Abstract
A scheme for reducing image distortion in photothermal microscopy is presented. In photothermal microscopy, the signal shape exhibits twin peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction. This causes a distortion when imaging a structured sample in the axial plane. Here, we demonstrate that image distortion caused by the twin peaks is effectively suppressed by providing a small offset between two the focal planes of the pump and the probe beams. Experimental results demonstrate improvement in resolution, especially in the axial direction, over conventional optical microscopy-even with the focal offset. When a dry objective lens with a numerical aperture of 0.95 is used, the full width at half the maximum of the axial point spread function is 0.6 μm, which is 50% (62%) smaller than the focal spot sizes of the pump (probe) beam. Herein, we present high-resolution three-dimensional imaging of thick biological tissues based on the present scheme.
Highlights
Photothermal (PT) microscopy’s ability to measure light-absorbing molecules at high sensitivity and spatial resolution has made it useful in detecting endogenous non-fluorescent chromoproteins in biological tissues
The signal shape exhibits twin peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction
We present highresolution three-dimensional imaging of thick biological tissues based on the present scheme
Summary
Photothermal (PT) microscopy’s ability to measure light-absorbing molecules at high sensitivity and spatial resolution has made it useful in detecting endogenous non-fluorescent chromoproteins in biological tissues. In the forward detection scheme, the nano-lens (thermal lens) effect produces positive and negative peaks corresponding to the focusing or defocusing of the probe beam when a sample is scanned in the axial direction [9,10,11]. This signal shape would be useful in the tracking or positioning of nanomaterials, but it causes a serious problem in biological imaging, as the twin peaks result create distortion when reconstructing an image of a structured sample
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