Multiphoton Microscope Alignment using Second Harmonics Generation Crystal


 From: delmarphotonics | March 03, 2010

Multiphoton Microscope Alignment using Second Harmonics Generation Crystal

Mavericks femtosecond Cr:Forsterite laser is aligned into Olympus FluoView FV1000 using Second Harmonic Generation (SHG) crystal, in this case BBO crystal, placed in the focal plane of the microscope.

The Mavericks-65 Cr:forsterite laser is tunable over wavelengths from 1230 to 1270 nm, making it ideal for imaging, condensed matter and biomedical
applications. These wavelengths are less damaging to biological samples than the shorter wavelengths
produced by Ti:sapphire and other femtosecond lasers. This allows in vivo imaging of cells and other biological samples.

The MFM uses pulsed long-wavelength light to excite fluorophores within the specimen being observed. The fluorophore absorbs the energy from two long-wavelength photons which must arrive simultaneously in order to excite an electron into a higher energy state, from which it can decay, emitting a fluorescence signal. It differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength, as the summed energies of two long-wavelength exciting photons will produce an emission wavelength shorter than the excitation wavelength.

Multiphoton fluorescence microscopy has similarities to confocal laser scanning microscopy. Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures, which give confocal microscopes their optical sectioning quality. The optical sectioning produced by multiphoton microscopes is a result of the point spread function formed where the pulsed laser beams coincide. The multiphoton point spread function is typically dumbbell-shaped (longer in the x-y plane), compared to the upright rugby-ball shaped point spread function of confocal microscopes.

The longer wavelength, low energy (typically infra-red) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than short-wavelength lasers, so cells may be observed for longer periods with fewer toxic effects. Many researchers are currently working toward better and higher resolution multiphoton imaging developments.


There was a recent discussion on that topic in Confocal Microscopy List with several very good example how it can be done.

I dissolved some KDP or KTP in hot water and let it evaporate onto slides and coverslips. The resulting crystals give spectacular SHG performance:
Scroll down and click on 'Second Harmonic Generation Imaging' to see my results! The pictures were generated by imaging using a 60x or 40x oil lens through a standard coverslip with crystals grown on the backside using the evaporating method. Also, there are some pictures of rat tail collagen and dorsal column membrane with SHG signal.

KDP and KTP are Potassium di- and tri-phosphate respectively. It usually comes in a powder. You can image the powder directly, but the grains are small. I prefer to dissolve the grains in hot water and grow larger crystals to get better pictures. Let the water evaporate on a coverslip, or wait for a supersaturated solution to cool; large crystals will precipitate out into solution.



Haven't tried it myself, but I've been told on good authority that a slice of  potato (lots of starch) will demonstrate SHG.  You can hardly get cheaper than
that.  I wonder if it still "performs" after it turns black from exposure to air.

Carl Boswell


A MP/SHG contact at Leica recommended urea crystals for SHG signal for measuring the instrument response function of our FLIM component. I don't know if you can buy this at the store (maybe online at, but you could produce it yourself.
Slides containing what Guy Cox suggested can probably be purchased from Carolina Biological Supply ( or Amazon.

George McNamara


There are biology shops which sell prepared slides for schools – skin would be a good choice, as would tooth or bone. If you don’t care what you are looking at and just want to see if you are detecting a signal, anything containing starch will give a good signal. Flour, cornstarch, laundry starch – they should all work. If you have any access to simple chemicals, either of the sodium phosphates should be good and very bright indeed.

Guy Cox


Del Mar Photonics Product brochures - Femtosecond products data sheets (zip file, 4.34 Mbytes) - Del Mar Photonics

Send us a request for standard or custom ultrafast (femtosecond) product

Pulse strecher/compressor
Avoca SPIDER system
Buccaneer femtosecond fiber lasers with SHG Second Harmonic Generator
Cannon Ultra-Broadband Light Source
Cortes Cr:Forsterite Regenerative Amplifier
Infrared cross-correlator CCIR-800
Cross-correlator Rincon
Femtosecond Autocorrelator IRA-3-10
Kirra Faraday Optical Isolators
Mavericks femtosecond Cr:Forsterite laser
OAFP optical attenuator
Pearls femtosecond fiber laser (Er-doped fiber, 1530-1565 nm)
Pismo pulse picker
Reef-M femtosecond scanning autocorrelator for microscopy
Reef-RTD scanning autocorrelator
Reef-SS single shot autocorrelator
Femtosecond Second Harmonic Generator
Spectrometer ASP-100M
Spectrometer ASP-150C
Spectrometer ASP-IR
Tamarack and Buccaneer femtosecond fiber lasers (Er-doped fiber, 1560+/- 10nm)
Teahupoo femtosecond Ti:Sapphire regenerative amplifier
Femtosecond third harmonic generator
Tourmaline femtosecond fiber laser (1054 nm)
Tourmaline TETA Yb femtosecond amplified laser system
Tourmaline Yb-SS femtosecond solid state laser system
Trestles CW Ti:Sapphire laser
Trestles femtosecond Ti:Sapphire laser
Trestles Finesse femtosecond lasers system integrated with DPSS pump laser
Wedge Ti:Sapphire multipass amplifier



Two Photon Microscopy and Second Harmonic Generation
Leif Gibb and David Matthews

SHG imaging: From molecules to tissues


Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms

Second-harmonic imaging microscopy of living cells

Second Harmonic Generation (SHG) Microscopy: The Forward-to-Backward (F/B) issue. Dr. Rebecca Williams


Interferometric Second Harmonic Generation microscopy

Second Harmonic Imaging of Plant Polysaccharides

Epi-third and second harmonic generation microscopic imaging of abnormal enamel

Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy


Del Mar Photonics, Inc.
4119 Twilight Ridge
San Diego, CA 92130
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