Abstract
We extend the subtractive imaging method to label-free second harmonic generation (SHG) microscopy to enhance the spatial resolution and contrast. This method is based on the intensity difference between two images obtained with circularly polarized Gaussian and doughnut-shaped beams, respectively. By characterizing the intensity and polarization distributions of the two focused beams, we verify the feasibility of the subtractive imaging method in polarization dependent SHG microscopy. The resolution and contrast enhancement in different biological samples is demonstrated. This work will open a new avenue for the applications of SHG microscopy in biomedical research.
Highlights
An alternative and less direct strategy for the resolution enhancement is based on the intensity subtraction between two scanned images under the illumination of a solid focal spot and a doughnut-shaped hollow focal spot
Additional considerations on the control of the polarization states in the beam engineering process are indispensable and a pair of bright and dark beams with matched intensity and polarization distributions are essentially needed if we apply subtractive imaging to Second harmonic generation (SHG) microscopy
This makes our system to be retrofitted in commercial microscopes
Summary
An alternative and less direct strategy for the resolution enhancement is based on the intensity subtraction between two scanned images under the illumination of a solid focal spot (bright spot) and a doughnut-shaped hollow focal spot (dark spot). This method exploits the smaller feature size of the dark spot and its validity has been well demonstrated in confocal and two-photon excitation fluorescence (TPEF) microscopy[15,16,17]. An apodized vortex phase modulated circularly polarized beam is used as the dark beam to form a doughnut-shaped intensity distribution in the focal field. Taking advantage of the flexibility of dynamic diffractive optical elements, we further implement the subtraction method in SHG microscopy to demonstrate the resolution and contrast enhancement in the SHG microscopy images
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