title: Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.
abstract: Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1 × 1.6 mm (3.4 mm(2)) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm(-1) used in conjunction with a 4 × 0.1 NA objective (v=0.165). This yielded an optical section thickness of 128 µm and an 8.9 × contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. henry.fu@duke.edu
authors.forename: Henry L
authors.initials: HL
authors.lastname: Fu
authors.forename: Jenna L
authors.initials: JL
authors.lastname: Mueller
authors.forename: Melodi P
authors.initials: MP
authors.lastname: Javid
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Mito
authors.forename: David G
authors.initials: DG
authors.lastname: Kirsch
authors.forename: Nimmi
authors.initials: N
authors.lastname: Ramanujam
authors.forename: J Quincy
authors.initials: JQ
authors.lastname: Brown
mesh_terms: D000465:Algorithms; D000818:Animals; D002467:Cell Nucleus / Q000473:pathology*; D004195:Disease Models, Animal; D007091:Image Processing, Computer-Assisted; D008029:Lighting / Q000295:instrumentation*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D018482:Muscle, Skeletal / Q000473:pathology*; D019047:Phantoms, Imaging; D012513:Sarcoma, Experimental / Q000473:pathology*; D017154:Stromal Cells / Q000473:pathology*
references.citation: Am Surg. 2005 Jan;71(1):22-7; discussion 27-8
references.pmid: 15757052
references.citation: J Microsc. 1999 Dec;196(Pt 3):317-31
references.pmid: 10594772
references.citation: J Biomed Opt. 2009 Jan-Feb;14(1):014004
references.pmid: 19256692
references.citation: J Biomed Opt. 2008 Mar-Apr;13(2):024021
references.pmid: 18465984
references.citation: J Microsc. 2001 Sep;203(Pt 3):246-57
references.pmid: 11555142
references.citation: Nat Methods. 2011 Sep 29;8(10):811-9
references.pmid: 21959136
references.citation: J Biomed Opt. 2011 Jan-Feb;16(1):016014
references.pmid: 21280920
references.citation: Am J Surg. 2006 Oct;192(4):538-40
references.pmid: 16978970
references.citation: Stereotact Funct Neurosurg. 2010;88(1):1-10
references.pmid: 19907205
references.citation: Lasers Surg Med. 2012 Apr;44(4):310-7
references.pmid: 22415634
references.citation: Cancer. 2012 Nov 1;118(21):5320-30
references.pmid: 22437667
references.citation: J Microsc. 2000 May;198(Pt 2):82-7
references.pmid: 10810003
references.citation: Opt Express. 2008 Feb 4;16(3):1975-89
references.pmid: 18542277
references.citation: IEEE J Sel Top Quantum Electron. 2010 Mar 1;16(3):530-544
references.pmid: 21544237
references.citation: Nat Med. 2007 Aug;13(8):992-7
references.pmid: 17676052
references.citation: Ann Surg Oncol. 2011 Nov;18(12):3506-13
references.pmid: 21509632
references.citation: Opt Lett. 1997 Dec 15;22(24):1905-7
references.pmid: 18188403
references.citation: Am Surg. 2007 Apr;73(4):337-43
references.pmid: 17439024
references.citation: Opt Express. 2010 Apr 12;18(8):8058-76
references.pmid: 20588651
references.citation: J Cancer Res Clin Oncol. 1985;109(3):241-4
references.pmid: 4008520
references.citation: Ann Surg Oncol. 2007 May;14(5):1618-28
references.pmid: 17443388
references.citation: Cancer Res. 2009 Nov 15;69(22):8790-6
references.pmid: 19910294
references.citation: J Am Coll Surg. 2008 Jan;206(1):76-82
references.pmid: 18155571
references.citation: Micron. 2008 Oct;39(7):808-18
references.pmid: 18337108
references.citation: Opt Express. 2007 Dec 10;15(25):16413-23
references.pmid: 19550931
references.citation: PLoS One. 2012;7(1):e30887
references.pmid: 22303465
references.citation: Opt Express. 2008 Sep 29;16(20):16227-39
references.pmid: 18825262
references.citation: J Biomed Opt. 2010 Mar-Apr;15(2):026027
references.pmid: 20459272
references.citation: Gastrointest Endosc. 2008 Oct;68(4):737-44
references.pmid: 18926182
references.citation: J Biomed Opt. 2012 Jun;17(6):066018
references.pmid: 22734774
references.citation: Am J Obstet Gynecol. 2010 Jan;202(1):90.e1-9
references.pmid: 19800605
references.citation: J Biomed Opt. 2009 May-Jun;14(3):034050
references.pmid: 19566342
references.citation: J Biomed Opt. 2009 May-Jun;14(3):030502
references.pmid: 19566286
references.citation: Am J Surg. 2006 Oct;192(4):509-12
references.pmid: 16978962
references.citation: J Biomed Opt. 2010 Nov-Dec;15(6):066019
references.pmid: 21198193
references.citation: Opt Express. 2007 Nov 26;15(24):16130-40
references.pmid: 19550902
references.citation: Opt Lett. 2009 Oct 15;34(20):3235-7
references.pmid: 19838284
references.citation: J Biomed Opt. 2009 Jul-Aug;14(4):044030
references.pmid: 19725741
references.citation: Opt Express. 2012 Jan 2;20(1):403-13
references.pmid: 22274364
references.citation: Arch Surg. 1991 Apr;126(4):490-3
references.pmid: 2009065
references.citation: J Microsc. 2009 May;234(2):124-9
references.pmid: 19397741
references.citation: J Biomed Opt. 2010 Jan-Feb;15(1):010506
references.pmid: 20210421
references.citation: Ann Surg Oncol. 2008 May;15(5):1271-2
references.pmid: 18320287
references.citation: J Pathol. 2013 Jan;229(1):132-40
references.pmid: 22951975