Spectral Selfinterference Fluorescence Microscopy

1
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
Cellular Imaging with 4pi microscope
Abstract
Biological Applications of SSFM
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.
Lipid Bilayers (Artificial cell membranes)
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
Probing fluorophore position in top or bottom
leaflet of an artificial cell membrane
Main lobe and side lobes marked for upper and
lower cell walls
Acomplishments up through Current Year
DNA Conformation Studies

• 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

average height of tags on hybridized DNA from SSFM
average height of tags on single-strand from SSFM
height of single-strand from white light
height of double-strand from white light
12 nm
10 nm
8 nm
6 nm
12 nm
4 nm
10 nm
2 nm
height of single-strand from white light
8 nm
6 nm
Future Plans
4 nm
2 nm
Hybridization
Current State of the Art

• 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.
  

Double strand DNA
Single strand DNA
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
SSFM in 4Pi Mode
Advantages of SSFM in 4pi mode
Contribution to CenSSIS Research Thrusts

• 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

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.
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
4Pi Microscope Setup
Technical Approach Spectral Self-interference
Fluorescence Microscopy (SSFM)
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. 
Wide field imaging with Koehler Illumination
Collection APD and Spectrometer/CCD
Interference Path
Reflection Self-interference Interference
fringes in spectrum
No reflection No self-interference Smooth
fluorescence envelope
Challenges

• 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

• Path length adjustment
• Right mirror in the interference path is
• on a piezo stage for path length and
• phase adjustments
• ?/60 stepping capability

Preliminary Results
Self-interference Reveals Axial Position
Information
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)
One Objective
Two Objectives
d2-d15nm
Main Lobe FWHM100nm
Contact Information Mehmet Dogan, PhD
Student Physics Department, Boston
University mdogan_at_bu.edu http//ultra.bu.edu
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)
FWHM660nm
Best fit to MODEL fitting parameter d, axial
position
Axial Position
Spectral Fringes