Dataset Information

High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy.

ABSTRACT: Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia. Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction. Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining). The wavelengths previously used have shown no phototoxicity on sperm. We used TPLSM to detect spermatogenetic foci in fresh mouse testicular parenchyma without disrupting the tunica albuginea. Fresh surgically retrieved testes were observed using TPLSM within 1 h after extraction. Contralateral testes for each animal were observed using standard histology. Using TPLSM we were able to observe and measure the diameter of seminiferous tubules through the tunica albuginea, similar to the histological control. Structures within epithelial tubules were also observed, although their nature has yet to be identified. TPLSM is a real-time microscopy technique that could detect spermatogenetic foci.

SUBMITTER: Achard V

PROVIDER: S-EPMC3739577 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy.

Achard Vincent V Weber Pascal P Mercier Georges G Blanchard Marie-Pierre MP

Asian journal of andrology 20110411 5

Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia. Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction. Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining). The wavelengths previously used ha ...[more]

PMID: 21478898

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Project description:Penile size is closely concerned and short penis contributes serious sexual dysfunction and tremendous psychological problems to couples. Androgen is essential for penile development and testosterone replacement is recommended to patients with micropenis. We previously proved that inhibiting activity of lysyl oxidase (Anti-lysyl oxidase, Anti-LOX) combined with vacuum erectile device (VED) lengthened penis by remodeling tunica albuginea. We thus explored whether HCG supplement could accelerate tunica albuginea remodeling (induced by Anti-LOX + VED) to promote penile growth. Forty-two SD male rats (4 weeks old) were purchased and divided into 7 groups: control, Anti-LOX, HCG, VED (with a negative aspirated pressure of - 300 mmHg), Anti-LOX + VED, HCG + VED, and Anti-LOX + HCG + VED. After an intervention for 4 weeks, all rats' penile length, exposed penile length, and erectile function were measured. Serum samples were collected to detect hormone levels and penile corpus cavernosum were harvested for histo-pathological analysis. All intervention groups showed significantly longer penis than controlled rats. Anti-LOX sharply increased penile length and exposed length by 15% and 9% respectively, this lengthening effect was more obvious in Anti-LOX + VED group (26% and 19%, respectively). Although HCG promoted penile length by 8%, this effect was slight for exposed length (3%). Moreover, Anti-LOX + HCG + VED dramatically increased penile length and exposed length by 22% and 18%, respectively, which was similar with that in Anti-LOX + VED (26% and 19%, respectively). HCG dramatically stimulated testosterone and dihydrotestosterone secretions than control group, whether with or without Anti-LOX and VED; while it induced more AR expression than other groups. Finally, all procedures did not improve or deteriorate normal erectile function. Although we verified that Anti-LOX + VED lengthened penis by inducing tunica albuginea remodeling, however, HCG supplement did not synergize with Anti-LOX + VED to accelerate albuginea remodeling to facilitate penile growth.

Project description:Significance: Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. Aim: We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging. Approach: We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity. Results: First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics. Conclusions: The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.

Dataset Information

High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy.

Publications

High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy.

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