Near-IR Pump-probe microscopy for label-free superresolution imaging

At present, most optical microscopy techniques provide sub-diffraction scale imaging based on fluorescence as the underlying contrast mechanism. Fluorescence introduces certain limitations such as a reliance on labels, photo-bleaching and a reduction of penetration depth. Two-photon excitation microscopy, which utilizes near IR femtosecond lasers as a light source, overcomes these limitations. As a novel approach based on similar laser source, we implement the proposed absorption/saturation (pump-probe) microscopy method [1, 2]. Its principles are borrowed from pump-probe spectroscopy, where in order to investigate dynamic properties of an object two femtosecond laser beams are typically used. The first beam called the ‘pump’ modifies the carrier density inside the sample. This beam is followed by intensity changes during the transmission of a second (probe) beam, thereby creating a transient contrast. The method can be further improved by introducing a third ‘doughnut’ shaped beam, which arriving together along with the pump beam and saturate the induced transition within the periphery of the focal spot. As a result the transient contrast is generated only within the central area of sub-diffraction range dimension. Our saturated transient absorption microscope (STAM) integrates this pump-probe spectroscope with a commercial Nikon microscope. The apparatus is based on a femtosecond laser coupled with an Optical Parametric Oscillator (OPO). The wavelengths of excitation and detection pulses can be tuned in accordance with experimental needs within the near IR region- a spectral range that is known to lie in the transparency window of biological tissues. By choosing specific wavelengths one can observe selected species and non-fluorescent markers. Our setup has the following capabilities: conventional confocal single and two-photon imaging both in reflection and in transmission, pump-probe imaging and saturated pump-probe. Combination of these techniques allows us to demonstrate explicit spatial and dynamic information for applications in cellular biophysics and nanochemistry