The work presented in this study was financially supported by the Slovenian Research Agency (research core funding nos. P3-0396 and I0-0029, as well as research projects nos. J3-9289, N3-0048, and N3-0133) and by the Austrian Science Fund / Fonds zur F&#246;rderung der Wissenschaftlichen Forschung (bilateral grants I3562--B27 and I4319--B30). We thank Maru&#353;a Ro&#353;er, Ma&#353;a &#268;ater, and Rudi Mlakar for excellent technical assistance.

The authors declare that the research was conducted in the absence of any commercial or financial interest.

The pancreatic tissue slice method is a fast experimental method to study the morphology and physiology of the endocrine and exocrine parts of the pancreas in a more conserved, in situ preparation. Many of the advantages have already been pointed out in the Introduction. It is worth pointing out that in general (i.e., not only for calcium imaging), the slice approach to study pancreatic physiology saves time because it does not involve a recovery period after isolation. The latter is not absolutely necessary with all types of experiments and uses of isolated islets from different species, but is typically employed to increase purity, restore viability and functionality, and sometimes to collect islets from several donors59,60,61,62,63,64. However, in the context of calcium imaging, beta cell responses have been found to depend on culture duration and conditions, and this is an important source of variation that should be taken into account when using isolated islets15,65. The same issue should be considered for tissue slices if their long-term culture becomes a widely used option in the future22,36. The tissue slice method also has a high yield and thus potentially reduces animal suffering and increases statistical power. Moreover, as many slices can be prepared from a single animal and because the slices survive for long periods, including the same animal or even the same islet in both the experimental and the control groups becomes feasible.
As the original architecture and cell-to-cell communications are preserved, and because it is compatible with a number of structural analyses, electrophysiological, imaging methods, and hormone secretion assays, this method is especially useful to study pancreatic functions that depend on undisturbed interactions between individual cells, e.g., sensitivity to secretagogues, paracrine and immune interactions between different cell types, patterns of electrical activity, properties of calcium dynamics, and the secretion of different hormones. For calcium imaging specifically, the main advantages of using slices are the exposure of the islet core and the possibility to acquire signals from many different cell types with high resolution. Depending on the requirements of the experiment and the age of animals, the thickness can be varied, the slices can be transfected, or obtained from animals with genetically encoded reporters. As explained in more detail below, the latter two approaches also enable specific functional identification and characterization of responses from non-beta cells 31,66. Moreover, islets from well-defined parts of the organ can be studied for differences in responsiveness or susceptibility to disease. Although they do not require a recovery incubation period, they can easily be incubated with different pharmacological agents, fatty acids, high glucose, and cytokines.
Most importantly, as high resolution is achievable in combination with single-cell or even subcellular resolution, confocal calcium imaging in slices is one of the most suitable methods for analyzing calcium waves, functional connectivity, and the different functional roles of cells in distinct parts of an islet54,67. Despite a number of advantages, the tissue slice approach has important limitations. First, it is still at least partly disruptive to islet and exocrine architecture, especially at the cut surface, and precautions, such as low temperature, frequent exchange of solutions, and gentle and quick manipulation, are needed during preparation to prevent additional mechanical and endogenous enzymatic damage. Second, the patterns of nutrient and secretagogue delivery are still inferior to the in vivo route, the preparation is detached from systemic innervation, and inter-organ feedback, such as between the islet and its target tissues, is impossible, in contrast to in vivo approaches. Third, maximum slice thickness is limited by oxygenation, nutrient delivery, and pH regulation at ~200 &#181;m9. Further, both preparation of slices and imaging need a lot of training, and in-depth analyses of calcium data from long time series and from many cells require specialized knowledge that is often not included in the toolkit of a classical physiologist and requires help from physicists or data scientists. The advantage that homo- and heterotypic interactions are preserved can also complicate the analysis of samples due to the presence of signals from other cells in regions of interest. Depending on protocols, activation of other cells can lead to indirect additional stimulation or inhibition of an observed cell.
This can only be resolved conclusively by deconvolution approaches, by more complex stimulation protocols including substances that block some of the indirect effects, by using specific knock-out animals, and by careful comparison of results with results from other studies employing more reductionist methodologies. Additionally, if secretion measurements are necessary, it should be kept in mind that some slices may lack islets, and the total mass of endocrine tissue in a single slice is typically low. The preparation of acute pancreatic tissue slices for imaging involves several critical steps discussed in the following sections and summarized in Table 1, where the reader can also find short, but important tips for troubleshooting. First, when preparing the agarose solution, the agarose powder must dissolve completely, otherwise the undissolved particles may obstruct the injection. Keep the homogeneous agarose solution at 37-45 &#176;C to prevent hardening of agarose due to too low a temperature on the one hand and to prevent tissue damage due to too high temperatures on the other hand. After use, the remaining agarose may be stored at 4 &#176;C and reheated, although repeated reheating can result in increased density due to water evaporation, eventually making the injection difficult or impossible.
The next critical step in the preparation is clamping the major duodenal papilla correctly. A white spot on the duodenum indicates the junction of the common bile duct and the duodenum. A clamp placed too proximally will result in obstruction of some lateral pancreatic branches of the common duct, disabling the injection of these parts, whereas a clamp placed too distally will result in agarose leaking through the lower resistance path directly into the duodenum. Before cannulation of the common bile duct, the surrounding adipose tissue can be carefully removed for better visualization of the duct and greater control during injection. Insufficient precision during removal of the surrounding tissue may result in perforation of the duct. The selection of the needle diameter used for agarose injection is also important. In mice, a 30 G needle is preferably used; smaller (32 or 33 G) needles require more effort due to high viscosity of the agarose solution and are more prone to obstruction. However, if used in combination with a lower-density agarose solution, they can be very helpful in smaller mouse strains and younger animals. During the initial postnatal days, agarose may alternatively be injected subcapsularly rather than intraductally2. Using needles with greater diameter in mice will most probably result in damaging the common bile duct. This can also happen with the correct needle diameter, and a forceps can help in keeping the needle in place during injection. Larger diameter needles may be the only solution in case of larger ducts, as found in rats. If the needle is too narrow to ensure a tight seal preventing back-leakage, a ligature may be placed around it upon successful entry into the duct.
Agarose injection takes some effort due to the solution&#39;s viscosity, and once the injection process has started, it should not be interrupted as the low-melting-point agarose solution may solidify in the needle or the largest parts of the ductal tree before the injection is completed. This will result in poor tissue penetration and worse support during cutting. The duct should always be cannulated at the point where the left hepatic duct and the cystic duct join to form the common bile duct.. If the common bile duct gets perforated, repeatedly try cannulating closer to the duodenum. When the pancreas is sufficiently stabilized with agarose solution and extracted from the peritoneal cavity, small pieces of well-injected tissue are cut. Before embedding them into the agarose, it is crucial to remove all the adipose and connective tissues as their residues make slicing more challenging. The same applies to blood vessels and duct residues, except when they are the focus of the experiment. In this case, make sure to position them in such a way that the desired cross-section will be obtained. When embedding the tissue in agarose, ensure that the temperature is appropriate (37 &#176;C), and that the tissue is completely surrounded by agarose, as forces during vibratome slicing can rip out the pancreas tissue from the agarose blocks.
Quickly drying the tissue blocks before placing them in agarose by placing them briefly on a paper tissue can help prevent poor contact between tissue and agarose during this step. During solidification of agarose blocks, place the Petri dish horizontally, and prevent contact between the pancreas tissue and the bottom of the Petri dish. If the pancreas is not fully injected, the cutting process will be challenging. Therefore, try to reduce the cutting speed to obtain tissue slices. To minimize cell damage during vibratome slicing, replace the ECS (and the ice cubes made of ECS) in the slicing chamber regularly. The latter will reduce the activity of pancreatic enzymes released from acinar tissue during slicing. The thickness of the slices is also of crucial importance. For calcium dynamics and electrophysiological experiments, 140 &#181;m slices are usually cut; however, according to the aim of the study, slice thickness can range from 90 &#181;m to 200 &#181;m. Keep in mind that in thicker slices, the diffusion of oxygen and nutrients will be limited, but they will include more tissue. Additionally, the proportion of uncut islets may be expected to increase with increasing slice thickness. Slices can be stored in a regularly exchanged ECS at room temperature for several hours or even cultivated in an appropriate cell medium for several days; however, this may eventually affect the normal islet cell physiology3,22.
When preparing the dye solution, ensure careful mixing of all components, and avoid exposure to ambient light. The pancreatic slice is composed of many cell layers, and the uptake of calcium dye is limited to the first few most superficial cell layers, as described previously for isolated islets58,68, and pituitary slices69. However, in contrast to isolated islets where the surrounding capsule and outer cell layers hinder the penetration of the dye into deeper layers, tissue slices permit access to the entire cross-sectional surface of the islet, enabling simultaneous measurement of calcium dynamics in hundreds of cells from all layers of an islet. Fluorescent Ca2+ indicators are the most widely used for measuring calcium dynamics, and together with CLSM, they enable recordings with high temporal resolution, reaching several hundred Hertz. When selecting the most appropriate fluorescent Ca2+ indicator, consider different factors, including the indicator form, which influences the cell loading method, measurement mode (qualitative or quantitative), and dissociation constant (Kd) that needs to be in the Ca2+ concentration range of interest and depends on pH, temperature, presence of Mg2+ and other ions, as well as protein binding. As cellular Ca2+ signals are usually transient, the Ca2+ binding rate constant should also be considered. For measuring [Ca2+]IC dynamics in pancreatic cells, this group mainly uses the cell-permeable Ca2+ indicator dye described in this protocol (Table of Materials) as it is a long wavelength indicator with the emission wavelengths in the spectrum where cellular autofluorescence is usually less problematic, and the energy of excitation light is low, which reduces the potential for cellular photodamage. Because this dye is fluorescent at low Ca2+ concentrations, this facilitates the determination of baseline [Ca2+]IC and increases cellular visibility before stimulation. After binding Ca2+, the fluorescence intensity of the dye increases 14-fold, enabling detection of even slight changes in [Ca2+]IC.
For successful live-cell calcium imaging, several crucial hardware parameters need to be considered, as described in the protocol section. For live-cell imaging wherein signal amplitudes are low and chances of phototoxicity are high, objectives with a higher NA are preferably used to collect more light from the specimen. If calcium dynamics must be recorded with a high temporal resolution, use the resonant scanner instead of linear galvanometers. Besides choosing the right objective, the use of highly sensitive detectors-such as hybrid detectors that require less laser power-avoids phototoxicity and photobleaching. This is of special importance for long-lasting calcium imaging. Other important steps in calcium imaging are parameter settings of image quality for time series acquisitions. The most important are the temporal and the spatial resolution. As the calcium dynamics per se determines the lowest acceptable temporal resolution, the sampling rate needs to be at least two-fold higher than the expected signal frequency to detect the signal or even 10 times higher to detect the shape of the signal reliably. In acute pancreatic tissue slices, calcium dynamics can be measured in hundreds of cells simultaneously and therefore, the spatial resolution is also important. This can be enhanced by increasing the number of pixels or by increasing the line averaging during live acquisition. However, because of the inverse relationship between the spatial and the temporal resolution, a trade-off between both settings is needed.
If calcium imaging has to be performed in a specific cell population within the pancreas, a stimulus able to functionally differentiate the cells within the slice is necessary. High glucose reliably and quickly activates beta cells to an oscillatory pattern that is superimposed on an elevated calcium level and is highly synchronized among all cells within an islet32,58,70. The beta cells are the most numerous cell type within an islet and are located mostly in the islet core in mice. The same stimulation protocol decreases and sometimes does not appreciably change the bursting in alpha cells30,32,58,70,71,72. To discriminate alpha cells functionally, low (3 mM) glucose, glutamate or adrenalin can be used to increase their frequency or basal [Ca2+]IC21,72,73,74,75. They represent 10-20% of islet cells and will be detected on the islet periphery1. Delta cells are also found on the periphery. They make up only ~5% of the total number of endocrine cells in an islet and are typically active in 6 mM glucose and respond to glucose stimulation with an increased irregular bursting activity from the baseline or a slightly elevated calcium level1,32,71,76. Ghrelin can be used for specific stimulation of delta cells21,77,78,79 in calcium imaging experiments. However, protocols for specific functional identification of PP and epsilon cells remain to be defined. Further, 25 nM acetylcholine reliably activates acinar cells into bursting activity35,80,81. Additionally, a number of other secretagogues, such as cerulein, cholecystokinin, and carbamylcholine, can be used to evoke calcium responses in acinar cells22,40,82,83.
Finally, 1 mM chenodeoxycholic acid reliably evokes calcium responses in ductal cells in tissue slices; angiotensin II, ATP, and some other secretagogues can also be used11,23,84,85. Whenever a functional identification based on characteristic responses to specific secretagogues and inhibitors is not sufficient, genetically labelled animals31, transfected cells73, or immunocytochemistry can be employed for the identification of different cell types9,22,71,86. During the last couple of years, the tissue slice method has been successfully adapted to human tissue, opening many new important research avenues in both exocrine41 and endocrine physiology9,36,37,39. Interestingly, a detailed assessment of calcium dynamics in human islets has been notoriously difficult and remains to be investigated in greater detail87. Combined with advanced confocal microscopy, the pancreatic tissue slice method has enabled many new insights into the calcium dynamics in mice and will hopefully do the same for human tissue.

The mammalian pancreas is a large exocrine and endocrine gland. The exocrine part makes up 96-99% of the total pancreas volume and consists of acini and ducts. The endocrine part is made up of a large number of islets of Langerhans accounting for the remaining 1-4% of the total pancreas volume1. The exocrine part secretes major digestive enzymes that break down energy-rich polymers in food, as well as a bicarbonate-rich fluid, which combines with other gastrointestinal secretions to provide an environment suitable for the action of enzymes. The endocrine part secretes hormones that regulate postprandial distribution, storage, and interprandial release of energy-rich nutrients. Although the exocrine tissue is relatively underdeveloped and the endocrine relatively well-developed at birth, the former quickly overgrows the latter upon weaning2,3,4. Early studies of pancreatic function marked the birth of modern physiology, and major methodological advancements in the field have been followed by major scientific breaktroughs5. Working with the pancreas is technically challenging due to the intricate structure of the gland, but is also a big motivation because of diseases such as pancreatic cancer, pancreatitis, and diabetes that present major threats to public health, and for which novel therapeutic approaches are needed.
Isolated islets6, acini7,8, and ductal fragments had been developed and used for decades as gold standard methods owing to their advantages compared with cell lines and primary dispersed endocrine, acinar, and ductal cells9,10. Despite the markedly improved function of isolated cell collectives, these methods still involve considerable mechanical and enzymatic stress, isolate cells from the surrounding tissue and thus lack paracrine interactions and mechanical support, and most importantly, are accompanied by significant changes to normal physiology11,12,13. The acute mouse pancreatic tissue slice was developed in 2001 out of a perceived need to develop an experimental platform similar to brain, pituitary, and adrenal slices with preserved intercellular contacts, paracrine interactions, mesenchyme, and tissue architecture, as well as without some of the most important shortcomings of the gold standard method in islet research of that time-the isolated islets12,14. Among these shortcomings are damage to the outermost layers, lack of accessibility of core islet areas, and the need for cultivation with possibly important effects on cell identity and physiology12,15. Moreover, the tissue slice method enables studies on animal models with grossly deranged islet architecture where it is impossible to isolate islets, or when islet yield is extremely low by traditional isolation16,17,18,19,20,21.
Additionally, the slice is more suitable for studying morphological changes during the development of diabetes and pancreatitis, for instance, as it enables a better overview of the whole tissue and is also compatible with studying regional differences. Importantly, despite the early focus on the endocrine part, the tissue slice method inherently enables the study of the exocrine components9,22,23. During the first decade after its introduction, the method was employed for electrophysiological studies of beta14,24,25,26,27,28,29 and alpha30,31 cells as well as for examining the morphological and functional maturation of the pancreas2,3. A decade later in 2013, the method was successfully adapted for live-cell calcium imaging of islet cells using CLSM to characterize their responses to glucose32, their functional connectivity patterns33, and the relationship between membrane potential and intracellular calcium by combining a fluorescent calcium dye with a membrane potential dye34. Later in the same year, the method was also used to assess calcium&#160;dynamics in acinar cells22,35. Over the following years, pancreatic tissue slices have been used in a number of different studies and successfully adapted to pig and human tissue9,36,37,38,39,40,41. However, taken together, calcium imaging-in mouse pancreatic tissue slices in general and in islets in particular-is still mostly performed by this group. One of the main reasons for this may lie in the combination of a technically challenging tissue slice preparation, the need for a confocal microscope, and rather complex data analysis. The main aim of the present paper is to make this powerful method more accessible to other potential users.
There are already some excellent methodological articles dealing in detail with tissue slice preparation and the use of slices for structural and secretion studies, but not for confocal calcium imaging9,42,43. Therefore, this paper focuses on some additional tips and tricks during the preparation of slices, on steps critical for successful dye loading, image acquisition, as well as on the main steps of basic calcium data analysis. Therefore, this contribution should be viewed as being complementary to rather than as an alternative for the abovementioned method. Similarly, calcium imaging in mouse pancreatic tissue slices shall be viewed as an experimental approach to be used to answer specific questions and is thus complementary to rather than an absolute alternative for other calcium imaging approaches in pancreatic physiology such as isolated ducts or acini, isolated islets, organoids, islets transplanted into the anterior chamber of the eye, and recordings in vivo11,44,45,46,47,48. The promise of calcium imaging in mouse pancreatic tissue slices is probably best illustrated by recent successful recordings of calcium dynamics in islet mesenchymal cells such as pericytes49 and macrophages50, as well as in ductal cells23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance KERN ALJ 120-4</td>
			<td>KERN &#38; SOHN GmbH</td>
			<td>ALJ 160-4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope Leica TCS SP5 II Upright setup</td>
			<td>Leica</td>
			<td>5100001578</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope Leica TCS SP5 AOBS Tandem II setup</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cork pad 15 cm x 15 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;15 mL centrifuge tubes</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>CLS430790</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Round Ice Bucket with Lid, 4 L</td>
			<td>Fischer Scientific, Leicestershire, UK</td>
			<td>432124</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge razor blade</td>
			<td>Personna, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>FST, Germany</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 0.5 mL</td>
			<td>Eppendorf</td>
			<td>0030 121.023</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask 200 mL</td>
			<td>IsoLab, Germany</td>
			<td>027.01.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - ToughCut</td>
			<td>FST, Germany</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat orbital shaker&#160; IKA KS 260 basic</td>
			<td>IKA</td>
			<td>Ident. No.: 0002980200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass lab bottle 1000 mL</td>
			<td>IsoLab, Germany</td>
			<td>091.01.901</td>
			<td></td>
		</tr>
		<tr>
			<td>Hartman Hemostat, curved</td>
			<td>FST, Germany</td>
			<td>13003-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HCX APO L 20x/1.00 W HCX APO L (water immersion objective, 20x, NA 1.0)</td>
			<td>Leica</td>
			<td>15507701</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder 25 mL</td>
			<td>IsoLab, Germany</td>
			<td>015.01.025</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator Control box SM-7, Keypad SM-7</td>
			<td>Luigs &#38; Neumann</td>
			<td>&#160;200-100 900 7311, 200-100 900 9050</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave owen</td>
			<td>Gorenje, Slovenia</td>
			<td>MO20MW</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer Gonotec 010</td>
			<td>Gonotec, Berlin, Germany</td>
			<td>OSMOMAT 010 Nr. 01-02-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td>Faber-Castell, No.2</td>
			<td></td>
			<td>Any thin soft round paint brush No.2, preferably black</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perifusion pumps</td>
			<td>Ismatec</td>
			<td>ISM 827</td>
			<td>Reglo Analog MS - 4/8</td>
		</tr>
		<tr>
			<td>Petri dish 100/20 mm</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35/10 mm</td>
			<td>Greiner bio-one</td>
			<td>627102</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35 x 10 mm Nunclon Delta</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>153066</td>
			<td>NON-STICKY for agarose blocks</td>
		</tr>
		<tr>
			<td>pH meter inoLab pH Level 1</td>
			<td>WTW, Weilheim, Germany</td>
			<td>E163694</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 1000 mL</td>
			<td>Eppendorf</td>
			<td>3121 000.120</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 50 mL</td>
			<td>Eppendorf</td>
			<td>3121 000.066</td>
			<td></td>
		</tr>
		<tr>
			<td>Push pins 23 mm</td>
			<td>Deli, Ningbo, China</td>
			<td>E0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 mL</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken Forceps</td>
			<td>FST, Germany</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilizing ring for Erlenmeyer flask</td>
			<td>IsoLab, Germany</td>
			<td>027.11.048</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope Nikon SMZ 745</td>
			<td>Nikon, Melville, NY USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Injekt Solo 5 mL</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4606051V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle 0.30 x 12 mm (30 G x 1/2&#34;)</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Luigs &#38; Neumann</td>
			<td>200-100 500 0150, 200-150-500-145</td>
			<td>Slice mini chamber, Temperature controller TC 07</td>
		</tr>
		<tr>
			<td>Tubings for perifusion system</td>
			<td>Ismatec</td>
			<td>SC0310</td>
			<td>Ismatec Pharmed 1.14 mm(ID) + silicone tubing 1.0 (ID) x 1.8 mm(OD)</td>
		</tr>
		<tr>
			<td>Ultrasonic bath Studio GT-7810A</td>
			<td>Globaltronics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome Leica VT 1000 S</td>
			<td>Leica, Nussloch, Germany</td>
			<td>14047235613</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask 1000 mL</td>
			<td>IsoLab, Germany</td>
			<td>013.01.910</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer Neolab 7-2020</td>
			<td>Neolab</td>
			<td>&#160;7-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Thermo Haake open-bath circulator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Z527912</td>
			<td></td>
		</tr>
		<tr>
			<td>Material/Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate - CaCl2.2H2O</td>
			<td>Sigma Aldrich, Germany</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma Aldrich, Germany</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D4540-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-lactic acid</td>
			<td>Sigma Aldrich, Germany</td>
			<td>L1250-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas mixture containing 95% O2 and 5% CO2 at barometric pressure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glue Wekem sekundenkleber WK-110</td>
			<td>Wekem GmbH, Bergkamen, Germany</td>
			<td>WK 110-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich, Germany</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(+)-ascorbic acid</td>
			<td>Sigma Aldrich, Germany</td>
			<td>A9,290-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate - MgCl2.2H2O</td>
			<td>Sigma Aldrich, Germany</td>
			<td>M2670-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Sigma Aldrich, Germany</td>
			<td>I5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oregon Green 488 BAPTA-1, AM</td>
			<td>Invitrogen (Thermo FisherScientific)</td>
			<td>O6807</td>
			<td>cell-permeable Ca2+ indicator (excitation/emission: 495/523 nm)</td>
		</tr>
		<tr>
			<td>Pluronic F-127 (20% Solution in DMSO)</td>
			<td>Invitrogen (Thermo Fisher Scientific)</td>
			<td>P3000MP</td>
			<td>polaxamer: nonionic triblock copolymer</td>
		</tr>
		<tr>
			<td>Potassium chloride - KCl</td>
			<td>Sigma Aldrich, Germany</td>
			<td>31248</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaPlaque GTG agarose</td>
			<td>Lonza, Rockland, USA</td>
			<td>50111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate - NaHCO3</td>
			<td>Honeywell, Germany</td>
			<td>31437-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride - NaCl</td>
			<td>Honeywell, Germany</td>
			<td>31434-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide - NaOH</td>
			<td>Sigma Aldrich, Germany</td>
			<td>30620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic- NaH2PO4</td>
			<td>Sigma Aldrich, Germany</td>
			<td>S0751-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich, Germany</td>
			<td>15990-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>FIJI is an open source project</td>
		</tr>
		<tr>
			<td>LASAF</td>
			<td>Leica microsystems, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Python</td>
			<td>Python Software Foundation</td>
			<td></td>
			<td>Python is an open source project</td>
		</tr>
	</tbody>
</table>

confocal laser scanning microscopy of calcium dynamics in acute mouse pancreatic tissue slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

The injection of the agarose solution into the pancreatic duct is the most critical step in pancreas tissue slice preparation. A successful injection can be recognized by a whitening of the pancreas tissue, as seen on the left side of Figure 3A, while an incompletely injected part of the pancreas is presented on the right side of Figure 3A. The islets of Langerhans can be recognized by the naked eye or under a stereomicroscope, and this aids in cutting the appropriate parts of the pancreas for subsequent embedding in agarose blocks (Figure 3B). In a freshly cut mouse pancreatic tissue slice, islets of Langerhans can be easily distinguished from the surrounding exocrine tissue and mesenchyme as white spots under the stereomicroscope (Figure 3C) or as brownish structures under the light microscope (Figure 3D). The pancreatic tissue slices can be used for distinct types of experiments for at least 12 h after slicing. In addition to the gross morphological assessment under the stereomicroscope, the light microscope, and the functional responses of cells during calcium imaging, the viability of the pancreatic tissue slices can be assessed (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62293/62293fig04.jpg" /> 
Figure 4: Viability of cells within the tissue slice. Viability of cells was determined with the Live/Dead assay. Live cells are stained by Calcein AM (shown in green), while dead cells are stained with ethidium homodimer-1 (shown in red). Yellow lines denote the position of the X-Y cross section of the Z-stack displayed at the bottom and the right. The full depth of the Z-stack is 88 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62293/62293fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For calcium imaging experiments, the fluorescent calcium indicator needs to penetrate through a few layers of cells. Figure 5A presents successful loading of the cell-permeable Ca2+ indicator dye into the pancreatic tissue slice in which individual islet and acinar cells can be recognized. In contrast, slices in Figure 5B-D are not optimal due to unsuccessful penetration of the dye (Figure 5B), lack of islet cells (Figure 5C), and a lot of necrotic tissue on the surface (Figure 5D). Such slices can be discarded, checked for the presence of additional islets that are cut or stained better (see Table 1 for troubleshooting), or used for recording the responses of exocrine cells.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62293/62293fig05.jpg" /> 
Figure 5: Examples of usable and unusable preparations. (A) An example of a successful preparation of the pancreas tissue slice with well-stained cells in the islets of Langerhans, as well as ductal cells and surrounding acinar tissue. (B) An example of a poorly stained tissue slice. (C) Example of an islet of Langerhans with structural discontinuations. (D) An example of an islet of Langerhans containing many dead cells and a lot of debris. The &#34;glow-over, glow-under&#34; lookup table on the right displays 0 intensity in green and saturation in blue. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62293/62293fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Representative results from calcium imaging using the cell-permeable Ca2+ indicator dye are shown in Figure 6. In Figure 6A, a high-resolution image of a pancreatic tissue slice is presented, containing an islet of Langerhans, acinar tissue, and a pancreatic duct. For better distinction, the endocrine, exocrine, and ductal part of the pancreatic tissue slice presented in Figure 6A are colored in Figure 6B. Using appropriate stimuli can functionally discriminate between different islet cells, or islet and non-islet cells51. Beta cells will typically respond to a square-pulse stimulation by glucose with a transient increase in [Ca2+]IC followed by fast calcium oscillations on a sustained plateau (Figure 6C, upper panel).
As all beta cells are coupled into a single, large, functional syncytium, these oscillations are also very well synchronized among different cells by means of spreading [Ca2+]IC waves32,34,52,53,54 (Figure 7C). Slower [Ca2+]IC oscillations with a period of 5-15 minutes may underlie the fast oscillations or even be the predominant type of reponse55,56. The same simple protocol may reveal other types of responses, especially at the periphery of islets (Figure 6C, lower panel). As these cells are not synchronized with beta cells and respond with faster and more irregular oscillations that are already present in low glucose conditions or with a decrease in activity, such responses are highly suggestive of non-beta cells21,32,57,58. However, their definitive functional characterization requires more complex protocols with additional stimulation steps or alternative approaches, which are discussed below. Typical responses of acinar and ductal cells are presented in Figure 6D and Figure 6E, respectively. Refer to the literature for more details on acinar and ductal cells22,23,35.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62293/62293fig06.jpg" /> 
Figure 6: Representative results of calcium dynamics in distinct types of pancreatic cells. (A) A high-resolution image of an islet of Langerhans with surrounding tissue. Scale bar = 100 &#181;m. (B) Delineation of distinct parts of pancreatic tissue with acinar tissue shown in yellow, an islet of Langerhans shown in red, and a segment of the ductal tree in blue. Scale bar = 100 &#181;m. (C) Typical traces of calcium dynamics in beta and putative non-beta cells during stimulation with 12 mM glucose; 3 mM glucose was used for non-stimulatory conditions. Protocols that can be used for more specific discrimination of non-beta cells are described in the discussion section. (D) A typical trace of calcium dynamics of acinar cells stimulated by 25 nM acetylcholine. (E) A typical trace of calcium dynamics of ductal cells stimulated by 1 mM chenodeoxycholic acid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62293/62293fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After successful calcium imaging, the data are first exported and corrected for bleaching by a combination of an exponential and linear fit, as described in the protocol section. A time series before and after bleaching correction is presented in Figure 7A. Thereafter, several parameters in the activation and deactivation phase of the response as well as the plateau phase can be analyzed. A delay in the onset of [Ca2+]IC increase after stimulation can be measured as represented by delayA in Figure 7B and the heterogeneity in delays among individual cells (delayA1). The same parameters (delayD and delayD1)&#160;can be used to describe the deactivation phase. Following the initial transient [Ca2+]IC increase, the plateau phase in most pancreatic beta cells in an islet is characterized by relatively regular high frequency [Ca2+]IC oscillations. The plateau phase can be described by analyzing the classical functional parameters. The schematic presentation of [Ca2+]IC oscillations duration, frequency, and percentage of active time are presented in Figure 7C. In calcium imaging with acquisition rates higher than 10 Hz, calcium waves repeatedly spreading across the islet can also be recognized clearly (Figure 7C).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62293/62293fig07.jpg" /> 
Figure 7: Analysis of time-series data. (A) Correction of time-series data for photobleaching. (B) Analysis of delays to activation after stimulation and to deactivation after cessation of stimulation with 12 mM glucose. Duration of stimulation is denoted by the light gray, shaded bar in the image. (C) Analysis of several parameters of the plateau phase: I) Duration of the oscillation determined at half-height, II) frequency of oscillations determined by inter-oscillation intervals. III) active time as a product of frequency and duration of oscillations. I-IV) Delays between oscillations in any given wave of oscillations that spread across the islet of Langerhans determined by the delays (&#916;t) in time at which a single cell reaches half-height of the oscillation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62293/62293fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62293/Table 1 (3).xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices at JoVE.com

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

confocal-laser-scanning-microscopy-calcium-dynamics-acute-mouse

Nanyang Technological University

Research

JoVE Journal

Biology

3.8K Views.  University of Maribor. We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

Video: Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

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Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples