Lasers for Fluorescence Microscopy

Over the last decade, fluorescence based life science research has been revolutionized by new imaging methods and the transitioning from bulky gas-laser sources into solid-state lasers with a smaller footprint, longer lifetime, and lower maintenance requirements.

One of the most important tools for microbiology research is high-resolution live-cell imaging through fluorescence microscopy, in which certain molecules, so-called fluorochromes or fluorophores, return low-energy light after excitation with light of a defined wavelength, i.e. light with a higher wavelength than the excitation light. Scientists are taking advantage of this physical effect to investigate ever smaller structures visible in biological processes. Striving to increase the resolution has led to the need not only to use special microscopes but also suitable light sources such as lasers of different wavelengths. In particular, the exact selection of suitable lasers enables the temporal and local resolution to be increased. Lasers are an integral part of modern fluorescence microscopy!

The development of compact, reliable solid-state lasers was an initial enabling technology for commercialization and expansion of high-resolution fluorescence microscopy techniques to new markets and applications, accompanied by parallel improvements in data storage and advanced camera systems, to name a few. While some microscope applications are able to utilize the advancements in LED and super-continuum white-light sources; the high-resolution, high-speed techniques like confocal microscopy still rely on the high-brightness and wavelength precision of lasers.

Modern fluorescence microscopy consists of an enormous variety of different techniques ranging from standard laser scanning confocal microscopy, TIRF and spinning-disc microscopy to light sheet microscopy and various approaches for super-resolution imaging using spatial manipulation of the fluorescence signal.

All these techniques put many different demands on the excitation sources being used, in terms of wavelengths, power levels, power modulation, beam quality and spectral characteristics. A common factor across most techniques is the typically need for many excitation wavelengths in order to address a continuously increasing number of fluorophores and to achieve multi-color imaging.

Most microscope set-ups with multi-color excitation capability typically use multiple individual lasers combined through optical elements and coupled into one or more output beams or optical fibers. Such a laser combiner offers the greatest flexibility in all respects, many wavelengths, many power levels, as well as fast and slow modulation. However, for systems and set-ups where flexibility is not the highest priority, a multi-line laser can offer a more permanently aligned, easier to use and maintenance free alternative.

An example of an image taken in a single-molecule localization microscopy
(SMLM) setup including Cobolt Skyra™ from Department of Biotechnology
& Biophysics at Julius-Maximilian-University of Würzburg.

At HÜBNER Photonics we have a very large selection of single and multi-line lasers by Cobolt, perfect for fluorescence microscopy applications. The fast modulation capabilities of our Cobolt 06-01 laser diodes in particular make them very suited to all confocal systems such as cLSM, Spinning Disc and TIRFM. In addition, the excellent on/off modulation, including true off, make them even more attractive for applications such as Photoactivation, Photoconversion, Optogenetics, Laser manipulation, FRET, FRAP, to name just a few. In addition, they are also suitable for super-resolution microscope techniques such as STORM, PALM and STED, since these lasers are available over a wide wavelength range even with high powers.

All Cobolt lasers can be integrated into very flexible and user-friendly laser combiner solutions on the C-FLEX platforms with up to 8 lasers in one combiner. The unique C-WAVE laser offers wide tunability across the almost the complete visible and near IR spectrum, enabling access to more exotic fluorophores. In addition, we can offer the Cobolt Skyra, which is an extremely compact, permanently aligned, multi-line laser that simplifies the integration of multi-color excitation in compact microscopy equipment by eliminating the need for in-field alignment and service, which reduces manufacturing cost and allows for more compact designs. The Cobolt Skyra can also be configured to make a perfect compact solid-state alternative to Ar-ion lasers.

Recent laser-based imaging methods try to combine different techniques such as cLSM and Raman spectroscopy. The Cobolt lasers of the 04/05 and 08-01 Series are well suited for Raman spectroscopy due to the very narrow linewidth and excellent spectral purity.  Our C-FLEX laser combiner is the perfect solution to combine both Cobolt 06-01 Series of plug & play diode lasers and the narrow linewidth lasers from the 04/05 and 08-01 Series, thus offering maximum flexibility for many Life science applications.

See our selection of lasers suited for fluorescence microscopy and bioimaging:

C-Wave

Cobolt 04-01 Series

Single frequency, CW diode pumped lasers

Wavelength: 457 nm – 1064 nm
Power: 25 mW – 400 mW

Read more
C-Wave

Cobolt 05-01 Series

High power, single frequency, CW diode pumped lasers

Wavelength: 355 nm – 1064 nm
Power: 10 mW – 3000 mW

Read more
C-Wave

Cobolt 06-01 Series

Plug & play modulated CW lasers

Wavelength: 375 nm – 975 nm
Power: 40 mW – 400 mW

Read more
C-Wave

Cobolt Skyra™

A revolutionary multi-line laser platform

Wavelength: 405 nm – 685 nm
Power: 50 mW, 100 mW

Read more
C-Wave

C-FLEX

The compact and flexible laser combiner

Wavelength: 375 nm – 1064 nm
Power: 50 mW – 1000 mW

Read more

Publication M. Haahr et al. Permanently aligned multi-line lasers SPIE 2020 Paper 11231

Publication M. Haahr et al. Fluorescence microscopy simplified using novel multi-line lasers SPIE 2019 Paper 10884-35

Publication A. Tengholm et al. Fluorescent protein vectors for pancreatic islet cell identification in live cell imaging 2016

Publication I. Rocha-Mendoza et al. Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters 2015

Publication M. Stefaniuk et al. Light sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene Nature 2015

Publication S. Barg et al. Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site Nature 2014

Publication T. Grotjohann et al, rsEGFP2 enables fast RESOLFT nanoscopy of living cells eLife 2013

Publication S. Doose et al Single molecule FRET. Biophysics Letter 2007

Publication M. Dueser et. al Single molecule FRET SPIE Vol 6092 2006

Publication N. Zarrabi et. al. Monitoring the rotary motors of single FF-ATP

Publication U. Krzic et al. Cobolt Mambo 594nm for 3D imaging of live specimen  (German version)

Editorial Multiline lasers for fluorescence microscopy PhotonicsViews June 2019

Editorial Highly stable multi-line lasers for next-generation imaging BioOptics 2018

Editorial CW DPSS Lasers make STED Microscopy More Practical BioPhotonics 2012

Editorial Fluorescence microscopy of living cells using new 515 nm DPSS lasers Photonik International Magazine 2009

Editorial Improved contrast with 473nm for confocal microscopy OSA CLEO CThII5 2003

White paper Multi-line lasers simplify boimedical imaging

Poster Novel 515 nm DPSS laser. Focus on Microscopy 2008

Apps note Cobolt Zouk 355 nm for fluorescence microscopy and flow cytometry

Apps note TRAST microscopy for measuring oxygen concentration

Apps note Cobolt Flamenco™ 660 nm for Stimulated Emission Depletion (STED)

References

References Application Publication
Light-sheet microscopy imaging of a whole clear rat brain with Thy1-GFP transgene Light sheet microscopy Nature
C.Hertlein et.al. TIRF microscopy TIRF microscopy  ACS 2007
Cobolt-Mambo-594nm-for-flow-cytometry Flow cytometry  Application note
Cobolt-Mambo-594nm-for-Raman Raman spectroscopy  Application note
E.Illy et.al Low noise orange lasers for flow cytometry Flow cytometry  CYTO 2010
Fluorescence microscopy of living cells 515 nm lasers Fluorescence microscopy  Photonik International Magazine 2009
G.Karlsson et.al. Improved contrast with 473nm Confocal microscopy  OSA CLEO CThII5 2003
G.Strîmqvist et. al. Suppression of Raman scattering Raman scattering  PPKTP. ECLEO 2009
K.Peneva et. al. FCS  Fluorescence microscopy  J. Am. Chemical Society 2007
L.Kaestner et.al. Novel 515 nm DPSS laser Microscopy  Focus on Microscopy 2008
Multiline DPSS lasers-true Ar+ alternative  Fluorescence microscopy/ Flow cytometry  Europhotonic, July 2004
M. Duser et. al Single molecule FRET  FRET microscopy  SPIE Vol 6092 2006
N. Zarrabi et. al. Monitoring the rotary motors of single FF-ATP  FRET microscopy
Orange Laser Sources for Life Sciences Research  Fluorescence microscopy/ Flow cytometry  BioPhotonics Magazine, Jan 2010
S.Doose et. al. Single molecule FRET.  FRET microscopy  Biophysics Letter 2007
 T.Grotjohann et al, rsEGFP2 enables fast RESOLFT nanoscopy of living cells  RESOLFT microscopy  2013

Literature 04-01

References Application Publication
A. Tengholm et al. Fluorescent protein vectors for pancreatic islet cell identification in live cell imaging TIRF microscopy Pflügers Arkiv 468:1765-1777 (2016)
S. Barg et al. Contact-induced clustering of syntaxin… TIRF microscopy Nature Communications 5:3914 (2014)
C.Hertlein et.al. TIRF microscopy TIRF microscopy ACS 2007
 Cobolt Mambo 594nm for 3D imaging of live specimen 3D Imaging  Photonik International, 2011
Cobolt-Mambo-594nm-for-flow-cytometry Flow cytometry  Application note
Cobolt-Mambo-594nm-for-Raman Raman spectroscopy  Application note
E.Illy et.al Low noise orange lasers for flow cytometry Flow cytometry  CYTO 2010
Fluorescence microscopy of living cells 515 nm lasers Fluorescence microscopy  Photonik International Magazine 2009
G.Karlsson et.al. Improved contrast with 473nm Confocal microscopy  OSA CLEO CThII5 2003
G.Strîmqvist et. al. Suppression of Raman scattering Raman scattering  PPKTP. ECLEO 2009
K.Peneva et. al. FCS  Fluorescence microscopy  J. Am. Chemical Society 2007
L.Kaestner et.al. Novel 515 nm DPSS laser Microscopy  Focus on Microscopy 2008
Multiline DPSS lasers-true Ar+ alternative  Fluorescence microscopy/ Flow cytometry  Europhotonic, July 2004
M. Duser et. al Single molecule FRET  FRET microscopy  SPIE Vol 6092 2006
N. Zarrabi et. al. Monitoring the rotary motors of single FF-ATP  FRET microscopy
Orange Laser Sources for Life Sciences Research  Fluorescence microscopy/ Flow cytometry  BioPhotonics Magazine, Jan 2010
S.Doose et. al. Single molecule FRET.  FRET microscopy  Biophysics Letter 2007
T.Grotjohann et al, rsEGFP2 enables fast RESOLFT nanoscopy of living cells  RESOLFT microscopy  2013