Spectral Self-interference Fluorescence Microscopy in 4Pi Mode
1
17500 18000 18500 19000 19500 0 200 400 600 800 1000 1200 1400 1600 1800 Intensity (a.u.) W avenum b e r (cm -1 ) L ip id S pe ctrum Fit S pectra w ith and w ithoutm irror e ne rg y (w ave num ber) intensity(au) Spectral Self-interference Fluorescence Microscopy in 4Pi Mode Mehmet Dogan, Lev Moiseev, Stephen B. Ippolito, Anna K. Swan, Bennett B. Goldberg, M. Selim Ünlü Boston University Technical Approach: Spectral Self-interference Fluorescence Microscopy (SSFM) Biological Applications of SSFM Future Plans Lipid Bilayers (Artificial cell membranes) DNA Conformation Studies No reflection – No self- interference Smooth fluorescence envelope Reflection – Self- interference Interference fringes in spectrum Glass slide objectiv e mirror space r objective Self-interference Reveals Axial Position Information 5.1nm (white light) 3.4 nm (fluorescen ce) 2 nm Film of surface- absorbed water Silicon oxide spacer layer Silicon mirror 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 10 Position on the chip (mm) Height (nm) wl bottom wl top fl bottom fl top Probing fluorophore position in top or bottom leaflet of an artificial cell membrane 17000 18000 19000 20000 d 1 d 2 d 2 -d 1 =5nm Spectral Fringes Axial Position Best fit to MODEL fitting parameter: d, axial position 4Pi Microscope Setup average height of tags on single- strand from SSFM 2 nm 4 nm 6 nm 10 nm 8 nm 12 nm height of single-strand from white light 8 nm 2 nm 4 nm 6 nm 10 nm 12 nm height of single-strand from white light height of double-strand from white light average height of tags on hybridized DNA from SSFM Single strand DNA Double strand DNA Hybridiza tion •High NA objectives •Increased lateral resolution : ~200nm •3-D high resolution confocal imaging capability •Nanometer precision position determination of sparse fluorescent layers with high lateral resolution Collection: APD and Spectrometer/CCD Interference Path Wide field imaging with Koehler Illumination Maintaining common focus • Two objectives must have common foci within 10-20 nm • Invar metal components used to minimize thermal drift • Ultra stable piezo stage for focus stability: 1nm stability in closed loop •Work in 4Pi-C mode: interference of both excitation and emission. •Spectral data acquisition with interference in collection for determining axial position . •Sub-cellular imaging. •Two-photon excitation for side lobe suppression. One Objective Two Objectives Path length adjustment • Right mirror in the interference path is on a piezo stage for path length and phase adjustments • λ/60 stepping capability Abstract A thin layer of AlexaFluor488 on glass cover slip was scanned through the focus along axial direction where coherent excitation of 488nm CW laser was used (No interference in the collection channel) Advantages of SSFM in 4pi mode Current State of the Art Confocal Microscopy : ~600nm axial resolution Microscopies with two collection arms: ~ 100nm axial resolution I5M Microscopy 4Pi Microscopy Stimulated Emission Depletion Microscopy (STED) :~30 nm lateral resolution SSFM in 4Pi mode provides the ability to do high resolution imaging in 3-D while having a position determination capability in axial dimension Fluorescence microscopy is a widely used method in biological imaging. It provides a nondestructive tool for the study of living cells. However, optical confocal microscopes have a limited axial optical resolution of ~0.6 µm while many sub-cellular processes require imaging/vertical sectioning with nanometer resolution. A method, Spectral Self-interference Fluorescence Microscopy (SSFM), was introduced to determine the location of fluorescent molecules above a reflecting surface with nanometer precision. The method utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. SSFM has been used to determine the position of fluorescent markers attached to sub-cellular structures such as lipid bilayer membranes and DNA strands revealing conformational information. Despite the unprecedented axial precision capability, SSFM lacks high lateral resolution for planar substrates when emission is collected from one side of the sample. In this configuration, low collection angle is required for sufficient fringe visibility to extract the position from the spectrum with high precision and confidence. However, higher lateral resolution is possible in SSFM by using two opposing high numerical objectives with two interference paths in 4pi mode where the sample is illuminated and the emission of fluorescence signal is collected coherently from both sides of the sample. In order to get the desired fringes in the spectrum for position determination, phase delay is introduced on one of the paths by adjusting the path length difference within the coherence length of the fluorescent markers. Challenges and Significance This work combines the Spectral Self-interference Fluorescence Microscopy that is capable of determining the axial position sparse fluorescent layers with nanometer precision and high 3-D resolution 4Pi confocal microscopy. There is an ongoing effort to build a 4Pi confocal microscope SSFM in 4Pi Mode Challenges Acomplishments up through Current Year This work was supported in part by CenSSIS, the Center for Subsurface Sensing and Imaging Systems, under the Engineering Research Centers Program of the National Science Foundation (Award Number EEC-9986821) Contribution to CenSSIS Research Thrusts R1 R2 O verview ofthe Strategic Research Plan O verview ofthe Strategic Research Plan Fundam ental Science Fundam ental Science Validating TestBEDs Validating TestBEDs L1 L1 L2 L2 L3 L3 R3 S1 S4 S5 S3 S2 Bio-M ed Enviro-Civil This work falls under CenSSIS Research Thrusts R1(multispectral imaging ). The development of the 4pi SSFM nanoscope relates the projret to the CenSSIS BioBED platform. Contact Information Mehmet Dogan, PhD Student Physics Department, Boston University [email protected] http://ultra.bu.edu Publications A.K. Swan, L.A. Moiseev, C.R. Cantor, B. Davis, S.B. Ippolito, W.C. Karl, B.B. Goldberg, and M. S. Ünlü, "Towards nm resolution in fluorescence microscopy using spectral self interference," IEEE Journal of Selected Topics in Quantum Electronics, Vol. 9, No. 2, March/April 2003, pp. 294-300. L.A. Moiseev, C.R. Cantor, I. Aksun, M. Dogan, B.B. Goldberg, A.K. Swan and M. S. Ünlü, “Spectral self- interference fluorescence microscopy” accepted to Journal of Applied Physics, 2004 Swan, A.K.; Unlu, M.S.; Tong, Y.; Goldberg, B.B.; Moiseev, L.; Cantor, C.; “Self- interference fluorescent emission microscopy 5-nm vertical resolution ”Lasers and Electro-Optics, 2001. CLEO '01. Technical Digest. Summaries of papers presented at the Conference on , 6-11 May 2001 ,pp. 360 -361 Lev Moiseev, Anna K. Swan, M. Selim Ünlü, Bennett B. Goldberg, Charles R. Cantor, “DNA Conformation on Surfaces Measured by Fluorescence Self-Interference”, to be submitted to Nature Biotechnology. Preliminary Results •4pi microscope designed and built. •Started characterizing the microscope with test samples •Cellular imaging done using 4pi microscope through collaboration with MPI for Biophysical Chemistry , Nanobiophotonics group in Germany 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 z-axis (um ) Intensity (arb.u.) 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 z-axis (um ) Intensity (arb.u.) FWHM=660nm Main Lobe FWHM=100nm Cellular Imaging with 4pi microscope x-z slices of Shigella strain KAL O- antigen labeled with Texas Red. The separation between the slices is 483 nm. The scale bar represents 1 micron 0.0 0.5 1 .0 1 .5 2.0 2.5 0 20 40 60 80 F lu o re sce n e z ( m) Main lobe and side lobes marked for upper and lower cell walls
description
objective. spacer. mirror. 16. 14. 12. 10. 5.1nm (white light). 8. 3.4 nm (fluorescence). Height (nm). wl bottom. 6. wl top. 4. fl bottom. 2. fl top. Film of surface-absorbed water. 0. 2 nm. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Position on the chip (mm). - PowerPoint PPT Presentation
Transcript of Spectral Self-interference Fluorescence Microscopy in 4Pi Mode
17500 18000 18500 19000 19500
0
200
400
600
800
1000
1200
1400
1600
1800
Inte
nsity
(a.u
.)
Wavenumber (cm-1)
Lipid Spectrum Fit
Spectra with and without mirror
energy (w ave num ber)
inte
nsity
(au)
Spectral Self-interference Fluorescence Microscopy in 4Pi Mode
Mehmet Dogan, Lev Moiseev, Stephen B. Ippolito, Anna K. Swan, Bennett B. Goldberg, M. Selim ÜnlüBoston University
Technical Approach:Spectral Self-interference Fluorescence
Microscopy (SSFM)
Biological Applications of SSFM
Future Plans
Lipid Bilayers (Artificial cell membranes)
DNA Conformation Studies
No reflection – No self-interference Smooth fluorescence envelope
Reflection – Self-interference Interference fringes in spectrum
Glass slide
objective
mirror
spacer
objective
Self-interference Reveals Axial Position Information
5.1nm(white light)
3.4 nm(fluorescence)
2 nmFilm of surface-absorbed waterSilicon oxide spacer layer
Silicon mirror 0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10
Position on the chip (mm)
Hei
gh
t (n
m)
wl bottom
wl top
fl bottom
fl top
Probing fluorophore position in top or bottom leaflet of an artificial cell membrane
17000 18000 19000 20000
d1
d2
d2-d1=5nm
Spectral Fringes Axial Position
Best fit to MODEL fitting parameter: d, axial
position
4Pi Microscope Setup
average height of tags on single-strand from SSFM
2 nm4 nm
6 nm
10 nm8 nm
12 nm
height of single-strandfrom white light
8 nm
2 nm4 nm6 nm
10 nm12 nm
height of single-strandfrom white light
height of double-strandfrom white light
average height of tags on hybridized DNA from SSFM
Single strand DNA Double strand DNAHybridization
•High NA objectives
•Increased lateral resolution : ~200nm
•3-D high resolution confocal imaging capability
•Nanometer precision position determination of sparse fluorescent layers with high lateral resolution
Collection: APD and Spectrometer/CCD
Interference Path
Wide field imaging with Koehler Illumination
Maintaining common focus• Two objectives must have common foci within 10-20 nm• Invar metal components used to minimize thermal drift• Ultra stable piezo stage for focus stability:
1nm stability in closed loop
•Work in 4Pi-C mode: interference of both excitation and emission.
•Spectral data acquisition with interference in collection for determining axial position .
•Sub-cellular imaging.
•Two-photon excitation for side lobe suppression.
One Objective Two Objectives
Path length adjustment
• Right mirror in the interference path is on a piezo stage for path length and phase adjustments• λ/60 stepping capability
Abstract
A thin layer of AlexaFluor488 on glass cover slip was scanned through the focus along axial direction where coherent excitation of 488nm CW laser was used
(No interference in the collection channel)
Advantages of SSFM in 4pi mode
Current State of the ArtConfocal Microscopy : ~600nm axial resolutionMicroscopies with two collection arms: ~ 100nm axial resolution
I5M Microscopy 4Pi MicroscopyStimulated Emission Depletion Microscopy (STED) :~30 nm lateral resolution
SSFM in 4Pi mode provides the ability to do high resolution imaging in 3-D while having a position determination capability in axial dimension
Fluorescence microscopy is a widely used method in biological imaging. It provides a nondestructive tool for the study of living cells. However, optical confocal microscopes have a limited axial optical resolution of ~0.6 µm while many sub-cellular processes require imaging/vertical sectioning with nanometer resolution. A method, Spectral Self-interference Fluorescence Microscopy (SSFM), was introduced to determine the location of fluorescent molecules above a reflecting surface with nanometer precision. The method utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. SSFM has been used to determine the position of fluorescent markers attached to sub-cellular structures such as lipid bilayer membranes and DNA strands revealing conformational information. Despite the unprecedented axial precision capability, SSFM lacks high lateral resolution for planar substrates when emission is collected from one side of the sample. In this configuration, low collection angle is required for sufficient fringe visibility to extract the position from the spectrum with high precision and confidence. However, higher lateral resolution is possible in SSFM by using two opposing high numerical objectives with two interference paths in 4pi mode where the sample is illuminated and the emission of fluorescence signal is collected coherently from both sides of the sample. In order to get the desired fringes in the spectrum for position determination, phase delay is introduced on one of the paths by adjusting the path length difference within the coherence length of the fluorescent markers.
Challenges and SignificanceThis work combines the Spectral Self-interference Fluorescence Microscopy that is capable of determining the axial position sparse fluorescent layers with nanometer precision and high 3-D resolution 4Pi confocal microscopy.
There is an ongoing effort to build a 4Pi confocal microscope
SSFM in 4Pi Mode
Challenges
Acomplishments up through Current Year
This work was supported in part by CenSSIS, the Center for Subsurface Sensing and Imaging Systems, under the Engineering Research Centers Program of the National Science Foundation (Award Number EEC-9986821)
Contribution to CenSSIS Research Thrusts
R1
R2
Overview of the Strategic Research PlanOverview of the Strategic Research Plan
FundamentalScienceFundamentalScience
ValidatingTestBEDsValidatingTestBEDs
L1L1
L2L2
L3L3
R3
S1 S4 S5S3S2
Bio-Med Enviro-Civil This work falls under CenSSIS Research Thrusts R1(multispectral imaging ). The development of the 4pi SSFM nanoscope relates the projret to the CenSSIS BioBED platform.
Contact Information
Mehmet Dogan, PhD StudentPhysics Department, Boston [email protected]://ultra.bu.edu
Publications A.K. Swan, L.A. Moiseev, C.R. Cantor, B. Davis, S.B. Ippolito, W.C. Karl, B.B. Goldberg, and M. S. Ünlü, "Towards nm resolution in fluorescence microscopy using spectral self interference," IEEE Journal of Selected Topics in Quantum Electronics, Vol. 9, No. 2, March/April 2003, pp. 294-300.
L.A. Moiseev, C.R. Cantor, I. Aksun, M. Dogan, B.B. Goldberg, A.K. Swan and M. S. Ünlü, “Spectral self-interference fluorescence microscopy” accepted to Journal of Applied Physics, 2004
Swan, A.K.; Unlu, M.S.; Tong, Y.; Goldberg, B.B.; Moiseev, L.; Cantor, C.; “Self-interference fluorescent emission microscopy 5-nm vertical resolution ”Lasers and Electro-Optics, 2001. CLEO '01. Technical Digest. Summaries of papers presented at the Conference on , 6-11 May 2001 ,pp. 360 -361
Lev Moiseev, Anna K. Swan, M. Selim Ünlü, Bennett B. Goldberg, Charles R. Cantor, “DNA Conformation on Surfaces Measured by Fluorescence Self-Interference”, to be submitted to Nature Biotechnology.
Preliminary Results
•4pi microscope designed and built.
•Started characterizing the microscope with test samples
•Cellular imaging done using 4pi microscope through collaboration with MPI for Biophysical Chemistry , Nanobiophotonics group in Germany
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3
z-axis (um)
Inte
nsi
ty (
arb
. u
.)
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3
z-axis (um)
Inte
nsi
ty (
arb
. u
.)
FWHM=660nmMain Lobe
FWHM=100nm
Cellular Imaging with 4pi microscope
x-z slices of Shigella strain KAL O-antigen labeled with Texas Red. The separation between the slices
is 483 nm. The scale bar represents 1 micron
0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
Flu
ore
sce
ne
z (m)
Main lobe and side lobes marked for upper and lower cell walls
