Orca ag

Cartilage tissues were placed in 2% paraformaldehyde in PBS for 30 min before mounting in Tissue-Tek OCT compound (Fisher Scientific, Pittsburgh, PA) and flash-freezing in liquid nitrogen. Frozen samples were cryosectioned (10 μm per slice), observed by NIR fluorescence microscopy, and also stained with Alcian Blue or hematoxylin and eosin (H&E), respectively. NIR fluorescence microscopy was performed on a 4-filter Nikon Eclipse TE300 microscope system as previously described.^{[21 (link),22 (link)]} The microscope was equipped with a 100 W mercury light source (Chiu Technical Corporation, Kings Park, NY), NIR-compatible optics, and a NIR-compatible 10X Plan Fluor objective lens and a 100X Plan Apo oil immersion objective lens (Nikon, Melville, NY). Images were acquired on an Orca-AG (Hamamatsu, Bridgewater, NJ). Image acquisition and analysis was performed using iVision software (BioVision Technologies, Exton, PA). Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were respectively used to detect C700-OMe and C800-OMe signals in the frozen tissue samples.

Bone tissues were placed in 2% paraformaldehyde in PBS for 30 min before mounting in Tissue-Tek OCT compound (Fisher Scientific, Pittsburgh, PA) and flash-freezing in liquid nitrogen. Frozen samples were cryosectioned (10 μm per slice), observed by NIR fluorescence microscopy, and also stained with hematoxylin and eosin (H&E). NIR fluorescence microscopy was performed on a 4-filter Nikon Eclipse TE300 microscope system as previously described.^{[9 (link),23 (link),24 (link)]} The microscope was equipped with a 100 W mercury light source (Chiu Technical Corporation, Kings Park, NY), NIR-compatible optics, and a NIR-compatible 10X Plan Fluor objective lens and a 100X Plan Apo oil immersion objective lens (Nikon, Melville, NY). Images were acquired on an Orca-AG (Hamamatsu, Bridgewater, NJ). Image acquisition and analysis was performed using iVision software (BioVision Technologies, Exton, PA). Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were respectively used to detect P700SO3 and P800SO3 signals in the frozen tissue samples.

For histological evaluations, lymph nodes were resected from the Man-ZW800-1 injected rats after scheduled imaging, embedded in Tissue-Tek OCT compound (Fisher Scientific, Pittsburgh, PA), and flash frozen in liquid nitrogen. Tissue was cryosectioned at 10 µm intervals and observed using a NIR fluorescence microscope. Consecutive sections were stained with hematoxylin and eosin (H&E). NIR fluorescence microscopy was performed on a 4 filter-set Nikon Eclipse TE300 epifluorescence microscope to confirm the fluorescence of lymph node as previously described [10 (link)]. The microscope was equipped with a 100 W mercury light source, NIR-compatible optics, and a NIR-compatible 4×, 10×, 20×, and 40X Plan Fluor objective lens (Nikon, Melville, NY). Custom filter sets (Chroma Technology Corporation, Brattleboro, VT) composed of 750 ± 25 nm excitation filter, 785 nm dichroic mirror and 810 ± 20 nm emission filter were used to detect the fluorescent signal in the frozen tissue samples. Images were acquired on an Orca-AG (Hamamatsu, Bridgewater, NJ) and QImaging 12-bit camera for color imaging (Surrey, BC, Canada). Image acquisition and analysis was performed using iVision software (BioVision Technologies, Exton, PA).

Cells were grown in multiwell glass bottom dishes (MatTek). MCF-10A or HeLa growth medium was used during image acquisition with a layer of mineral oil on top of the medium to prevent evaporation. All images were collected with a Nikon TE2000E or Nikon Ti motorized inverted microscope equipped with Perfect Focus System for continuous maintenance of focus. The microscopes were enclosed in incubators that provided both 37°C temperature control and 5% CO₂. Both microscopes were equipped with 20× Plan Apo 0.75 NA or 60× Plan Apo 1.4 NA objective lenses. Histone H2B-GFP or GFP-tubulin fluorescence was excited with a mercury halide light source using a 480/40 excitation filter (Chroma) and collected with a 535/50 emission filter (Chroma). Images were acquired with a Hamamatsu ORCA-ER cooled charge-coupled device (CCD) camera controlled with MetaMorph 7 software or Hamamatsu ORCA-AG cooled CCD camera controlled with NIS-Elements image acquisition software. Time-lapse microscopy images were collected every 3 min, using an exposure time between 100 and 200 ms, with illumination light shuttered between acquisitions. Gamma, brightness, and contrast were adjusted on displayed images (identically for compared image sets) using MetaMorph 7 software or NIS-Elements.