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To add to an old thread:
We did some single molecule imaging using homebuilt sample-scanning
confocals equipped with APD detectors and this definitely works. The main
differences between the TIRF and the confocal are:
1) The confocal scans point by point while the TIRF with EMCCD camera
detects all pixel simultaneously. When you use 20-100 ms integration time in
the TIRF you need to match these on the confocal using the slowest scan
speed and some averaging. It takes quite long to image a 512 x 512 pixel
image (for 1 ms integration time/pixel 5 min.).
2) The TIRF has about twice the electric field at the surface (incoming and
reflected light), which gives four times higher intensity for the
fluorophore excitation but does also work for detection of the emitted light
(optical reciprocity).
3) The EMCCD has >90% quantum efficiency while the typical PMTs in the
confocals have less than 20%.

A good sample to start with is Alexa 647 labelled IgG (5 fluorophores /
molecule) excited with 633 nm, a drop 50 pM concentration on a coverslip,
add 10 mM CaCl to pin the IgG down and wash excess away. 
You need to match the pixel size (100nm is good), laser power and
integration time of the confocal to the TIRF conditions. The lower
sensitivity of the detector can be compensated by increasing the laser power
at the expense of fluorophore lifetime. After bleaching of the majority of
fluorophores and background, there will be a few molecules left with longer
lifetime, which give a good indication of the signal of a single
fluorophore. I managed to image single Alexa 647 IgG’s on a Fluoview 1000
using the PMT detector with bandpass filter but would not recommend this for
imaging (too slow). However this would be an interesting benchmark to
compare S/N between microscopes.

Customer inquiry:

we are looking for a CW dye laser for single molecule spectroscopy. We need linewidth < 1 MHz, longterm stability better than 100 kHz/second and a very good pointing stability.



Related Bibliography

"Optical Detection and Spectroscopy of Single Molecules in a Solid," by W. E. Moerner and L. Kador, Phys. Rev. Lett. 62, 2535 (1989). This is the first report of single-molecule detection and spectroscopy in condensed phases.
"Fluorescence Spectroscropy and Spectral Diffusion of Single Impurity Molecules in a Crystal," by W.P. Ambrose and W. E. Moerner, Nature 349, 225 (1991).
"Spectroscopy of Single Impurity Molecules in Solids," by W. E. Moerner and Th. Basche', Angew. Chem. 105, 537 (1993); Angew. Chem. Int. Ed. Engl. 32, 457 (1993).
"Examining Nanoenvironments in Solids on the Scale of a Single, Isolated Impurity Molecule," by W. E. Moerner, Science 265, 46 (1994).
"High-Resolution Optical Spectroscopy of Single Molecules in Solids, by W. E. Moerner, in "Single Molecules and Atoms," Special Issue of Accounts of Chemical Research, December 1996.
"Fundamentals of Single-Molecule Spectroscopy in Solids," Chapter 1 of Single Molecule Optical Detection, Imaging, and Spectroscopy, T. Basche, W. E. Moerner, M. Orrit, and U. P. Wild, eds. (Verlag Chemie, Munich, 1997).
W. E. Moerner, "Those Blinking Single Molecules," Science 277, 1059 (1997).
W. E. Moerner and M. Orrit, "Illuminating Single Molecules in Condensed Matter," Science 283, 1670-1676 (1999).
B. Lounis and W. E. Moerner, "Single Photons on Demand from a Single Molecule at Room Temperature," Nature 407, 491-493 (2000).
H. Sosa, E. J. G. Peterman, W. E. Moerner, and L. S. B. Goldstein, "ADP-Induced Rocking of the Kinesin Motor Domain Revealed by Single-Molecule Fluorescence Polarization Microscopy," Nature Structural Biology 8, 540-544 (2001).
W. E. Moerner, "Thirteen Years of Single-Molecule Spectroscopy in Physical Chemistry and Biophysics," in Single-Molecule Spectroscopy: Nobel Conference Lectures, R. Rigler, M. Orrit, Th. Basche, Editors, Springer Series in Chemical Physics, Volume 67 (Springer-Verlag, Heidelberg, 2001), pp. 32-61.
W. E. Moerner, "A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics," J. Phys. Chem. B 106, 910-927 (2002).
W. E. Moerner, "Single-Molecule Optical Spectroscopy of Autofluorescent Proteins," J. Chem. Phys. 117, 10925 (2002).
W. E. Moerner and D. P. Fromm, "Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy," Rev. Sci. Instrum. 74, 3597-3619 (2003).
E. J. G. Peterman, H. Sosa, and W. E. Moerner, “Single-Molecule Fluorescence Spectroscopy and Microscopy of Biomolecular Motors,” invited review, Ann. Rev. Phys. Chem. 55, 79-96 (2004).
P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the Mismatch Between Light and Nanoscale Objects with Gold Bowtie Nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).
K. A. Willets, S. Y. Nishimura, P. J. Schuck, R. J. Twieg, and W. E. Moerner, "Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy," invited review, Accounts Chem. Res. 38, 549-556 (2005)
S. Y. Kim, Z. Gitai, A. Kinkhabwala, L. Shapiro, and W. E. Moerner, “Single Molecules of the Bacterial Actin MreB Undergo Directed Treadmilling Motion in Caulobacter crescentus,” Proc. Nat. Acad. Sci. (USA) 103, 10929-10934 (2006)..
A. E. Cohen and W. E. Moerner, “Suppressing Brownian Motion of Individual Biomolecules in Solution,” Proc. Nat. Acad. Sci. (USA) 103, 4362-4365 (2006).
W. E. Moerner, “New Directions in Single-Molecule Imaging and Analysis,” Invited Perspective, Proc. Nat. Acad. Sci. (USA) 104, 12596-12602 (2007).
J. S. Biteen, M. A. Thompson, N. K. Tselentis, G. R.Bowman, L. Shapiro, W. E. Moerner, “Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable EYFP,” Nature Meth. 5, 947-949 (2008).
W. E. Moerner, “Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances,” in Single Molecule Spectroscopy in Chemistry, Physics and Biology: Nobel Symposium 138, Springer Series in Chemical Physics Vol. 96, A. Gräslund, R. Rigler, J. Widengren, Eds. ( Springer-Verlag, Berlin, 2009).
A. Kinkhabwala, Z. Yu, S. Fan, Y. Avlasevich, K. Müllen, and W. E. Moerner, “Large Single-Molecule Fluorescence Enhancements Produced by a Bowtie Nanoantenna,” Nature Photonics 3, 654-657 (2009)..
S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells," Anal. Chem. 82, 2192-2203 (2010) .

CW single-frequency ring Dye laser DYE-SF-077 - request a quote

Frequency-stabilized CW single-frequency ring Dye laser, model DYE-SF-077, is a further development of model DYE-SF-07. It now includes a system of frequency stabilization on the basis of a thermo-stabilized interferometer and a fast electronic driver.
Laser DYE-SF-077 features exceptionally narrow generation line width, which amounts to less than 100 kHz. DYE-SF-077 sets new standard for generation line width of commercial lasers. Prior to this model, the narrowest line-width of commercial dye lasers was as broad as 500 kHz - 1 MHz. It is necessary to note that the 100-kHz line-width is achieved in DYE-SF-077 without the use of an acousto-optical modulator, which, as a rule, complicates the design and introduces additional losses. A specially designed ultra-fast PZT is used for efficient suppression of radiation frequency fluctuations in a broad frequency range

570-700 nm, output > 1.5 W (10 W pump), linewidth < 100 kHz rms, frequency drift < 30 MHz/hour, smooth scanning 6/20 GHz.

The DYE-SF-077 laser cavity has horizontal orientation, the optical mounts of the cavity elements are attached to a rigid base plate, which is further stabilized by a volumetric framework with three invar rods underneath. Additional passive stability of the position of cavity elements is provided by the vibration isolating design of the laser base.

Dye laser DYE-SF-077 is the first representative of the new generation of dye lasers that offer to the user virtually the same level of convenience and simplicity of operation as with a solid-state tunable laser. As a result we are able to offer an option of combined configuration of DYE-SF-077 with Ti:Sapphire laser.

Laser DYE-SF-077 may be equipped with a USB compatible interface to remotely scan the generation line of the laser and to perform multi-channel data acquisition. Laser DYE-SF-077 also may be shipped together with an atom cell and a system for reduction of long-term generation line drift. Besides, laser DYE-SF-077 in combination with highly-efficient resonant frequency doubler FD-SF-07 delivers several hundreds milliwatts of narrow-band UV radiation within the 285–350-nm range.

CW single-frequency ring Dye laser DYE-SF-077 - request a quote


 Wavelength range  570-620 nm
 620-700 nm
 Output  > 1 W at 6 W pump
 >1.5W at 10W pump
 Linewidth  < 100 kHz rms1
 Frequency drift  < 30 MHz/hour
 Smooth scanning  > 6 GHz3
 Spatial mode  TEM00
 Polarization  horizontal

1. relative to the reference cavity
2. < 1 MHz/hour with frequency stabilization to an atomic/molecular line (option)
3. up to 20 / 40 GHz (option)

1. 20 / 40 GHz smooth scanning;
2. 285-350 nm wavelength range with Resonant Frequency Doubler FD-SF-07
3. Absolute Frequency stabilization to an atomic/molecular line
4. + Ti:Sapphire laser (linewidth < 5 kHz) in the same Laser head


Dye circulation system of the CW single-frequency ring Dye laser DYE-SF-077Dye circulation system of the CW single-frequency ring Dye laser DYE-SF-077

Dye Circulation System


CW single-frequency ring Dye laser DYE-SF-077 (Standard quotation) - request a quote

Actively frequency-stabilized, continuous-wave, single-frequency ring Dye laser, model DYE-SF-077

Unique DYE-SF-077 laser has more narrow linewidth for Dye lasers on the present market. DYE-SF-077 laser has super-narrow linewidth (< 70 kHz) and unique Auto Re-lock function which is extremely useful in a work with frequency stabilized laser.

General description
•Ring design, three wavelength selectors: birefringent filter, thin and thick etalons, electronically controlled thick etalon to ensure laser operation in a single longitudinal-cavity mode.
•Ultra-narrow line width up to < 70 kHz rms: active frequency stabilization to an external reference cavity, special fast PZT actuator with extended response bandwidth.
•Unique function Smart Auto-Relock that allows uninterrupted laser operation in the frequency stabilization mode under arbitrary external perturbations.
•Smooth scanning capability: up to 20 GHz.
•Actively thermostated high-finesse reference cavity, frequency drift <40MHz/hr.
•External lock capability for stabilization to an absolute reference (e.g. iodine saturated absorption line).

Wavelength range, power
•Spectral ranges 570-620 / 620-700 nm
•Output power at max gain 590 / 650 nm 1W or better with 6W pump (DPSS, 532 nm, TEMoo).
•TEMoo mode, linear polarization.

•Absolutely dry dye-jet laser: the laser has a reliable shutter for the dye solution during powering up and switching off the circulation system.
•Most of the mounts are placed on a horizontal base plate, which is also the top element of the compact rigid 3-D frame made from three invar rods that ensures high stability of the optical element positions.
•Ease of adjustment, simplified laser alignment in the ring configuration because of preliminary optimization of the elements in the linear cavity, exceptionally accurate alignment of the pump beam position.
•Premium sapphire nozzle.
•Enclosed cavity with nitrogen purge port.
•Ergonomical and reliable electronic control unit featuring a built-in generator for smooth scanning of the laser frequency.

Dye Circulation System
•Compact and powerful system of dye solution circulation with a leak-free magnetically coupled pump.
•Efficient system for suppression of pressure fluctuations in the dye circulation loop.
•The dye solution circulation system of DYE-SF-077 is designed to make the procedure of dye change convenient and clean. Laser DYE-SF-077 may be shipped with several circulation systems, in which case switching between working spectral ranges will be extremely fast and simple.

Actively frequency-stabilized, continuous-wave, single-frequency ring Dye laser, model “DYE-SF-077”:
Wavelength range: 570-620 nm
Linewidth: < 100 kHz rms
Output power: > 1W@590nm at 6W pump (532 nm, TEMoo)
Smooth scanning: > 6 GHz
Frequency drift: < 40 MHz/hour
Polarization: horizontally polarized output
Auto-Relock function, external lock capability for stabilization to an absolute reference


Installation of the T&D Scan high resolution Laser Spectrometer based on broadly tunable CW laser at the Drexel University




DYE-SF-077 datasheet (1,2 Mb)


Del Mar Photonics, Inc.
4119 Twilight Ridge
San Diego, CA 92130
tel: (858) 876-3133
fax: (858) 630-2376
Skype: delmarphotonics