Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences.

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.

HPM live µ for a full CLEM workflow.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information-known as multimodal imaging or correlative microscopy (CM)-has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

High-resolution two-photon excitation microscopy of ocular tissues in porcine eye.

BACKGROUND AND OBJECTIVE: Two-photon excitation laser scanning microscopy (TPM), based on nonlinear optical (NLO) response under high irradiance, is currently being extensively employed for diagnostic purposes in biomedical fields and becomes more and more an interesting imaging technique in the intact bulk tissue examination. In this study, this nonlinear-excitation imaging technique including two-photon-mediated autofluorescence (2PF) and second harmonic generation (SHG) was employed to investigate the microstructures in the whole-mount scleral, retinal, and corneal tissues of porcine eyes with intracellular spatial resolution and high signal-to-noise ratio. MATERIALS AND METHODS: Image acquisition was based on the intense 80 MHz femtosecond (fs) near-infrared (NIR) laser pulses, emitted from a mode-locked solid-state titanium:sapphire system. By integrating a high-numerical aperture diffraction-limited objective, the whole-mount ocular specimens could be viewed from the surface of eye globes further to a 200 microm depth. Under high light irradiance at the order of MW-GW/cm2, more than one photon was simultaneously absorbed by endogenous molecules in ocular tissues. RESULTS: The cellular and fibrous components of whole-mount scleral and corneal tissues were selectively displayed in situ by in-tandem detection of 2PF and SHG with high efficiency without the assistance of any exogenous dye. NLO images of fibroblasts and mature elastic fibers in sclerae as well as of the retina radial Müller glial cells, ganglion cells, bipolar cells, photoreceptors, and retina pigment epithelial (RPE) cells were acquired with subcellular spatial resolution. In particular, the microstructural topography of cells and extracellular components in the whole-mount ocular tissues was elucidated in situ. CONCLUSION AND OUTLOOK: The combination of the sensitive image acquisition technique allows to selectively studying of three-dimensional (3-D) architecture of cellular microstructures and extracellular matrix arrangement in situ at substantial depths in bulk tissues. The data obtained provided the primary knowledge for further studies of imaging entire eye globes based on two-photon excitation microscopy.

Suspension (S)-FISH, a new technique for interphase nuclei.

We describe a versatile method for performing fluorescence in situ hybridization (FISH) in suspension instead of on a slide as usually done. This so-called suspension-FISH (S-FISH) opens new possibilities for the analysis of shape and functions of the human interphase nucleus. The procedure is described and the first results using this approach are presented.