Reduction of distortion in photothermal microscopy and its application to the high-resolution three-dimensional imaging of nonfluorescent tissues

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 highresolution three-dimensional imaging of thick biological tissues based on the present scheme.

Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

“…The wavelength of the pump beam overlapped the absorption spectrum of the gold nanoparticles due to the plasmon resonance peak at 524 nm. It has been also demonstrated that either of the twin peaks is suppressed by an offset between the focal planes of the pump and probe beams (Figure 4a) [35]. Figure 4b shows PT images of a 20 nm gold nanoparticle with a variety of ∆z.…”

“…It has been also demonstrated that either of the twin peaks is suppressed by an offset between the focal planes of the pump and probe beams (Figure 4a) [35]. Figure 4b shows PT images of a 20 nm gold nanoparticle with a variety of z.…”

“…The spatial resolution in the pump-probe microscopy is equivalent to the confocal microscopy with zero pinhole. PT microscopy is a form of a pump-probe microscopy and imaging of a single 20 nm gold nanoparticle shows that the lateral PSF in PT microscopy is close to the products of the pump and probe beam intensities at the focus ( Figure 3b) [35]. …”

Photothermal imaging of skeletal muscle mitochondria

“…Thus, it does not involve the distortion of mitochondria due to the labeling process. In addition, its advantages include simplification of sample preparation due to the absence of a requirement for labeling and its superior resolution (lateral, ~200 nm; depth, ~500 nm) achieved by the quadratic dependence of the point spread function in pump-probe configuration [18][19][20]. To date, PT imaging of mitochondria has been performed only in limited tissue types, including nematodes [17], HeLa cells [16,21], and kidney cells [21,22].…”

Further resolution enhancement of high-sensitivity laser scanning photothermal microscopy applied to mouse endogenous

“…[1][2][3][4][5] The photothermal microscopy has been applied to various materials 6,7 including several biological samples such as cytochrome c in mitochondria, [8][9][10][11] hemoglobin in blood vessels, 12 and skin cancers. [13][14][15] Recently, our group reported a new photothermal microscope system with high detection sensitivity with low noise by a balance detection method [16][17][18] and with high-speed scanning utilizing a Galvano mirror. 19 Further resolution improvement of the pump-probe microscope applied to biological samples was achieved by non-linear detection scheme using a supercontinuum white light laser taking advantages of its high intensity with the piezo-driven stage system.…”