Optical Information Processing and Holography Volume 4 - 2023 | https://doi.org/10.3389/fphot.2023.1096294 This article is part of the Research TopicEditors' Showcase: Frontiers in PhotonicsView all 9 articles The purpose of the article is to explore the need and advantages of using the incoherent color holography lattice light-sheet (ICHLLS) to provide multiwavelength quantitative monitoring of 3D cellular dynamics in live tissue to further understand complex functions of cells and cellular compartments We have explored the use of incoherent color holography lattice light-sheet to investigate colocalization of fluorescent markers in live cells in intact tissue Neuronal structures provide an attractive target for incoherent color holography lattice light-sheet The cells show a complex architecture in 3D space in which signaling both between cells and within subcellular structures requires colocalization of proteins and lipids to function During activity and over long periods it is important in understanding these signaling functions in Parkinson’s Alzheimer’s and motoneuron diseases within live cells in intact tissue As a proof of concept this article recalls the key aspects in lattice light-sheet imaging and provides a description of the incoherent detection system configuration to actively control dual diffractive lenses phase-shifting at multiple excitation wavelengths sequentially The incoherent color holography lattice light-sheet system will allow simultaneous recording of multidimensional object waves that contain intensity in 3D space We measure colocalization of fluorescence indicators introduced into live cells in intact neural tissue fixed tissue loses that very dynamic activity which defines cell function We seek to develop live cell 3D imaging approaches that can capture multiple wavelengths of fluorescence information in 3D space sufficiently rapidly to examine dynamic co-localizations between structures and fluorescent molecules An extreme example of colocalization necessary for function in the central nervous system is in the structural complexity of neurons To understand neuronal function at all levels it is necessary to image the spatiotemporal properties of axons and somata in 3-dimensional space simultaneously and with high resolution because the LLS system excites with a lattice with regions of positive and negative interference This variance is smoothed by dithering the beam in the x plane with a similar x-galvo While LLS beams are coherent and produce stronger modulation depth than incoherent patterned light sheets they work by causing fluorescence in the sample which results in emission light that is incoherent We explore here the self-interference property of this emitted fluorescent light in which interference patterns are created to generate holograms of a 3D object with the depth information encoded in these patterns We built a single-wavelength incoherent holography lattice light-sheet (IHLLS) (Potcoava et al., 2021a; Potcoava et al., 2021b; Rosen et al., 2021) but here we combine IHLLS with multiwavelength information at 488 nm and 561 nm termed incoherent color holography lattice light-sheet (ICHLLS) to provide quantitative monitoring of 3D cellular dynamics in live tissue The system could be developed further for all four excitation laser lines (405 and 642 nm) of the existing LLS system by configuring a phase spatial light modulator placed in the incoherent detection arm of the instrument to actively control dual diffractive lens phase-shifting at all colors sequentially The ICHLLS system is built as a new detection module on an existing LLS system This holographic technique gives us the possibility to access the complex amplitude of the object’s wavefront which describes both the amplitude and phase of a light wave Amplitude and phase measurements acquired with our new instrument provide intrinsic instrumental simplicity and higher resolution when compared to the original LLS schemes Phase imaging provides quantitative information on the state and size of subcellular structures In this paper we focus on assessing the colocalization between dual-color excited structures and fluorescent molecules detected in incoherent holographic images The rest of the paper is structured as follows: in the next section the principle of the ICHLLS system is provided with a short theory on the colocalization approach Section 3 covers the materials and methods Section 4 covers a series of ICHLLS experiments using calibration beads and neuronal cells The interference patterns created by the two beams at each excitation wavelength create Fresnel holograms The beam splitter of an interferometer is replaced by the phase SLM (Meadowlark Inc.; 1920 × 1,152 pixels The SLM transparency for the two beams has the expression: The two diffractive lenses focus on the planes fp1 and fp2 When fd1,λ=∞ , Figure 1C with an uneven distribution of the two constants, in front of each term in Eq. (1) with only one the phase factor of θ=0 This case refers to the technique called ICHLLS 1L and it is used for calibration purposes. The z-scanning principle in ICHLLS 1L, same as in LLS, is that both the z-galvanometric mirror (z-galvo) and the detection objective (z-piezo), synchronize in motion to scan the sample in 3D, Figures 1D, E but the axial resolution could be lower than the axial resolution in LLS due to the blurring effect of the constant phase lens added on the SLM that focusses to infinity The ICHLLS 2L technique is used for the actual 3D sample imaging with the condition that the height of the two beams for each excitation wavelength was equal in size at the camera plane for a perfect overlap In this case the focal distances of the two diffractive lenses for each excitation wavelength are fd1,488 nm=220 mm fd2,488 nm=2356 mm fd1,561 nm=228 mm and fd2,561 nm=2444 mm where: Au,v is the amplitude of the image, Figure 2D left panel, IH are the hologram intensities, Figure 2B, and phase, Figure 2D right panel: (A) The 9 z-galvo position hologram intensity images: zgalvo=±40 μm ±30 μm; ±20 μm; ±10 μm,; 0 μm; (B) Hologram intensity images at θ=0 for each z-galvo level; (C) Example of hologram reconstruction at different z-galvo positions; (D) The reconstructed amplitude and phase at two z-galvo positions; zgalvo=−40 μm and 0 μm The FOV is 208 μm2 × 208 μm2 and pixel size 0.101 µm The depth of the object points is encoded by the density of the rings in the Fresnel holograms meaning that each plane in the image space reconstructed from the Fresnel hologram is in focus at a different axial distance In this manuscript the phase data represent the object phase retrieved at certain axial position of z-galvo mirror We intend to include extra variables for future work to obtain biomedically relevant information on the state of cells The spatio-temporal performance of a single-wavelength incoherent holography lattice light-sheet was already demonstrated in our previous work (Potcoava et al., 2021b) The technology provides the imaging resolution comparable or better than the conventional LLS’s resolution in dithering mode and with maximum detector FOV The LLS resolution in the z-axis is limited by the light sheet illumination depth of about 400 nm but LLS microscopy enables diffraction limited resolution in the x and y The axial resolution in IHLLS 2L and ICHLLS 2L can be improved by recording and reconstructing images at multiple z-galvo steps and minimizing the hologram reconstruction increment to achieve better localization of the sample points Pearson’s correlation coefficient (PCC), Manders’ overlap coefficient (MOC), and Manders’ colocalization coefficients (M1, M2) are all generally accepted methods to assess the spatial overlap of multi fluorescent labels recorded at different emission wavelengths when imaged at a particular spatial resolution (Manders et al., 1992; Manders et al., 1993; Costes et al., 2004; Li et al., 2004) These coefficients underline two aspects of the co-localization: correlation (PCC) and co-occurrence (MOC represent the fraction of co-localizing objects in each component of a dual-spectral channel image above threshold intensity values M1 is the percentage of pixels above threshold pixels in image recorded at 488 nm that overlap above threshold pixels in image recorded at 561 nm and M2 is the percentage of pixels above threshold pixels in image recorded at 561 nm that overlap above threshold pixels in image recorded at 488 nm This method is not intended for analyzing molecular interaction, which requires super-resolution techniques such as electron (Pastorek et al., 2016) or fluorescence resonance energy transfer microscopy (Day et al., 2001) but are useful first step to show that our IHLLS technique is capable of imaging biological samples in multiple spectral channels Fluorescent latex beads of 500 nm (λexc=488 nm,λem∼520 nm) and 1 μm (λexc=561 nm,λem∼575 nm) The bead solution (2% solids) was diluted 1:4000 with distilled water and briefly centrifuged in a desktop centrifuge for 1 min Clean coverslips were prepared by applying 1 µL as a thin layer that was left to dry the cover slip was mounted in the sample holder under distilled water Lamprey motoneurons were imaged in live intact isolated spinal cords Twenty-four hours prior to isolation of the spinal cord the animal was anesthetized with tricaine methanesulfonate (MS-222; 100 mg/L; Sigma Under anesthesia the myotomal muscle wall (1–3 segments) was injected with 2 µL of 10 mM 10,000 MW dextran conjugated to each of Alexa 488 and Alexa 555 The animal was allowed to recover and maintained in an isolated aquarium at 10C for 24 h The animals were again anesthetized with (MS-222; 100 mg/L) and dissected in a cold saline solution (Ringer) of the following composition (in mM): 100 NaCl adjusted to a pH of 7.60 with NaOH and to a final osmolarity of 270 ± 5 mOsm The spinal cord was isolated and placed ventral side up to expose the motoneurons closest to the imaging system in a cooled The chamber and spinal cord were then transferred onto the customized stage of the LLS microscope The recording chamber was continually superfused with cold oxygenated Ringer (8°C–10°C) for the duration of the experiment while maintained under the dual lenses of the LLS system The myotomal injection of dye allowed retrograde transport of dye throughout the axons somata and dendritic fields of motoneurons in the spinal ventral horn with labeling with both Alexa 488 and Alexa 555 allowing a demonstration of colocalized dye imaging of neurons in a complex 3D space The entire system was controlled by the original LLS software based on LabView platform (National Instruments) with the diffractive SLM (Meadowlark Inc.) synchronized with the ORCA camera for the IHLLS module The diffractive lenses with different phase factor were generated in MATLAB (MathWorks Inc.) for each of the two emission wavelengths Data acquisition is performed in two steps: a) move the z-galvo mirror to a specific z-galvo position (offset) and record four phase-shift holographic intensity images per z-galvo position ±30; ±20; 0 μm ; b) switch the excitation wavelength and perform step a) for the same offset z-galvo positions The image acquisition time for recording an intensity image is 50 ms in the middle of the FOV or at z-galvo position of 0 μm increasing progressively to 100 ms at edges of the z-galvo mirror image processing steps are the same for most of the diffraction techniques Each hologram processing can be sub-divided as follows: 1) Image pre-processing in which a background correction is performed by subtracting an average background level obtained by measuring the mean intensity of each stain outside the cells; 2) Hologram reconstruction in which the complex hologram is propagated and reconstructed at the best focal plane using a custom diffraction method routine in MATLAB (MathWorks Inc.); and 3) Object feature extraction performed on the reconstructed amplitude and phase images using FIJI (ImageJ) Reaching certain z-galvo levels is possible by combining the two diffractive lenses of focal lengths and fd2,561 nm=2444 mm IHLLS 2L beads volume reconstruction of a mixture of fluorescent beads; (A,C,E,G) 500 nm (488 nm) diameter fluorescent beads and (B,D,F,H) 1 µm (561 nm) diameter fluorescent beads z-galvo levels ±40; 20 μm are displayed at the phase-shift θ = 0; The xy views of beads after the hologram reconstruction represent the z-max projection of all the best z-reconstructed planes; and (I) The colocalization map of the xy planes in (G) and (H) When imaging individual beads with both wavelengths, shorter wavelength excitation, 488 nm, bleeds through when imaging 561 nm beads, Figure 3I. The superposition of the z-max projections of selected z-reconstructed planes for each of the two laser beams are shown in Figure 3G, H and the colocalization map of the beads at both wavelengths is shown in Figure 3I We zoomed in an area of the colocalization map to show the 488 nm beads are superposed with the 561 nm beads color 3D sensing of multiple beads and cells was performed To examine the effects of applying ICHLLS holography, neurons fluorescently labeled live in situ in the central nervous system (lamprey spinal ventral horn neurons), Figure 4 Alexa Fluor™ 488 nm and 568 nm hydrazide were injected by microinjection into the neuronal lamprey cell IHLLS-2L colocalization of two colors of dye Alexa 488 nm and 561 nm throughout the complex 3D space of the motoneuron dendritic trees; The xy volume map projection FOV 208 μm2 × 208 μm2 in an ICHLLS 1L with only one diffractive lens of focal length 400 mm for 488 nm (A) and 415 mm for 561 nm (B) without deconvolution; The xy projections were built from 400 z-axis planes in the range −30μm to 30μm in 0.101μm steps The excitation Bessel beams are displayed in the upper-left corner of (A) to show the orientation of the beams (FOV 208 μm2 in yellow for the ICHLLS and FOV 52 μm2 in red for the original LLS) The xy volume map projection from ICHLLS -2L reconstructed amplitude images with only 9 z-axis planes in the range −40 μm to 40 μm in 10 μm steps exciting sequentially with 488 nm (E,I) and 561 nm (F,J); The colocalization maps (C,G,K) and the scatter plot (D,H,L) for each case are displaced on the right A goal of this study is to enable colocalization or separation of probes of separable fluorescent wavelength to be identified in the 3D structure of neurons during live cell recording in intact tissue in situ Photodamage from excessive excitation will destroy the neurons the tissue is dynamic and can move during extended periods of recording and the tissue is complex and diffractive limiting the lattice beam penetration depth as well as the clarity of the emitted light The first step in showing that the location of neurons in dual-channels are closely related is to overlay the dual-channel data 488 nm in red and 561 nm in green and then to state that anything that shows up as white shows colocalisation The second step in the colocalization process is to use a scatter plot diagram of the pixel values of the dual-channel images against each other to calculate the colocalization parameters The data information of a given pixel in the 488 nm image is used as the y-coordinate of the scatter plot and the data information of the corresponding pixel in the 561 nm image as the x-coordinate A pixel distribution along a straight line and a Pearson’s or Mander’s coefficients close to 1 means that both channel distributions are linked and colocalized Colocalization coefficients in the phase data from IHLLS-2L demonstrates less colocalization This reflects the very high sensitivity of this approach to z-plane offset and the fact that in live tissue small movements occur throughout the recording also demonstrates the capability of this approach to detect faint data in the periphery of the recording This indicates the possibility of extracting data from larger volumes of 3D structure TABLE 1. Quantitative analysis of colocalization using all three imaging techniques, IHLLS 1L and IHLLS 2L reconstructed amplitude and phase, shown for the neuron in Figure 4 We have demonstrated that 3D amplitude and phase information can be extracted from fluorescent samples scanned using a lattice light-sheet and that this can be achieved at two or more wavelengths This also enables an increase in resolution compared to the original LLS approach of moving the sample or the objective lens we have demonstrated that this can be achieved with 9 z-galvo positions of the ICHLLS 2L system compared to 300 z-galvo positions of the original LLS or IHLLS 1L In the present configuration single image frames can be obtained in between 50 and 100 ms This will allow complete volumes of up to 100 μm2 × 100 μm2 × 80 µm with 9 z-galvo positions and 4 phase shifts to be obtained in as little as 1.8 s in single colors With smaller volumes these rates can be improved even further Bandpass filters with multiple transmission notches can be used emission bands must be narrow to prevent blurring of the hologram then bleed-through from shorter wavelengths to longer is an issue This can be prevented by sufficiently separated wavelengths On our system with excitation lasers at 405 emission bands can be combined with centers at 520 and 660 nm and at 420 and 580 nm to ensure no bleed-through If these two multiple bandpass filters are combined with two cameras then switching of emission filters can be eliminated and the Bessell beam excitation and emission SLMs can be coordinated allowing two color emission to be captured by a simple doubling of the time for each volume a color camera with specific bandpass filters over the photosensitive elements would allow single camera detection Another solution is to use a mechanical filter switcher with just one camera and the dual bandpass approach described above Filter switching with a fast device would slow acquisition by about 100 ms for each two-color changes Our goal is to enable automated and repetitive capture of similar volumes over time to achieve spatial and temporal resolutions to track dynamic movements of cellular structure in 3D over time to enable very high-resolution phase and amplitude images we will need to automate 3D scanning and ICHLLS 2L imaging in up to all 4 colors of the original LLS system by sweeping excitation through hundreds of z-axis planes Our approach will enable high temporal resolution of the spatial relationships between synaptic and cellular structures and retain both amplitude and phase information in the reconstructed images Understanding colocalization of different labeled proteins and cellular elements and their interaction is crucial to understanding brain function and the low light excitation of LLS coupled with enhanced sensitivity of ICHLLS-2L will enable this The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding author The animal study was reviewed and approved by Animal Care and Use Committee (ACC) and CM; writing—original draft preparation MP and SA; writing—review and editing All authors have read and agreed to the published version of the manuscript This research was funded by the NIH RO1 NS111749 to SA and the NIH R21 DC017292 to JA The authors gratefully thank to Zack Zurawski and Kuei Tseng for very helpful discussions and recommendations The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: 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Abstract | CrossRef Full Text | Google Scholar Keywords: Incoherent Color Holography Lattice Light-Sheet Art J and Potcoava M (2023) Incoherent color holography lattice light-sheet for subcellular imaging of dynamic structures Received: 11 November 2022; Accepted: 18 January 2023;Published: 08 February 2023 Copyright © 2023 Alford, Mann, Art and Potcoava. 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Advertise on Cineuropa Logos and Banners PRODUCTION / FUNDING Romania by Ştefan Dobroiu 22/01/2024 - The drama film tells the story of a man who reconnects with a long-lost love after he dies and goes to heaven which seems to be located on a deserted beach with perfect weather and a cold beer always within reach the girl he fell in love with at secondary school And what if Dan now has the opportunity to right mistakes from the past The €200,000 project was made without any funds from the Romanian National Film Center. 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multiplied by many thousands of birds the astronomical figure was once easily supplied by Delaware Bay New Jersey’s beaches contained 100,000 eggs per square yard The mere 5,000 or 6,000 eggs laid per square yard in recent years are hardly enough to feed hungry shorebirds fishermen took as many as two million horseshoe crabs each year to use as bait in eel and whelk traps biomedical companies were catching the animals to protect public health Horseshoe crabs’ blue blood is the key ingredient in an all-important test that detects endotoxin a potentially life-threatening bacterial contamination that can occur in drugs and medical devices Approximately 70 million endotoxin tests are performed each year after the horseshoe crab Limulus polyphemus—bleed horseshoe crabs Academic researchers and industry scientists differ on how many die in the process: estimates range from five percent to 30 percent And the loss of blood has longer term effects such as loss of fitness and increased susceptibility to infection that may also contribute to population declines. thin scientist who seems to disappear in his lab coat He works in a sparkling new lab at Eli Lilly’s sprawling technology development center in Indianapolis he’s been steadily working to develop a product that will take biomedical pressure off horseshoe crabs Building on research carried out in Singapore he’s been compiling evidence that a synthetic enzyme recombinant factor C—rFC for short—can replace horseshoe crab blood in endotoxin tests rFC works just as well as LAL, is more efficient and cost-effective This synthetic alternative has been on the U.S but institutional and regulatory barriers have hindered its acceptance and he’s collected enough data to convince his employer that it’s time to make the switch If rFC is taken up by the rest of big pharma and both horseshoe crabs and shorebirds will likely feel the difference. LAL had been the gold standard for endotoxin testing for two decades pharma companies tested their products on rabbits waiting to see if they developed a fever after injection and their skittishness—they sometimes spiked fevers when strangers walked into the room—made them imperfect test subjects After LAL proved more sensitive to endotoxin The problem solved by the crab’s blue blood goes something like this: When gram-negative bacteria like E Danger arises when high concentrations of the potent poisons enter a person’s spinal fluid or bloodstream injected drugs (for people and their pets) or implanted medical devices that come into contact with blood must be tested for endotoxin along with millions of doses of flu vaccine and intravenously delivered antibiotics and chemotherapies Manufacturers also must test the water and raw ingredients used in their manufacturing companies that make LAL capture and release some 500,000 horseshoe crabs along the eastern seaboard of the United States every year most bled horseshoe crabs are ultimately killed and hoped her discovery would usher in a new era of endotoxin testing Ding’s work piqued the interest of scientists at Cambrex Bio Science, now part of the Swiss biotechnology company Lonza which already produced and sold LAL Ding and the National University of Singapore licensed the cloned synthetic rFC to Lonza which in the early 2000s began manufacturing and selling it to drug companies “We built it,” says Lonza’s Allen Burgenson then manager for regulatory affairs at the company Burgenson led Lonza’s work in making the case for rFC on the grounds that rFC isn’t a blood product like LAL and therefore wasn’t under its jurisdiction Pharmacopeia—a compendium of drug information—and its Japanese equivalent biomedical use of horseshoe crabs increased 85 percent the FDA formally announced that the biomedical community could use rFC The European Pharmacopeia followed suit in 2016 drug companies had little incentive to spend time and money testing a newer But Bolden had a compelling reason: “I’m a birder,” he says “so this hit close to home.” He’d seen Red Knots one in Delaware’s Bombay Hook National Wildlife Refuge and then another in Ireland’s Ballycotton Marsh at an endotoxin summit in Delaware hosted by Lonza Bolden witnessed the arrival of horseshoe crabs suddenly filled with horseshoe crabs coming in from the sea It was a religious experience for me.” whether current takes of horseshoe crabs for endotoxin testing “can be sustained much less meet the projected future demands of this rapidly growing market.” aware that Asian horseshoe crabs taken for biomedical use are often bled to death became concerned about “supply problems down the road” when he learned that Eli Lilly was planning to build a second manufacturing plant in China I can be part of conservation.” His vocation and avocation came together If Ding in Singapore had started this relay to end the practice of bleeding horseshoe crabs then Bolden was ready for his turn at the track He pitched an Eli Lilly vice president on using rFC then sought approval from two of the company’s governance committees: the specifications committee Tremendous quantities of pharmaceutical-grade water—some of Eli Lilly’s water tanks are 12 feet wide and two stories tall—are required to manufacture injectable drugs and vaccines Hyglos (now owned by bioMérieux) had entered the market so the duo tested both compounds to compare their efficacy to LAL The effort required their undivided attention and a painstaking level of precision and they needed to be certain that rFC detects 100 percent of the endotoxin in a sample They published a paper in 2017 demonstrating that it does was making a similar recombinant and leading similar comparative studies Decades after Ding began her work to create a better a growing body of evidence points to rFC’s equivalency But the race is not over: Who among the other major drug manufacturers will pick up the baton and Ring-billed—practically carpeted the beach before him were hundreds of horseshoe crabs: large females had two and up to 11 males attached as they spawned in the surf “Just being here—it’s the culmination of five years of effort,” he says I effected conservation of these animals.” and representatives from local conservation groups After opening remarks from New Jersey First Lady Tammy Murphy president and co-founder of Revive and Restore he announced that Eli Lilly plans to make a major transition to rFC His work found that not only does rFC detect endotoxin as well as LAL it yields fewer time-consuming false positives and then we save what’s left over for the next run,” he says “And using rFC complies with European Union and U.S government directives to reduce and replace animals in medical testing and products.” The FDA is now reviewing Eli Lilly’s application for a new migraine-prevention drug it will be the first FDA‑approved drug to be released to the market with rFC as its endotoxin test is a less glamorous transition already underway at Eli Lilly’s facilities Two of its biggest manufacturing plants now test their pharmaceutical water with rFC only a few hours drive north of Delaware Bay the company aims for all eight of its manufacturing sites to test water with rFC the company will have reduced its LAL use by approximately 90 percent. After 14 years of trying to find a market for rFC Lonza’s Burgenson says he’s “over the moon.” “We hope that companies will act swiftly,” says Stiles of New Jersey Audubon “There’s a time factor here.” Shorebird populations are crashing only 20 percent gained the weight necessary to make it to the Arctic and successfully breed Stiles and Niles are both hoping that other large pharmaceutical companies “It’s a win-win,” Stiles says. “These companies can do well by doing good.” the biomedical industry takes some 500,000 to 600,000 horseshoe crabs from the sea If the rest of the pharmaceutical industry follows Eli Lilly’s lead—tests its manufacturing water with rFC and reduces its use of LAL by about 90 percent—that’d potentially leave an extra 450,000 to 540,000 horseshoe crabs untouched every year but the Ruddy Turnstones and Semipalmated Sandpipers that also desperately need those eggs to admire flocks of shorebirds feeding at Delaware Bay he could take great satisfaction knowing that in running his leg of the relay he’d set an enormous and important precedent. Now so Delaware Bay can once again be awash in horseshoe crabs and shorebirds Deborah Cramer is the author of The Narrow Edge: A Tiny Bird, An Ancient Crab, and An Epic Journey in which she accompanied Red Knots on their extraordinary migration from Tierra del Fuego to the Arctic. The book received the Rachel Carson Book Award from the Society of Environmental Journalists and the Best Book Award from the National Academy of Sciences Timothy Fadek is a photographer based in New York. He covers a wide range of subjects including social issues, world events, and science and nature. Some of his work can be found at www.TimothyFadek.com and on Instagram: @timothyfadek and Facebook: Timothy.Fadek.Photography Pledge to stand with Audubon to call on elected officials to listen to science and work towards climate solutions You don't have permissions to access this page 2023 marked a milestone in the company’s development: the opening of the Potcoava Farm the first 100% organic chicken farm in Romania highlights the company’s main achievements of the year Carmistin is one of the fewest Romanian holdings that has an integrated business on the local market To what extent has this business model contributed to the company’s success in Romania What were the biggest challenges Carmistin faced in 2023? the Carmistin Organisation faced challenges that were typical of the general business landscape and the need to navigate regulatory changes adapting to emerging technologies and addressing shifting consumer preferences also represent ongoing challenges for the company What were the most important projects the company carried out in 2023 Carmistin reached a significant milestone with the opening of the Potcoava Farm indicating a notable commitment to sustainability It is the first 100% organic chicken farm in Romania and it is aligned with Carmistin’s plan to produce organic food in Romania like the Organic Chicken from La Provincia indicates a maturing market that’s focused on high-quality plans a medium- to long-term expansion of its organic segment Carmistin also introduced its organic egg range under the La Provincia brand The brand’s portfolio includes vegetarian chicken the poultry division accounted for over 50% of Carmistin’s business the group ranks among the top food producers in Romania and community engagement initiatives were key achievements Carmistin implemented projects focusing on renewable energy and biodiversity conservation to further solidify its position as a leader of the agrozootechnical market in Romania What’s the current state of the Romanian agribusiness sector and what changes should we expect to see in the coming years The agribusiness sector is currently experiencing steady growth driven by increased investments in technology and sustainable practices Trends suggest a shift towards digitalization and a heightened focus on environmental sustainability Government policies supporting innovation and international collaborations are expected to play a pivotal role in shaping the industry’s trajectory in the coming years How important are ESG and sustainability criteria for Carmistin ESG and sustainability criteria are paramount for the Carmistin Organisation in today’s business landscape Adhering to these principles not only aligns the company with global sustainability goals but also enhances brand reputation attracts socially responsible clients and partners and ensures long-term viability in an increasingly conscientious market Integrating ESG practices is a strategic imperative for Carmistin to thrive amid evolving market expectations and contribute positively to the broader environmental and social ecosystem What were 2023’s main challenges from a management point of view The biggest challenges for Carmistin’s management in 2023 revolved around global supply chain disruptions and adapting to evolving consumer preferences Navigating the complexities of geopolitical uncertainties and maintaining operational resilience were also critical points The company strategically addressed these challenges through proactive risk management and continuous efforts to enhance operational efficiency and human capital management What is the outlook for the local agribusiness sector in 2024 with anticipated opportunities in technology adoption and the ongoing impact of global events may necessitate agile strategies Success in 2024 for companies like Carmistin will likely hinge on proactive adaptation to emerging trends and a continued commitment to sustainability to meet the evolving demands of the sector We use cookies for keeping our website reliable and secure providing social media features and to analyse how our website is used BERGAMO 2024 by Camillo De Marco 14/03/2024 - Radu Potcoavă delivers a surreal romantic comedy offering a rushed reflection on the consequences of our choices and an overly sentimental ending he plays the card of surreal romantic comedy Dan (the charismatic Bogdan Dumitrache) finds himself on the splendid sands of a golden beach (the film’s luminous photography is by Andrei Butică) alongside a campervan decked out with all kinds of comforts Mysterious character Petru (Sergiu Costache) who has accompanied him into what we assume to be Purgatory explains that he has 40 days to think about the actions he’s carried out during his lifetime to potentially repent and to stand in the presence of the Almighty with a ticket to Paradise ready and waiting in his pocket In addition to ice-cold beer and cigarettes Dan is given a bonus gift: a tablet which allows him to visit anyone he wants to on Earth just by typing their name he meets his friend Cipi (Silviu Pintileasa) then he briefly sees his wife Ada (Florentina Tilea) But Dan hasn’t come to terms with another “guest” on that particular beach: Laura (Cosmina Stratan) the girl he was in love with at high school and who died on account of a tumour The two look back on all the sliding doors which drove them apart forever Dan declares himself an idiot and a coward admitting to being the only person responsible for the car accident which killed him (I put the left indicator on but turned right”) asking her to take good care of their 13-year-old daughter Zoe in the only scene in the film which packs any real emotional punch He’s even allowed to meet his elderly parents (they’re already in Paradise) who give him their “blessing” the film loses itself in a saccharine series of “I’ve always loved you”s replete with evocative sunsets on the beach lovemaking in the campervan and splashing around and laughing in crystal clear waters given the subtle irony which initially reigned over this film made brilliant by Catalin Cristutiu’s nervy and “apprehensive” editing Any pretence of nigh-on metaphysical reflection on the consequences of our choices vanishes in favour of a distinctly male-flavoured form of romantic entertainment The ending – featuring a cameo by popular actor Șerban Pavlu – directs the film back towards the puerile immature and narcissistic dream of an Eden where men are platonically (in the sense of Plato’s Symposium) reunited with the ideal woman under the obliging eye of an ambiguous (and male) god The director himself admitted he was surprised by the film’s selection at the historically picky Bergamo FM because he considered Good Guys Go to Heaven to be mainstream rather than a festival film But the selectors chose Good Guys Go to Heaven for exactly what it is: a paradisiacal comedy for a wider audience Good Guys Go to Heaven is produced by Wearebasca together with Chainsaw Europe. The Open Reel are managing international sales Radu Potcoavă delivers a surreal romantic comedy offering a rushed reflection on the consequences of our choices and an overly sentimental ending 14/03/2024 | Bergamo 2024 Between 9 and 17 March, the festival’s 42nd edition will be showcasing European fiction feature films and documentaries, and hosting two industry days on audience development strategies 29/02/2024 | Bergamo 2024 The drama, which sees four actors playing 12 characters, will discuss “film, fear and fiction” 26/01/2024 | Production | Funding | Romania The drama film tells the story of a man who reconnects with a long-lost love after he dies and goes to heaven 22/01/2024 | Production | Funding | Romania 02/05/2025goEast 2025 30/04/2025Films / Reviews – Italy Review: San Damiano 30/04/2025Hot Docs 2025 Review: King Matt the First 29/04/2025Films / Reviews – Italy Review: Storia di una notte 29/04/2025Films / Reviews – Peru/Spain Review: Kayara. La guerrera del Imperio Inca 29/04/2025Hot Docs 2025 Review: Supernatural