We used Olympus IX81 inverted, epifluorescence, wide‐field microscope. For each time‐lapse movie, we collected images once every 10 min for every field of view. The temperature during microscope imaging was maintained by an incubator cage (OKO Lab) that enclosed the microscope. We acquired each image with an EM‐CCD Luca R camera (Andor) and IQ3 software (Andor). We used a wide‐spectrum lamp (AMH‐600‐F6S, Andor) for exciting fluorescent proteins.
Em ccd luca r camera
Manufactured by Oxford Instruments
The EM-CCD Luca-R camera from Oxford Instruments is a scientific imaging device designed for low-light applications. It features an electron-multiplying charge-coupled device (EM-CCD) sensor that provides high sensitivity and low noise performance. The camera is capable of capturing high-quality images and videos under challenging lighting conditions.
Automatically generated - may contain errors
5 protocols using em ccd luca r camera
1
Time-Lapse Imaging of Fluorescent Proteins
2
Time-lapse Imaging of Cell Adaptation and Aging
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All time-lapse experiments have been performed at least two times.
Freshly thawed cells were grown overnight at various final cell densities. In the morning, log phase cells were allowed to grow a few divisions and were transferred into the microfluidic device. Cells were imaged using an inverted Zeiss Axio Observer Z1 (adaptation assay) or a Nikon Tie (aging experiments). Focus was maintained using dedicated hardware throughout the assays. Fluorescence illumination was achieved using LED light (precisExcite, CoolLed or Lumencor) and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor; adaptation experiments) or an Hamamatsu Orca Flash 4.0 (Aging experiments).
We used automated stages in order to follow up to 20 (adaptation experiments) or 60 (aging experiments) positions in parallel over the course of the experiment. Images were acquired every 3 min (adaptation experiments) or 10 min (aging experiments).
Temperature control was achieved using custom sample holder with thermoelectric modules and an objective heater with heating resistors. Temperature control was achieved using a PID controller (5C7-195, Oven Industries).
Freshly thawed cells were grown overnight at various final cell densities. In the morning, log phase cells were allowed to grow a few divisions and were transferred into the microfluidic device. Cells were imaged using an inverted Zeiss Axio Observer Z1 (adaptation assay) or a Nikon Tie (aging experiments). Focus was maintained using dedicated hardware throughout the assays. Fluorescence illumination was achieved using LED light (precisExcite, CoolLed or Lumencor) and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor; adaptation experiments) or an Hamamatsu Orca Flash 4.0 (Aging experiments).
We used automated stages in order to follow up to 20 (adaptation experiments) or 60 (aging experiments) positions in parallel over the course of the experiment. Images were acquired every 3 min (adaptation experiments) or 10 min (aging experiments).
Temperature control was achieved using custom sample holder with thermoelectric modules and an objective heater with heating resistors. Temperature control was achieved using a PID controller (5C7-195, Oven Industries).
3
Inverted Microscope Wide-Field Epifluorescence Imaging
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Cells were imaged using an inverted microscope (Zeiss Axio Observer Z1, or Nikon Tie). Wide-field epifluorescence illumination was achieved using an LED light source (precisExcite, CoolLed), and light was collected using a 100× N.A. 1.4 objective and an EM-CCD Luca-R camera (Andor).
4
Microscopy Techniques for Cell Imaging
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For all experiments except pH measurements, cells in the observation device were imaged using an inverted widefield microscope (Zeiss Axio Observer Z1). Fluorescence illumination was achieved using LED lights (precisExcite, CoolLed) and the light was collected using a ×63 (N.A. 1.4) objective and an EM-CCD Luca-R camera (Andor). Standard GFP and mCherry filters were used.
For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390 /18 and 475/28 nm, beamsplitter 495 nm, and emission filter 525/50 nm). Emitted light was collected using a ×60 N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0).
We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane images were acquired every 15 , 30 , 60 , or 240 min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390 /18 and 475/28 nm, beamsplitter 495 nm, and emission filter 525/50 nm). Emitted light was collected using a ×60 N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0).
We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane images were acquired every 15 , 30 , 60 , or 240 min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
5
Multiparametric imaging of live cells
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For all experiments except pH measurements, cells in the observation device were imaged using an inverted widefield microscope (Zeiss Axio Observer Z1). Fluorescence illumination was achieved using LED lights (precisExcite, CoolLed) and the light was collected using a 63× (N.A. 1.4) objective and an EM-CCD Luca-R camera (Andor). Standard GFP and mCherry filters were used. For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390nm/18nm and 475/28nm, beamsplitter 495nm, emission filter 525/50 nm). Emitted light was collected using a 60x N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0). We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane-images were acquired every 15 min, 30min, 60min or 240min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
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