IMSI—Guidelines for Sperm Quality Assessment

Intracytoplasmic sperm injection (ICSI) is a widely used and accepted treatment of choice for oocyte fertilization. However, the quality of sperm selection depends on the accurate visualization of the morphology, which can be achieved with a high image resolution. We aim to correct the conviction, shown in a myriad of publications, that an ultra-high magnification in the range of 6000×–10,000× can be achieved with an optical microscope. The goal of observing sperm under the microscope is not to simply get a larger image, but rather to obtain more detail—therefore, we indicate that the optical system’s resolution is what should be primarily considered. We provide specific microscope system setup recommendations sufficient for most clinical cases that are based on our experience showing that the optical resolution of 0.5 μm allows appropriate visualization of sperm defects. Last but not least, we suggest that mixed research results regarding the clinical value of IMSI, comparing to ICSI, can stem from a lack of standardization of microscopy techniques used for both ICSI and IMSI.

Exploring the hidden depth by confocal Raman experiments with variable objective aperture and magnification

The article analyzes experimentally and theoretically the influence of microscope parameters on the pinhole-assisted Raman depth profiles in uniform and composite refractive media. The main objective is the reliable mapping of deep sample regions. The easiest to interpret results are found with low magnification, low aperture, and small pinholes. Here, the intensities and shapes of the Raman signals are independent of the location of the emitter relative to the sample surface. Theoretically, the results can be well described with a simple analytical equation containing the axial depth resolution of the microscope and the position of the emitter. The lower determinable object size is limited to 2–4 μm. If sub-micrometer resolution is desired, high magnification, mostly combined with high aperture, becomes necessary. The signal intensities and shapes depend now in refractive media on the position relative to the sample surface. This aspect is investigated on a number of uniform and stacked polymer layers, 2–160 μm thick, with the best available transparency. The experimental depth profiles are numerically fitted with excellent accuracy by inserting a Gaussian excitation beam of variable waist and fill fraction through the focusing lens area, and by treating the Raman emission with geometric optics as spontaneous isotropic process through the lens and the variable pinhole, respectively. The intersectional area of these two solid angles yields the leading factor in understanding confocal (pinhole-assisted) Raman depth profiles. Graphical abstract

Performing Microdissection Testicular Sperm Extraction: Surgical Pearls from a High-Volume Infertility Center

Microdissection testicular sperm extraction (mTESE) has been demonstrated to be the gold-standard surgical technique for retrieving testicular sperm in patients with non-obstructive azoospermia (NOA) as it enables the exploration of the whole testicular parenchyma at a high magnification, allowing the identification of the rare dilated seminipherous tubules that may contain sperm, usually surrounded by thinner or atrophic tubules. MTESE requires a skilled and experienced surgeon whose learning curve may greatly affect the sperm retrieval rate, as demonstrated in previous reports. The present review is intended to offer a precise and detailed description of the mTESE surgical procedure, accompanied by an extensive iconography, to provide urologists with valuable information to be translated into clinical practice. Advice about the pre-surgical and post-surgical management of patients is also offered.