Calcein – AM (calcein acetoxymethyl ester) upon internalization by live cells is converted to calcein (a green fluorescent dye) due to the hydrolytic removal of acetoxymethyl ester group by intracellular esterases. Therefore, calcein selectively stains live cells that can be visualized using a fluorescence microscope. After 1, 3, and 5 days, media was removed and scaffolds were washed with PBS. 1 ml of Calcein-AM dye (4μM) was added to each well and incubated at 37°C in dark for 20 minutes. The scaffolds were transferred to 35 mm glass bottom dishes (Mattek Corporation, Ashland, MA) and imaged by a confocal laser-scanning microscope (Zeiss LSM 510 Meta NLO Two-Photon) using Zeiss LSM Image Browser software (version 4.2, Carl Zeiss Microimaging, Thornwood, NJ).
Lsm 510 meta nlo two photon
Manufactured by Zeiss
The LSM 510 Meta NLO Two-Photon is a high-performance laser scanning microscope system designed for advanced imaging applications. It combines the capabilities of a confocal microscope with the benefits of two-photon excitation, enabling deep tissue imaging and improved signal-to-noise ratio. The system features a tunable femtosecond pulsed laser for non-linear optical (NLO) excitation, allowing for the visualization of fluorescent samples with enhanced contrast and reduced photodamage.
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Lab products found in correlation
3 protocols using lsm 510 meta nlo two photon
1
Calcein-AM Live Cell Imaging
2
Depth Mapping of Cell Infiltration
3
Visualizing Cell Attachment and Infiltration on Scaffolds
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To visualize cell attachment on scaffolds, glutaraldehydefixed cells on PLGA, MWCNT, and SWCNT scaffolds were subjected to dehydration steps using graded ethanol washes (70%-100%), air dried, and vacuum dried for 24 h. Scaffolds were then sputter coated with 3 nm of silver (Ag) and imaged using a JOEL 7600 F Analytical high-resolution SEM (Center for Functional Nanomaterials, Brookhaven National Laboratory, New York) at an accelerating voltage of 2 kV.
Image processing to assess cellular infiltration Z-stacks of calcein-stained ADSCs on PLGA, MWCNT, and SWCNT scaffolds were acquired using a confocal laserscanning microscope (Zeiss LSM 510 Meta NLO Two-Photon). Individual Z-stacks were then imported to ImageJ (Bethesda, MD, USA) and subjected to spectral coding using a time-lapse color coder plugin to false-color each slice as a function of depth (Z height, i.e., depth of cellular infiltration). The multiple spectrally color-coded slices of Z-stacks were then compressed to form one composite image and reported.
Image processing to assess cellular infiltration Z-stacks of calcein-stained ADSCs on PLGA, MWCNT, and SWCNT scaffolds were acquired using a confocal laserscanning microscope (Zeiss LSM 510 Meta NLO Two-Photon). Individual Z-stacks were then imported to ImageJ (Bethesda, MD, USA) and subjected to spectral coding using a time-lapse color coder plugin to false-color each slice as a function of depth (Z height, i.e., depth of cellular infiltration). The multiple spectrally color-coded slices of Z-stacks were then compressed to form one composite image and reported.
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