Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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 routine imaging (too slow). However this would be an interesting benchmark to compare S/N between microscopes.
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.
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CW single-frequency ring Dye laser DYE-SF-077 -
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
|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|
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
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.
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