Patient tumours were observed at × 100 magnification using a highly modified Olympus microscopy system. The microscope was attached to a cantilevered arm to extend over the patient. Given the high magnification required to observe individual vessels and flow, vibrations in the microscope were dampened using a weighted marble base of over 360 kg. Fluorescein was illuminated using a 467–498-nm excitation, 513–556 emission dichronic filter set (Spectra Services, Rochester, NY) with an X-Cite 120 Led light source (Lumen Dynamics, Ontario, Canada). Images were captured using a Luca EMCCD camera (Andor Technology Ltd., Belfast, Northern Ireland) controlled through the Solis acquisition and analysis programme. Images were acquired with a minimum of a 0.05-s exposure at 20 frames a second. Offline quantification of vessel diameter and blood velocity was performed using the ImageJ software suite.
Luca emccd camera
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Luca EMCCD camera is a sensitive imaging device designed for low-light scientific applications. It utilizes electron-multiplying charge-coupled device (EMCCD) technology to amplify signals, enabling high-resolution imaging with improved signal-to-noise ratio. The camera is capable of capturing detailed images and video in challenging lighting conditions.
Automatically generated - may contain errors
5 protocols using luca emccd camera
1
Intravital Microscopy of Tumor Vasculature
2
Comet Assay for Oxidative DNA Damage
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Cells were cultured into a 12-well plate (1 × 105 cells/well) and incubated O/N to reach cell attachment. Cells were treated with different HT–SQ combinations. Then, cells were scraped and washed twice with cold 1X PBS (300× g 10 min, 4 °C). They were resuspended in 1 mL of cold 1x PBS. Cells were treated with 50 µM H2O2 for 10 min at 4 °C to assess the ability of HT (100 μM)-SQ (100, 10 and 1 μM) in protecting against oxidative DNA damage. Then, the comet assay was conducted as described in Warleta et al. [17 (link)]. Each experiment was repeated independently at least 3 times.
DNA strand breaks were screened using the Komet 5.5 software package (Kinetic Imaging Ltd., Liverpool, UK) in a fluorescence microscope (Zeiss Axiovert 200) equipped with a Luca EMCCD camera (Andor Technology, Belfast, UK) (Ex. 494 nm/Em; 521 nm wavelength). We randomly characterized 25 cell images per sample at a magnification of 20×. Relative fluorescence between head and tail, determined through the olive tail moment (Olive_TM), was used to determine DNA damage. Olive_TM is defined as the product of the Tail Moment Length and the fraction of DNA in the tail.
DNA strand breaks were screened using the Komet 5.5 software package (Kinetic Imaging Ltd., Liverpool, UK) in a fluorescence microscope (Zeiss Axiovert 200) equipped with a Luca EMCCD camera (Andor Technology, Belfast, UK) (Ex. 494 nm/Em; 521 nm wavelength). We randomly characterized 25 cell images per sample at a magnification of 20×. Relative fluorescence between head and tail, determined through the olive tail moment (Olive_TM), was used to determine DNA damage. Olive_TM is defined as the product of the Tail Moment Length and the fraction of DNA in the tail.
3
Vagal Afferent Calcium Imaging
4
Comet Assay for DNA Damage Evaluation
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To estimate the state and wholeness of DNA, 5x104 cells/well (for MCF10A cells) or 1x105 cells/well (for MCF7 and MDA-MB-231 cells) were allowed to attach to a 12-well plate and treated with increasing PINO concentrations for 24 h. The cells were then detached and centrifuged twice in PBS. To evaluate whether PINO had the ability to protect against oxidative DNA damage, cells were also exposed to H2O2. The comet assay was carried out according to Warleta et al. [3 (link)]. Analysis of the DNA strands was performed by examining twenty-five random cell images per sample in a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss; Jena, Germany) equipped with Luca EMCCD camera (Andor Technology, Belfast, UK) and using the Komet 5.5 software package (Kinetic Imaging Ltd., Liverpool, UK). DNA damage was calculated by determining the relative fluorescence between the head and tail using the olive tail moment (Olive_TM), which was defined as:
5
Fura-2 Imaging of Astrocyte Calcium
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Almost confluent cultures of astrocytes grown on fibronectin‐coated eight‐well imaging slides (Thistle Scientific Ltd, Glasgow, UK) were loaded with fura‐2 by incubation with fura‐2 AM (2 μM) in HBS containing 2.5 mM probenecid (45 min, 20°C). After a further 45 min in the same medium without fura‐2 AM, the cells were used for experiments at 20°C in HBS without probenecid. Imaging was performed using an Olympus IX71 inverted fluorescence microscope with alternating excitation (340 nm and 380 nm) provided by a Xe‐arc lamp at 1‐s intervals. Emission was recorded at 510 nm using a Luca EMCCD camera (Andor Technology, Belfast, UK) and MetaFluor software (Molecular Devices, Sunnyvale, CA, USA). Background‐corrected ratios of F340/F380 fluorescence were used to determine whether ligands evoked increases in [Ca2+]i.
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