B
/%
%
.
L-$>4>)>#% 2*% M4*#% N94#-9#$% :-6% O:==22-% CP9#==#-9#% 4-% M4*#% $94#-9#$% :-6% Q42>#93-2=2DR%
SOCMQLTU0%V-4"#+$4>W%9:>32=4X)#%6#%M2)":4-0%F+24P%6)%N)6%BAE0%H>#%MY'ZY'ZI'0%QA.<B[%M2)":4-A
=:A\#)"#0%Q#=D4)]'%
7
(#G:+>]#->%2*%^3R$49$0%_:-:@:`:%V-4"#+$4>R0%_:-:@:`:%a7ZA..a70%J:G:-'%
<
L-$>4>)>2%6#%F4#-94:%6#%?:>#+4:=#$%6#%?:6+460%FNLF0%N2+%J):-:%L-W$%6#%=:%F+)@%<0%7[ZBa%
?:6+460%NG:4-'!
B
(#G:+>]#->%2*%Q42$R$>#]$%N94#-9#%:-6%C-D4-##+4-D0%C46D#-b$$4$93#%1#93-4$93#%c293$93)=#%
SC1cU%dK+4930%?:>>#-$>+:$$#%7[0%BZEI%Q:$#=0%N`4>@#+=:-6'!
E
(#G:+>]#->%2*%Q42\:-2$94#-9#0%(#=*>%V-4"#+$4>R%2*%1#93-2=2DR0%e:-%6#+%O::=$`#D%[0%7I7[%
Fc%(#=*>0%13#%\#>3#+=:-6$'%
I
N`4$$%\:-2$94#-9#%L-$>4>)>#0%V-4"#+$4>R%2*%Q:$#=0%_=4-D#=H#+D$>+:$$#%[Z0%BZEY%Q:$#=0%
N`4>@#+=:-6'!
/#A]:4=f%R"#$'6)*+#-#g)9=2)":4-'H#h%6:-4#='])#==#+gH$$#'#>3@'93%
!

7%
!
!
"8,#)0'#!
O4>34-% >3+##% 6#9:6#$0% :>2]49% *2+9#% ]49+2$92GR % S5&?U% 3:$% H#92]#% :% G2`#+*)=%
])=>4*)-9>42-:=%4]:D4-D%G=:>*2+]0%#-:H=4-D%>3#%"4 $):=4@:>42-%:-6%]:-4G)=:>42-%2*%H42=2D49:=%
$:]G=#$0%*+2]% $4-D=#%]2=#9)=#$%>2%=4"4-D%9#==$'%N22-%:*>#+%4>$%4-"#->42-0%4>%`:$%+#92D-4@#6%>3:>%
>3#% *)==% G2>#->4:=% 2*% 5&?% 4]:D4-D% 4-% H42=2DR% +#X)4+#$% i#R% >#93-2=2D49:=% 6#"#=2G]#->$% 4-%
2+6#+%>2%$2="#%:%-)]H#+%2*%=4]4>:>42-$%:-6%6+:`H:9i$'% 134$%-##6%3:$%H##-%:%i#R%6+4"4-D%*2+9#%
>2`:+6$%6#"#=2G4-D%-#`%4]:D4-D%]26:=4>4#$0%G)$34-D%92->4-)2)$=R%>3#%*4#=6%:3#:6'%c#+#0%`#%
$)+"#R%>3#%H:$49%G+4-94G=#$0%:6":->:D#$%:-6%=4]4>:>42-$%2 *%>3#%]2$>%92]]2-%5&?%H424]:D4-D%
]26:=4>4#$%:":4=:H=#%>26:R0%$>:+>4-D%*+2]%>3#%G2G)=:+%92->:9>%:-6%6R-:]49%]26#$0%>2%-#`=R%
6#"#=2G#6% ]26#$0% 4-9=)64-D% ])=>4G:+:]#>+490% ]2=#9)=:+% +#92D-4>42-0% ])=>4*+#X)#-9R% :-6%
34D3A$G##6% 4]:D4-D'%O#% 64$9)$$%+#9#->% #P:]G=#$% >3:>%34D3=4D 3>% >3#% )-4X)#% 9:G:H4=4>4#$% 2*%
>3#$#%#]#+D4-D%-#`%]26:=4>4#$'%O#%:->494G:>#%>3:>0%4-%>3#%-#P>%6#9:6#0%>3#$#%-:-2>22=$%`4==%
3:"#% :% G+2*2)-6% 4-*=)#-9#% 2-% >3#% `:R% +#$#:+93#+$% =22i% :>% H42=2D49:=% $R$>#]$0% >3#+#HR%
3#=G4-D% >3#]% >2% $2="#% *)-6:]#->:=% X)#$>42-$% >3:>% 92)=6% -2>% 3:"#% H##-% :66+#$$#6% `4>3%
>+:64>42-:=%>#93-4X)#$'%

<%
!
!
/2#)$35'#&$2!
13#%#]#+D#-9#%2*%:>2]49%*2+9#%]49+2$92GR%S5&?U%<Z%R#:+$%:D2
.
%4-%>3#%>3#-%*=#6D=4-D%*4#=6%2*%
-:-2>#93-2=2DR
7
%3:$%2G#-#6%-#`%:"#-)#$%4-%G3R$49$0%93#]4$>+R0%H42=2DR0%:-6%]#6494-#0%:-6%
$4-9#%>3#-%3:$%92->4-)2)$=R%4-$G4+#6%+#$#:+93#+$%:==%2"#+%>3#%`2+=60%:$%>#$>4*4#6%HR%]2+#%>3:-%
<BZ0ZZZ%$94#->4*49%:+>49=#$%4-%G##+%+#"4#`#6%j2)+-:=$%S`#H%2*%$94#-9#U'%13#%i#R%4-"#->42-%`:$%
>2%92->2)+%-2-92-6)9>4"#%$)+*:9#$%])93%H#=2`%>3#%64 **+:9>42-%=4]4 >%2*%=4 D3>% HR%92->+2==4-D%:%
92-D=2]#+:>#%2*%*2+9#$%:9>4-D%H#>`##-%:%>4-R%G+2H#%:-6%>3#%2Hj#9>'%O3#+#:$%*4+$>%+#$)=>$%2-%
>3#% :>2]49% $9:=#% `#+#% 2H>:4-#6% `4>34-% :% R#:+
<
0% 4>% > 22i% :-2>3#+% *#`% R#:+$% >2% :992]G=4$3%
:>2]49%4]:D4-D%2*%-2- 92-6)9>4"#%$)+*:9#$%4- %":9))]'%?#:-`34=#0% >3#%>#93-4X)#%$ >:+>#6%>2%
H#%:6:G>#6%>2%`2+i%2"#+%:%" :$>%>#]G#+:>)+#%$9:=#%:-6%H:$49:==R%4-%#"#+R%#-"4+2-]#->
70B0E0I
'%
13#% :H4=4>R% >2% 4-"#$>4D:>#% $)+*:9#$% `4>3% #P9#G>42-:=%$4 D-:=A>2A-24$#% +:>42% :>%$)HA-:-2]#>#+%
+#$2=)>42-%>+4DD#+#6%:%`#:=>3%2*%5&?A+#=:>#6%>#93-4X)#$%)$4-D%:%":+4#>R%2*%G+2H#$%>2%=29:==R%
$#-$#% 4->#+:9>42-$% :-6% ]:-4G)=:>#% ]:>>#+% *+2]% >3#% :>2]49% >2% ] 49+2$92G49% $9:=#
70Y
'% 134$%
)-4X)#% *=#P4H4=4>R% 2*% 5&?% >2% 4]:D#0% G+2H#% :-6% ]:-4G)=:>#% ]:>#+4:=$% ]:6#% 4>% >3#% ]2$>%
"#+$:>4=#%>22=i4>%4-%-:-2$94#-9#%:-6%A>#93-2=2DR0% 93:-D#6% 2)+%G#+9#G>42-%2*%3:+6% :-6% $2*>%
]:>>#+% :-6% $>4])=:>#6% +#"2=)>42-:+R% 64$92"#+4#$% :-6% >#93-2=2D4#$
7
'% 13#% G2$$4H4=4>R% >2%
2G#+:>#%4-%*=)4649%#-"4+2-]#->$%:-6%:>%:]H4#->%>#]G#+:>)+#%]2"#6%5&?%>2`:+6$%H42=2DR0%
2G#-4-D% >3#% 622+% >2% 4]:D#% :-6% G+2H#% ]2=#9)=#$% :-6% 9#==$% :>% S$)HAU-:-2]#>#+%
+#$2=)>42-
B0E0I0[0a
'%12%:66+#$$%>3#%`46#%92]G=#P4>R%2*%H42=2D49:=%$R$>#]$0%+:-D4-D%*+2]%=4G46$0%
-)9=#49%:946$0%G+2>#4-$0%:$$#]H=4#$%>3#+#2*0%>2%9#==$%:-6%>4$$)#$0%:%`#:= >3%2*%5&?%]26:=4>4#$%
3:"#%H##-%6#"#=2G#6%2"#+%>3#%R#:+$%S(&19!:U'%?:j2+%:6":-9#$%4-%34D3A+#$2=)>42-%4]:D4-D%3:"#%
:=$2%H##-%:934#"#6%4-%92]G=#]#->:+R%]#>326$%4-9=)64-D%$)G#+%+#$2=)>42-%]49+2$92GR%:-6%
9+R2A#=#9>+2-% ]49+2$92GR0% `3493% $4D-4*49:->=R% #-+493%>3#% 4]:D4-D%>22=H2P% -2`% :":4=:H=#% >2%
]2=#9)=:+%:-6%9#==%H42=2DR%S;084*!:U'%%
?:-R%+#"4#`$%3:"#%H##-%G)H=4$3#6%4-% >3#% G:$>%>`2% 6#9:6#$%>3:>%6#$9+4H#%>3#%)$#%2*%9#+>:4-%
5&?% 4]:D4-D% ]26:=4>4#$% >2% 93:+:9>#+4@#% H42=2D49:=% $R$>#]$'% c2`#"#+0% :$% 4>% 4$% 64**49)=>% *2+%
-#`92]#+$%:-6%2*>#-%#"#-%*2+%:6":-9#6%)$#+$%>2%2"#+"4#`%>3#%G+4-94G=#$%2*%>3#$#%X)49i=R%
6#"#=2G4-D% :-6% 64"#+$#% 4]:D4-D%]26:=4>4#$% :-6% >2% #" := ):>#% >3#4+% :GG=4 9:H4=4>R0% :6":->:D#$%
:-6%=4]4>:>42-$0%`#%3#+#%$)+"#R%>3#%]2$>%$4D-4*49:->%$>#G$%>3:>%3:"#%=#6%>2%#$>:H=4$3%5&?%:$%:%
G2`#+*)=%>22=H2P%4-%]2=#9)=:+%:-6%9#==%H42=2DR'%O#%2)>=4-#%*2+%#:93%5&?%4]:D4-D%]26:=4>R%
>2%`3493%i4-6%2*%H4 2=2D4 9:=% $R$>#]$%4>%9:-%H#%G+#*#+:H=R%:GG=4#60%>3#4+%9)++#->%=4]4>:>42-$%:-6%
*)>)+#%G#+$G#9>4"#$'%%
!
"!<$5)2*.!&2#$!"(+!&%01&21!#*'=2&>5*,!
/%01&21!20#&?*!8&$4$1&'04!,.,#*%,!&2!4&>5&3!
13#% i#R% H+#:i>3+2)D3% >3:>% =#6% >2% H42=2D49:=% 5&?% `:$% >3#% 6#"#=2G]#->% 2*% :-% 2G>49:=%
6#>#9>42-% $R$>#]0% *2==2`#6% HR% >3#% 6#$4D-% 2*% :% *=)46% 93:]H#+0% #-:H=4-D% 4]:D4-D% 4-% H)**#+%
$2=)>42-% :-6% >3)$% ]:4->:4-4-D% >3#% -:>4"#% $>:>#% 2*% >3#% H42=2D49:=% $R$>#]
B0E
'% 13#% *4+$>% 5&?%
4]:D4-D% ]26#% 4-"#->#60%contact&mode0%+:$>#+%$9:-$%:%>4G%2"#+%>3#%$:]G=#%:-6%:6j)$>$%G4P#=A
HRAG4P#=% >3#% 3#4D3>% 2*% >3#% >4G% $2% >3:>% >3#% *2+9#% :GG=4#6% >2% >3#% $:]G=#% 4 $% i#G>% 92-$>:->%
S(&19!@0U'%13#%+#$)=>4-D%3#4D3>%4]:D#%+#$#]H=#$%>3#% $:]G=#%>2G2D+:G3R%`4>3%>3#%+#$2=)>42-%
6#G#-64-D%2-%>3#%+:64)$%2*%>3#%>4G0%>3#%$:]G=#%92++)D:>42-0%>3#%G3R$49:=%G+2G#+>4#$%2*%>3#%
$:]G=#0% :-6% 32`% G+#94$#=R% >3#% *##6H:9i% $R$>#]% 92->2)+$% >3#% >4G% 2"#+% >3#% $2*>% H42=2D49:=%

B%
!
!
$:]G=#'%%
N32+>=R%:*>#+%4->+26) 94-D%>3#%*4+$>%92]]#+94:==R%:":4=:H=#%5&?0%H42=2D49:=%$G#94]#-$%4]:D#6%
4-9=)6#6% :-4]:=% 9#==$
.Z0..
0% 9#==% ]#]H+:-#%G:>93#$% :-6%]#]H+:-#%G+2>#4-$
.70.<0.B
0% (\5% :-6%
8\5
.E
0% :$% `#==% :$% =4G46% *4=]$
.I0.Y
'% &2+% *=:>0% $]22>3=R% 92++)D:>#6% $)+*:9#$% $)93% :$% G+2>#4-$%
G+2>+)64-D% k.%-]% *+2]% ]#]H+:-#$% 92->:9>% ]26#% 5&?% 9:-% G+2"46#% >2G2D+:G3$% 2*% $4-D=#%
]#]H+:-#% G+2>#4-$% :>% =:>#+:=% :-6% "#+>49:=% +#$2=)>42-% 2*% l.%-]% :-6% lZ'.%-]0% +#$G#9>4"#=R%
S(&19!@8U
.B0.[0.a
'%134$%#P9#G>42-:==R%34D3%+#$2=)>42-%:-6%$4D-:=A>2A-24$#%+:>42%2*%5&?%:==2`#6%*2+%
#P:]G=#%>2%)-+:"#=%>3#%*)-9>42-:==R%+#=#":->%2=4D2]#+49%$>:>#%2*%":+42)$%`:>#+A$2=)H=#%:-6%
]#]H+:-#%G+2>#4-$
7Z07.07707<07B
'%TG#+:>#6%4-%>3#%>4]#A=:G$#%92->:9>%]26#%5&?%"4$):=4@#6%>3#%
]2+G32=2D49:=% 6R-:]49$% 2*% 9#==$
.Z0..
0% >3#% D+2`>3% 2*% G:>32=2D49:=% :]R=246% *4H+4=$
7E
0% >3#%
#-@R]:>49%6#D+:6:>42-%2*%(\5
7I
%2+%=4G46%]#]H+:-#$
7Y
0%:-6%G+2"46#6%4-$4D3>%4->2%>3#%`2+i4-D%
G+4-94G=#$% 2*% H:9> #+4:=% 2)>#+% ]#]H+:-#% G2+#$
7[
0% D:G% j)-9>42-$% #-:H=4-D% 4->#+9#==)=:+%
92--#9>42-$% H#>`##-% 9#+>:4-% :-4]:=% 9#==$
7a
% :-6% -)9=#:+% G2+#% 92]G=#P#$
<Z
'% T>3#+% #P94>4-D%
#P:]G=#$% ]2-4>2+#6% >3#% 4-$#+>42-% 2*% G:>32=2D49:=% >2P4-$% 4->2% ]#]H+:-#$
<.
% :-6% >3#%
$)G+:]2=#9)=:+%:+934>#9>)+#%2*%G32>2$R->3#>49%]#]H+:-#$%93:-D4-D%4-%+#$G2-$#%>2%=4D3>
<7
'%
N)93% 4-$4D3>% :==2`#6% $>:>49% $>+)9>)+:=% ]26#=$% >2% H#% 92]G=#]#->#6% `4>3% *)-9>42-:=%
6R-:]49$
<<
'%
5=>32)D3%92->:9>%]26#%5&?%4$%`46#=R%)$#6%>2%93:+:9>#+4@#%$2=46%$)H$>+:>#$0%4>$%:GG=49:>42-%>2%
$2*>%H42=2D49:=%$R$>#]$%+#X)4+#$%#PG#+>%$i4==$%>2%:6j)$>%>3#%*2+9#%:GG=4#6%>2%>3#%>4G'%5$%:%+)=#%2*%
>3)]H0%*2+9#$%m.ZZ%G\%$32)=6%H#%:"246#6%:$%>3#R%9:-%9:)$#%+#"#+$4H=#%2+%#"#-%4++#"#+$4H=#%
6#*2+]:>42-$
<<
'%Dynamic&mode&imaging%S2+4D4-:==R%>#+]#6%>:GG4-D%2+%2$94==:>42-%]26#U%`:$%
4-"#->#6%>2%]4-4]4@#%>3#%*+49>42-%:-6%>3#%* 2+9#%:GG=4#6%H#>`##-%>4G%:-6%$:]G=#%S(&19!@0A'U'%L-%
4>$% $4]G=#$>%:GG=49:>42-0% >3#%9:->4=#"#+%4$% 2$94==:>#6%9=2$#%>2%+#$2-:-9#%`34=#%$9:--4-D%:9+2$$%
:% $:]G=#
.
'% L6#:==R% >3#% >4G% 2-=R% >2)93#$% >3#% $:]G=#% :>% >3#% "#+R% #-6% 2*% 4>$% 62`-`:+6%
]2"#]#->%>3)$%92-$46#+:H=R%]4-4]4@4-D%*+49>42-'%L-%9=2$#%G+2P4]4>R%>2%>3#%$:]G=#% $)+*:9#0%
>3#% 4->#+:9>42-$% H#>`##-% >4G% :-6% $:]G=#% 93:-D#% H2>3% >3#% 9:->4=#"#+% :]G=4>)6#% :-6%
+#$2-:-9#% *+#X)#-9R% :==2`4-D% >3#]% >2% H#% )$#6% :$% *##6H:9i% G:+:]#>#+$% *2+% 92->2)+4-D%
*+:D4=#% H42=2D49: =% $:]G=#$
<B0<E0<I
'% V$4-D% >3#% :]G=4>)6#% :$% *##6H:9i% 4$% >#93-49:==R% $4]G=#+%
H#9:)$#% 4>% +#X)4+#$% 2-=R% 2-#% *##6H:9i% =22G% 92]G:+#6% >2% )$4-D% *+#X)#-9R% :$% *##6H:9i%
+#X)4+4-D%>3+##%$)93%=22G$'%13)$0%:]G=4>)6#%]26)=:>42-%5&?%4$%9)++#->=R%]2+#%2*>#-%:GG=4#6%
>3:-%*+#X)#-9R%]26)=:>42-%5&?'%Q#$46#$%>3#$#%>`2%`#== Ai-2`-%5&?%4]:D4-D%]26#$0%2>3#+%
6R-:]49% ]26#$% 3:"#% H##-% 6#"#=2G#6% `3493% #]G=2R% 64**#+#->% $4D-:=$% :$% *##6H:9i%
G:+:]#>#+$% 2+% #P94>#% >3#% 9:->4=#"#+% :>% 64**#+#->% *+#X)#-94#$% $4])=>:-#2)$=R% S$##% $#9>42-%
?)=>4*+#X)#-9R% L ]:D4-DU
<Y
'% L]G2+>:->=R0% :$% 6R-:]49% ]26#$% 92-$46#+:H=R% +#6)9#% *2+9#% :-6%
*+49>42-%H#>`##-%>4G%:-6%$:]G=#0%>3#R%9:-%H#%:GG=4#6%>2%4]:D#%H42=2D49:=%2Hj#9>$0%`3493%:+#%
2-=R%`#:i=R%:6$2+H#6%>2%$)GG2+>$0%=4i#%(\50%$4-D=#%G+2>#4-$0%:-6%*4=:]#->$
<[0<a0BZ0B.
'% (R-:]49%
]26#$% :=$2% :==2`% 34D3=R% 92++)D:>#6% 2Hj#9>$0% =4i#% =4"4-D% 9#==$0% >2% H#% 6#G49>#6% 4-% >3#4+%
)-G#+>)+H#6%$>:>#
B7
'%c2`#"#+0%>3#%>2G2D+:G349%92->+:$>%+#=4#$%2-%+:>3#+%92]G=#P%4->#+:9>42-%
]#93:-4$]$% H#>`##-% >3#% 5&?% >4G% :-6% $:]G=#'% N>4**-#$$0% +2)D3-#$$0% $)+*:9#% 93:+D#% :-6%
93#]4$>+R0%2+%*+49>42-%2*%>3#%$:]G=#%9:-%93:-D#% >3#% 2$94==:>42-%2*%>3#%>4G%:-6%>3)$%:=>#+%2+%
#"#-% 4-"#+>% >3#% 92->+:$>
<Y
'% 12% +#92+6% *:4>3*)=% 34D3A+#$2=)>42-% 4]:D#$% 4>% 9:-% >3#+#*2+#% H#%
3#=G*)=% >2% 4]:D#% )-i-2`-% H42=2D49:=% $R$>#]$% 4-% >3#% G+#$#-9#% 2*% $>+)9>)+:==R% `#==A
93:+:9>#+4@#6%+#*#+#-9#%$:]G=#$
<Y0B<0BB
'%%
5GG=4#6% >2% 9#==)=:+% $R$>#]$% 92->:9>% :-6%
6R-:]49%]26#%5&?%+#"#:=% >2G2D+:G3$%H#=2`%>3#%
+#$2=)>42-%=4]4>%2*%92-"#->42-:=%=4D3>%]49+2$92GR'%13#%#:$#%2*%)$#%:-6%>3#%#P9#G>42-:=%$4D-:=A

Frequently asked questions

Q1. What are the contributions in this paper?

The authors discuss recent examples that highlight the unique capabilities of these emerging new modalities.