Multiparametric Single Molecule Fluorescence
Microscopy
Home built, open frame, inverted confocal imaging microscope based on a Olympus IX81
and featuring a nanopositioning piezo
scanning-stage (Physics Instrumente 100x100 microns), high NA objective lens
(oil 100x, 1.40 NA), 4 single photon
counting avalanche photodiodes (100 picoseconds response time), a
time-correlated single photon counting detection system based on a PicoHarp300
and an integrated spectrograph system (Acton SP2300i
spectrograph
combined with PIXIS 100 back-illuminated CCD
camera with QE 90%).
This single molecule microscope is coupled to various
lasers, including a femtosecond laser system (350-490nm and 580-670nm,
variable repetition rate, 80Mhz, 8MHz and lower)
and two synchronized picosecond diodes of 440nm and 532nm. Various dichroic mirrors
and band pass filters are available for the visible range, 400nm to
700nm.
The instrument can be operated in one
of the following modes:
-
Single molecule fluorescence lifetime
imaging (SM-FLIM), point measurements of time-resolved fluorescence
intensity and fluorescence lifetimes, time-resolved anisotropy and
polarization, FRET by two-color detection (intensity and lifetime), photon
pair correlation (antibunching).
-
Simultaneous confocal fluorescence/back scattering
for plasmon-exciton studies.
-
Single molecule fluorescence
spectroscopy (back-illuminated CCD camera with integration times as short as
100ms/spectrum .
-
Confocal Raman imaging and spectroscopy with CW lasers (457-514nm,
543nm, 632nm). Limited range for Raman optics.
-
combined FLIM / AFM with
a NanoSurf AFM microscope (90/90 microns scan area).
-
Symphotime software for FLIM and FLIM-FRET imaging,
post processing (data analysis) including Maximum Likelihood Estimation lifetime
analysis for single molecules, FRET analysis, on/off (blinking) statistics, antibunching.
Multiphoton Laser-Scanning Imaging Microscope (Leica SP5 TCS)
Leica SP5 laser-scanning microscope based on a
DM6000 frame for fast imaging (two-photon fluorescence and second-harmonic
generation). Includes several objective lenses (63x 1.4 NA oil,
63x 1.2 NA water, 10x 0.2 NA air).
Features 3-photomultiplier channels for simultaneous imaging of three colors and two photon counting
photomultipliers for tow-color FLIM (with NIR laser). The Leica SP5 is interfaced with an
ultrafast laser (MaiTai broadband, 710-980nm)
for multiphoton FLIM and Second Harmonic Generation Imaging (forward and
backward). Detection range 460-750 nm,
unrestricted by optics.
This instrument is capable of operation in one of the following modes:
- laser-scanning confocal fluorescence imaging including
FRAP, FRET, colocalization of up to three colors.
- multiphoton fluorescence imaging and FLIM, FLIM-FRET.
- transmission, differential interference contrast and reflectance imaging.
- new !!! Second Harmonic Generation Imaging.
Two-Color
Fluorescence Correlation Spectroscopy
A home built, open frame confocal microscope based on Olympus IX71 featuring
a high NA
objective (water, 1.2NA, 60x), two single photon
counting avalanche photodiodes (45 ps response time), time-correlated single photon counting
detection system based on a PicoHarp300.
The microscope is coupled to various CW lasers lines (Ar-ion 457-514nm tunable,
He Ne lines 543nm, 594nm, 632nm, possibility for
two-color excitation two-color detection. Dedicated exclusively to solution samples.
Fluorescence correlation spectroscopy (FCS) can be measured in autocorrelation mode, cross-correlation mode, antibunching in solution, two-color excitation (488/632nm), two color
cross-correlation. Symphotime software for FCS and
time-resolved FCS data analysis including 3D diffusion and lifetime analysis.
Symphotime analysis software.
Total Internal Reflection Fluorescence Microscopy
Olympus IX81 microscope featuring a 1.45 NA oil lens for
through-objective TIRFM, with alternate three-laser excitation
(488/561/632nm) and fast imaging EM-CCD
camera (down to 1 ms acquisition time per frame) . Through-objective TIRF excitation offers the advantage of easy access to sample. A Dual-view optics attachment
is installed for two-color colocalization / FRET imaging.
TIRF dichroic is optimized for
the following detection ranges:
- 530/50nm (FITC)
- 600/40nm (TRITC)
- 670/30nm (Cy5)
- Dual View optics: 565nm dichroic with 530/30nm (green channel) and
590/40nm (red channel)
The microscope has several additional lenses (40x, 20x and 10x air) and it can be
operated in one of the following modes:
- TIRFM mode with 100x 1.45 NA oil lens
- conventional epifluorescence imaging (FITC, TRITC dichroics)
- darkfield imaging (10x, 20, 40x
magnification)
- new !!! Combined optical microscopy/electrical probing and
electroluminecence spectroscopy for characterization of OPVs, LEDs. Dedicated sample holder for 18x18 mm
square photovoltaic devices (PVs, LEDs).
Time-correlated single photon counting cuvette spectroscopy
A state-of-the art time-resolved photoluminescence cuvette spectrometer based on the time-correlated single
photon counting method. The instrument is based on a Fluotime Picoquant 200 lifetime spectrometer
and includes excitation/emission polarizers, sample cuvette/thin films
compartment,
step-monochromator and a microchannel plate photomultiplier (Hamamatsu, Peltier
cooled, 25 ps response time).
This lifetime spectrometer is
coupled to a femtosecond laser system (85-200 fs pulses) providing excitation colors from 350-490nm
and 580-670nm with various repetition rates (80Mhz, 8MHz and lower).
Variable repetition rate allows to measure both short (few ps) and long
(hundreds of ns) fluorescence lifetimes. Detection range for the Hamamatsu PMT is 350-850nm.
Response time 40 ps.
The lifetime spectrometer can be operated in one of the following modes:
- acquisition of fluorescence decays at a given wavelength, with up to 4
ps time resolution / channel
- acquisition of time-resolved fluorescence anisotropy decay, with up to 4
ps time resolution / channel
- time-resolved fluorescence spectra by automatic scanning of the
detection monochromator
- Fitting software with various models including multiexponential, Gauss,
Lorentz.
Lasers available in the Single Molecule Spectroscopy Facility
- Ultrafast laser system based on a Ti:Sapphire broad-band (Tsunami
Spectra Physics, 690-1050nm) oscillator, an Optical Parametric Oscillator
(OPAL, Spectra Physics), two pulse-pickers, two frequency doublers, system
output 350-490nm & 580-670nm, 80MHz, 8MHz, 85-200 fs pulses depending on
color.
- Argon-ion tunable CW laser (457, 488, 514nm)
- He Ne CW lasers (543nm, 594nm, 632nm)
- PDL 800 picosecond diodes of 440nm and 530nm, 90 ps pulses, can be run
synchronous or alternatively, variable repetition rate from 40 MHz and
lower.
- MaiTai BroadBand Spectra Physics, 700-980nm, 200 fs pulses, dedicated to
Leica SP5
- Diode lasers 488nm Coherent, 561nm, dedicated to TIRFM.
- Frequency Doubler for Tsunami oscillator (MiniOptics) covers 350nm-480nm
at 80 MHz
* As part of the
Soft Bio Nano
theme, we have access to wet-chemistry labs,
conventional spectrophotometers (UV-VIS, fluorescence, circular dichroism),
Dynamic Light Scattering, glove box for sample preparation, labs for protein
expression and purification, tissue culture, cold room, low voltage TEM for soft
(polymers, proteins) samples.
How to Contact Us
Mircea Cotlet, PhD
Bus: 631-344-7778
Fax: 631-344-7765
Email: cotlet@bnl.gov
