Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

• Published in: Journal of microscopy (Oxford) 2015-10, Vol.260 (1), p.62-72
• Main Authors: VOGEL, MARTIN, WINGERT, AXEL, FINK, RAINER H.A., HAGL, CHRISTIAN, GANIKHANOV, FERUZ, PFEFFER, CHRISTIAN P.
• Format: Article
• Language: English
• Subjects: Coherent anti‐Stokes Raman scattering; condenser; multimodal nonlinear microscopy; third harmonic generation; third‐order sum frequency generation; ultraviolet transmission
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/jmi.12267

Summary Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm. Summary for nonexpert readers In modern microscopy, images from samples are often generated by scanning the spot of a pulsed laser over a sample, line by line. One, two or three photons of the laser beam will then excite the sample to emit light at other wavelengths, which we can collect with photo detectors. The signal from the photo detector will be fed into a computer where we then get the final image. Unfortunately, these multiphoton laser scanning microscopes are expensive, and many labs can therefore buy only a very basic configuration, which does not allow the measurement of signals excited by three or more photons – these are yet effects that are very useful for the reliable detection of certain structures in samples. We have therefore set out to customize our standard multiphoton microscopes in such a way that the measurement of these additional signals is possible. We describe the customization process in detail and, as we used only optical elements that are commonly available on the market, we hope that other labs can make use of this relatively low‐cost solution to get more experimental information out of their laboratory equipment.