Laboratory of High Resolution Optical Imaging (HROI)

The NIH intramural research program has shifted all non-mission-critical laboratory operations to a maintenance phase in order to promote physical distancing and diminished transmission risk of COVID-19. Effective Monday, March 23, 2020, only mission-critical functions within NIH research laboratories will be supported.

The Laboratory of High Resolution Optical Imaging (HROI) develops novel technologies for studying biological processes at unprecedented speed and resolution. Research includes improving the performance of 3D optical imaging microscopes, particularly with respect to resolution and depth (e.g. multifocal structured illumination microscopy, MSIM) and speed and phototoxicity (e.g. inverted selective plane illumination microscopy, iSPIM). We collaborate closely with intra- and extramural researchers (both academic and commercial) to ensure that our microscopes are both easily and widely used. Along with researchers at Sloan-Kettering (Zhirong Bao) and Yale University (Daniel Colon-Ramos), we are using one of our technologies (iSPIM) to construct the first atlas of 4D neurodevelopment in an animal.

Hari Shroff lab group photo
Photo: C. Chang for NIBIB.


Furthering development of superresolution optical imaging techniques, particularly 3-D structured illumination microscopy(SIM)

Thi is a picutre of fluorescently labeled microtubules fixed in a cell. The microtubules are less than 150 nm.

Structured illumination microscopy techniques use patterned excitation light and post-processing to double the resolution of a conventional microscope. Unlike other super-resolution techniques, they provide resolution enhancement at a relatively low illumination dose, thus enabling high resolution imaging over tens – hundreds of volumes. Previous implementations of SIM were limited to samples of < 10 um, but we have developed a multifocal version (MSIM)1 that permits resolution-doubling at depths exceeding 50 um. Most recently, we have extended the speed of MSIM 100x, facilitating super-resolution imaging at hundreds of frames per second.1

Improving the usability and resolution of selective plane illumination microscopy (SPIM)

This is a picture of GFP-histones in a live nematode embryo.  It shows both a top and bottom view of the embryo.

Selective plane illumination microscopy techniques combine a perpendicular excitation/detection geometry with light-sheet excitation, drastically reducing photobleaching and damage while providing higher signal-to-noise ratio and acquisition rates than confocal microscopy. Nevertheless, widespread adoption of SPIM has been slow because (1) the geometry is cumbersome, usually requiring special sample preparation; (2) Axial resolution is usually 3x worse than confocal microscopy. To address the usability issue, we have developed inverted selective plane illumination microscopy (iSPIM)2, a module that can be mounted onto an epifluorescence microscope, thus allowing conventional sample mounting on glass coverslips while retaining the advantages of SPIM. iSPIM offers an approximate 30-fold improvement over spinning disk confocal microscopy in terms of acquisition speed, photobleaching, and total acquisition length when imaging live samples. We have recently developed a variation of iSPIM that enables isotropic imaging with 330 nm spatial resolution (~4x better axial resolution than previous implementations) and temporal resolution down to 3 ms.

Construction of a 4-dimensional digital atlas of neuronal development in C. Elegans in collaboration with researchers at Yale University and Memorial Sloan-Kettering Cancer Center

Along with extramural neuroscientists, scientists, and developmental biologists, we are using iSPIM to follow all neurons in the developing nematode embryo. We intend to create the first digital atlas of neurodevelopment. Further information on this project may be obtained at

1. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. York AG, Parekh SH, Dalle Nogare D, Fischer RS, Temprine K, Mione M, Chitnis AB, Combs CA, Shroff H. Nat Methods. 2012 May 13;9(7):749-54. doi: 10.1038/nmeth.2025.

2. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Wu Y, Ghitani A, Christensen R, Santella A, Du Z, Rondeau G, Bao Z, Colón-Ramos D, Shroff H. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17708-13. doi: 10.1073/pnas.1108494108. Epub 2011 Oct 17.

This is an image of the laser configuration in the Section for High Resolution Optical Imaging

The Shroff lab currently houses:

  • A photoactivated localization microscope (PALM) for 3D super-resolution (25-50 nm in XY and 100 nm in z).
  • A multifocal structured illumination microscope with 150 nm lateral, 350 nm axial resolution and a time resolution of 5 ms.
  • Two selective plane illumination microscopes, with isotropic spatial resolution down to 330 nm and temporal resolution of 5 ms.
Guo MLi YSu YLambert TNogare DDMoyle MWDuncan LHIkegami RSantella ARey-Suarez IGreen DBeiriger AChen JVishwasrao HGanesan SPrince VWaters JCAnnunziata CMHafner MMohler WAChitnis ABUpadhyaya AUsdin TBBao ZColón-Ramos DLa Riviere PLiu HWu YShroff H
Nat. Biotechnol.
2020 Jun 29

Trcek TDouglas TEGrosch MYin YEagle WVIGavis ERShroff HRothenberg ELehmann R
Mol. Cell
2020 Jun 04

Rey-Suarez IWheatley BAKoo PBhanja AShu ZMochrie SSong WShroff HUpadhyaya A
Nat Commun
2020 Jan 23

Bhagwat ARLe Sage VNturibi EKulej KJones JGuo MTae Kim EGarcia BAWeitzman MDShroff HLakdawala SS
Nat Commun
2020 Jan 07

Peluso EAUpdegrove TBChen JShroff HRamamurthi KS
Proc. Natl. Acad. Sci. U.S.A.
2019 Oct 22

Chandler TShroff HOldenbourg RRivière P
J Opt Soc Am A Opt Image Sci Vis
2019 Aug 01

Chandler TShroff HOldenbourg RLa Rivière P
J Opt Soc Am A Opt Image Sci Vis
2019 Aug 01

Colón-Ramos DALa Riviere PShroff HOldenbourg R
Nat. Methods
2019 Aug

Duncan LHMoyle MWShao LSengupta TIkegami RKumar AGuo MChristensen RSantella ABao ZShroff HMohler WColón-Ramos DA
J Vis Exp
2019 Jun 06

Vogt EJTokuhiro KGuo MDale RYang GShin SWMovilla MJShroff HDean J
Nat Commun
2019 May 22

Guo MChandris PGiannini JPTrexler AJFischer RChen JVishwasrao HDRey-Suarez IWu YWu XWaterman CMPatterson GHUpadhyaya ATaraska JWShroff H
Nat. Methods
2018 Jun

Zhu GLynn GMJacobson OChen KLiu YZhang HMa YZhang FTian RNi QCheng SWang ZLu NYung BCWang ZLang LFu XJin AWeiss IDVishwasrao HNiu GShroff HKlinman DMSeder RAChen X
Nat Commun
2017 Dec 05

Zhu GMei LVishwasrao HDJacobson OWang ZLiu YYung BCFu XJin ANiu GWang QZhang FShroff HChen X
Nat Commun
2017 Nov 14

Wu YKumar ASmith CArdiel EChandris PChristensen RRey-Suarez IGuo MVishwasrao HDChen JTang JUpadhyaya ALa Riviere PJShroff H
Nat Commun
2017 Nov 13

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Mol. Cell
2017 Aug 17

Sardo LLin AKhakhina SBeckman LRicon LElbezanti WJaison TVishwasrao HShroff HJanetopoulos CKlase ZA
J. Cell. Sci.
2017 Sep 01

Laissue PPAlghamdi RATomancak PReynaud EGShroff H
Nat. Methods
2017 Jun 29

Zheng WWu YWinter PFischer RNogare DDHong AMcCormick CChristensen RDempsey WPArnold DBZimmerberg JChitnis ASellers JWaterman CShroff H
Nat. Methods
2017 Sep

Trcek TLionnet TShroff HLehmann R
Nat Protoc
2017 Jul

Ardiel ELKumar AMarbach JChristensen RGupta RDuncan WDaniels JSStuurman NColón-Ramos DShroff H
Biophys. J.
2017 May 09

Combs CAShroff H
Curr Protoc Neurosci
2017 Apr 10

Ogawa MTomita YNakamura YLee MJLee STomita SNagaya TSato KYamauchi TIwai HKumar AHaystead TShroff HChoyke PLTrepel JBKobayashi H
2017 Feb 07

Lakdawala SSWu YWawrzusin PKabat JBroadbent AJLamirande EWFodor EAltan-Bonnet NShroff HSubbarao K
PLoS Pathog.
2016 Dec

Wu YChandris PWinter PWKim EYJaumouillé VKumar AGuo MLeung JMSmith CRey-Suarez ILiu HWaterman CMRamamurthi KSLa Riviere PJShroff H
2016 Aug 20

Kumar AChristensen RGuo MChandris PDuncan WWu YSantella AMoyle MWinter PWColón-Ramos DBao ZShroff H
Biol. Bull.
2016 Aug

Dundon SEChang SSKumar AOcchipinti PShroff HRoper MGladfelter AS
Mol. Biol. Cell
2016 Jul 01

Christensen RPBokinsky ASantella AWu YMarquina-Solis JGuo MKovacevic IKumar AWinter PWTashakkori NMcCreedy ELiu HMcAuliffe MMohler WColón-Ramos DABao ZShroff H
2015 Dec 03

Trcek TGrosch MYork AShroff HLionnet TLehmann R
Nat Commun
2015 Aug 05

Curd ACleasby AMakowska KYork AShroff HPeckham M
2015 Oct 15

Santella ACatena RKovacevic IShah PYu ZMarquina-Solis JKumar AWu YSchaff JColón-Ramos DShroff HMohler WABao Z
BMC Bioinformatics
2015 Jun 09

Winter PWYork AGNogare DDIngaramo MChristensen RChitnis APatterson GHShroff H
2014 Sep 20

Kumar AWu YChristensen RChandris PGandler WMcCreedy EBokinsky AColón-Ramos DABao ZMcAuliffe MRondeau GShroff H
Nat Protoc
2014 Nov

Eswaramoorthy PWinter PWWawrzusin PYork AGShroff HRamamurthi KS
PLoS Genet.
2014 Aug

Ingaramo MYork AGWawrzusin PMilberg OHong AWeigert RShroff HPatterson GH
Proc. Natl. Acad. Sci. U.S.A.
2014 Apr 08

Lakdawala SSWu YWawrzusin PKabat JBroadbent AJLamirande EWFodor EAltan-Bonnet NShroff HSubbarao K
PLoS Pathog.
2014 Mar

Ingaramo MYork AGHoogendoorn EPostma MShroff HPatterson GH
2014 Mar 17

Wu YWawrzusin PSenseney JFischer RSChristensen RSantella AYork AGWinter PWWaterman CMBao ZColón-Ramos DAMcAuliffe MShroff H
Nat. Biotechnol.
2013 Nov

York AGChandris PNogare DDHead JWawrzusin PFischer RSChitnis AShroff H
Nat. Methods
2013 Nov

Wu YChristensen RColón-Ramos DShroff H
Curr. Opin. Neurobiol.
2013 Dec

Shroff HWhite HBetzig E
Curr Protoc Cell Biol
2013 Mar

York AGParekh SHDalle Nogare DFischer RSTemprine KMione MChitnis ABCombs CAShroff H
Nat. Methods
2012 May 13

Fischer RSWu YKanchanawong PShroff HWaterman CM
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2011 Dec

Wu YGhitani AChristensen RSantella ADu ZRondeau GBao ZColón-Ramos DShroff H
Proc. Natl. Acad. Sci. U.S.A.
2011 Oct 25

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2009 Jun 16

Ji NShroff HZhong HBetzig E
Curr. Opin. Neurobiol.
2008 Dec

Vaziri ATang JShroff HShank CV
Proc. Natl. Acad. Sci. U.S.A.
2008 Dec 23

Shroff HWhite HBetzig E
Curr Protoc Cell Biol
2008 Dec

Shroff HGalbraith CGGalbraith JABetzig E
Nat. Methods
2008 May

Manley SGillette JMPatterson GHShroff HHess HFBetzig ELippincott-Schwartz J
Nat. Methods
2008 Feb

Shroff HGalbraith CGGalbraith JAWhite HGillette JOlenych SDavidson MWBetzig E
Proc. Natl. Acad. Sci. U.S.A.
2007 Dec 18

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Biophys. J.
2008 Mar 15

Pauzauskie PJRadenovic ATrepagnier EShroff HYang PLiphardt J
Nat Mater
2006 Feb

Shroff HReinhard BMSiu MAgarwal HSpakowitz ALiphardt J
Nano Lett.
2005 Jul