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Laser scanning third-harmonic-generation microscopy in biology

D. Yelin and Y. Silberberg

Open Access Open Access

Abstract

A laser scanning microscope using third-harmonic generation as a probe is shown to produce high-resolution images of transparent biological specimens. Third harmonic light is generated by a tightly focused short-pulse laser beam and collected point-by-point to form a digital image. Demonstrations with two biological samples are presented. Live neurons in a cell culture are imaged with clear and detailed images, including organelles at the threshold of optical resolution. Internal organelles of yeast cells are also imaged, demonstrating the ability of the technique for cellular and intracellular imaging.

©1999 Optical Society of America

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Figures (7)
Fig. 1.
Fig. 1. Optical setup for laser scanning THG microscopy.

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Fig. 2.
Fig. 2. THG images of neurons in a cell culture. The size of the cell’s soma is about 15 µm.

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Fig. 3.
Fig. 3. Sectioning of live neurons in a cell culture. Each image is a horizontal section of the neuron’s soma. The sections are separated by 0.5 µm, where the top-left section is closer to the glass substrate and the bottom-right section is the top of the cell. The dimensions of each image are 20×20 µm.

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Fig. 4.
Fig. 4. Vertical sectioning of the neurons in Fig. 3. The bright nucleolus, the dark nucleus and organelles outside the nucleus can be seen.

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Fig. 5.
Fig. 5. Sectioning of live neurons. This cell has two nucleoli. The dimensions of each image are 20×20 µm.

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Fig. 6.
Fig. 6. THG images of three dendritic spines on a single dendrite.

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Fig. 7.
Fig. 7. THG images of yeast cell.

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