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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 artefacts 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 further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live μ instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep 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.

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.

The White-vented Storm-petrel Oceanites gracilis is one of the world's least known seabirds and is listed as Data Deficient by the IUCN. To date, only one breeding colony, with less than ten pairs, is known from the Chungungo Islet in Chile. To understand the breeding habitat and the threats faced by the species, it is crucial to search for other colonies. We carried out field surveys of this species in the Atacama Desert between 2013 and 2020, searching for nests along c. 870 km of coastline, from sea level to 1700 m.a.s.l. We also revisited the breeding colony at the Chungungo Islet. To detect nests, we used endoscopic cameras and scent-trained dogs. We found two new breeding colonies in Pampa Hermosa and Pampa del Indio Muerto in the Atacama Desert, and additionally, we found two non-active breeding sites in Tocopilla and Sierra Miranda. Colonies were on different substrates, including crevices in saltpetre formations, gypsum and rocks (only Chungungo Islet). The threats identified for each breeding site include light pollution and habitat destruction by mining and energy projects. Further surveys are needed to estimate the population size and to assess the conservation status of this species.

Markham's Storm-Petrel Oceanodroma markhami is one of the least known seabirds in the world. Between 2013 and 2017, we conducted an extensive ground based survey to locate nests, confirm nesting colonies and, whenever possible, characterize Markham's Storm-Petrel breeding phenology. The survey was conducted in the Atacama Desert, northern Chile, covering the area between Arica (18.240°S, 70.509°W) and Taltal (25.050°S, 70.486°W). Our study led to the discovery of two new colonies and to a six-fold increase in the estimate of the breeding population at the previously known Arica colony: a more accurate estimate of 55,308 pairs. Prior to this study, 9362 breeding pairs were known to occur in two colonies. Currently, the colonies of Arica (34,684 nests), Pampa La Perdiz (624 nests) and Salar Grande (20,000 nests) represent almost 95% of the known breeding population. We found that the colony of Arica has a different breeding phenology to that of the Pampa Perdiz and Salar Grande colonies, which could be due to differences in their food phenology. None of the colonies lie within designated protected areas and they strongly overlap with current and planned mining, wind farms and power plants in northern Chile. Apart from the disturbance caused by destruction of habitat and nests, these developments are also important sources of artificial light that cause the grounding of fledglings, after which many of them probably die from the collision impact or predation. It is vital to ensure the conservation of this species by protecting their breeding grounds and by reducing the light pollution not only in the colonies, but also at the access points to the colonies and at the big cities nearby.

Se confirma con fotografías la presencia del Playerito Menor (Calidris minutilla) en Argentina, y particularmente en la provincia de La Pampa. Previamente, su presencia había sido reportada por única vez en las Islas Orcadas del Sur, Argentina. Para Chile se amplía su rango de distribución hasta los 36°44'S, en la provincia de Concepción. The presence of the Least Sandpiper (Calidris minutilla) in Argentina, and particularly in La Pampa Province, is confirmed with photographs. Its presence had only been recorded previously with one observation at South Orkney Islands, Argentina. The distribution range in Chile is extended to 36°44'S in Concepción Province.