Researchers should complete our publication form to report publications with data acquired or quantified using CoRE equipment, expertise, or services. Language to be used in the publications are noted below.

Members of The Tisch Cancer Institute
This research was supported in part by The Tisch Cancer Institute at Mount Sinai P30 CA196521 – Cancer Center Support Grant.

Olympus FV1000 MPE
Multiphoton microscopy was performed in the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai, supported with funding from NIH Shared Instrumentation Grant (1S10RR026639).

Spinning Disk Confocal
Spinning disk confocal microscopy was performed in the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai, supported with funding from NIH Shared Instrumentation Grant (1S10RR024745).

Leica SP8 STED 3X
Confocal and/or stimulated emission-depletion microscopy was performed in the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai, supported with funding from NIH Shared Instrumentation Grant (FAIN: S10OD021838).

Electron Microscopy
Electron Microscopy tissue/cell processing and imaging was completed by the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai.

All Other Instruments
Microscopy and/or image analysis was performed at the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai.

Modern microscope-generated data is expected to be quantitative and reproducible, which often confuses biologists, as they do not know what exactly needs be reported from the design and implementation of an imaging study. This challenge is compounded by the fact that in designing a study, there are many configurations of a similar instrument as well as many different microscopy modalities to select. Downstream image analysis and data extraction can also add to the challenge of underreporting image manipulations, as biologists do not necessarily understand how image analysis algorithms and pipelines work, or which adjustments should be reported.

The CoRE adheres to Open Science Foundation’s Transparency and Openness Promotion reporting standards. For a primer of image integrity and standards, please see Nature Portfolio’s guidelines.

To accurately convey the information of acquisition set up, figure legends, text, and methods, researchers should collectively include, at minimum, the following information:

• Modality (e.g., widefield, confocal)
• Instrument model and manufacturer
• Acquisition software version and manufacturer
• Lens model, numerical aperture, magnification, manufacturer, correction collar, and/or iris type and setting
• Applied optical zoom
• Space and time resolution (xyz pixel/voxel dimensions and t intervals)
• Pinhole sizes
• Image bit depth
• Fluorophores and excitation/emission set up (light source, excitation and emission filter ranges, beam splitters)
• Detector setup (type and manufacturer, pixel size, frame size, binning and/or gain settings, acquisition mode)
• Scanner settings (speed and mode, such as line/frame/spot, accumulation/averaging, zoom/rotation)
• Other notable experimental conditions such as temperature, media and substrates, gas concentrations.

In addition to the above, per our policy, image manipulation and adjustments for analysis and publication should be kept to a minimum. For example, using raw uncompressed files for analysis is always preferred. Brightness and contrast should be adjusted throughout the whole image, equally to all images in the cohort, and only linear operations should be performed (e.g., no gamma manipulations).

All operations (preprocessing, filtering, deconvolution, thresholding, segmentation, etc.) performed during analysis should be reported in detail including software and package versions, and all values of parameters in algorithmic inputs. Ideally complete analysis code, or an analyzed file (e.g., FIJI macro, Imaris or Cellprofiler project file) should be provided, along with raw image files. If using open source or commercial AI classifiers relying on pre-trained neural networks, researchers should provide the version and architecture of the used network.

For publication quality images and for quantification, the images acquired with the microscopes have to be carefully handled and processed according to the Open Science Foundation’s Transparency and Openness Promotion reporting standards.

Finally, users can check the MicCheck, which is a microscopy metadata checklist generator tool designed to help simplify and customize the metadata list, according to the specific microscope you used for image acquisition.

For additional consideration, the CoRE provides the following instructions and ethical guidelines based on the article, “Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images” (10.1007/s11948-010-9201-y):

• The digital images acquired should represent the information accurately and not be altered to deceive the readers or obscure any information that can change the interpretation of the data. Images must be acquired following the appropriate design strategies, including controls where necessary, using well-maintained and aligned instrumentation.
• Simple adjustments such as brightness and contrast, levels, and gamma settings are acceptable, if it has been applied to the entire image. Cropping an image is acceptable for centering an object of interest, trimming “empty” space around the edges of the image, or excluding a piece of debris. Images cannot be cropped for removing information to manipulate the information present in the raw image. An example of changing the context of an image would be cropping out dead or dying cells to only display a healthy cell, or cropping out gel bands that might disagree with the hypothesis being proposed in the paper.
• For intensity quantification experiments, care should be taken to acquire the images under identical conditions, using the same microscope and the same settings, and should be closely timed (if possible) to avoid any drift in the instruments. The image processing should also be identical for all the images in the cohort. This includes techniques such as background subtraction or white-level balancing, which should be documented in the methods section. Individual images within a figure should only be processed differently if there are compelling reasons to do so. In such cases, the differences must be explained in the methods section or the figure legend. In conclusion, honesty and completeness are the best policies.
• Manipulations to a specific area of an image should be avoided. Selective enhancement of an image should be reported in the figure legend and if not reported, would most likely be viewed as research misconduct.
• Always maintain a copy of the raw file. Any manipulations to the images should be performed on the copy of the file.
• Use of software filters to enhance the quality of the images should also be avoided. Unmindful usage of the filters can cause artifacts in the image, and can lead to misinterpretation of the data. If filters must be used, they should be noted in the methods and figure legend.
• Copying objects from other parts of the image or from a different image is not acceptable. The use of cloning or copying techniques specifically to manipulate the image is research misconduct.
• Intensity measurements should be performed on uniformly processed image data, and the data should be calibrated to a known standard with appropriate scale bars.
• To maintain the integrity of the data, it is recommended to save the processed files using a loss-less compression format such as tiff. Loss compression techniques like JPEG reduces the quality of the images.
• Care should be taken when scaling the image size. Modifying the number of pixels in X and Y can introduce aliasing artifacts and alter the spatial resolution of the image. When enlarging or reducing an image in size, users should insert a magnification scale bar prior to changing the total number of pixels in an image.

Axio Imager Fluorescence Without ApoTome

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. An Axio Imager.Z2 widefield microscope (ZEISS Microscopy, Germany) was equipped with a Plan-Apochromat 10x/0.45 (Part Number 440639-0000-000; ZEISS Microscopy, Germany) objective lens. An Excelitas X-Cite 120 with a 120-watt mercury vapor short arc lamp provided illumination; no attenuation was used. The microscope was controlled by ZEN blue 2.0 (ZEISS Microscopy, Germany). Multiple fluorescence filter sets controlled by the software were used to separate fluorophore emission: 4',6-diamidino-2-phenylindole (DAPI) using Chroma Filter Set 49000 (Chroma Technology GmbH, Germany), captured at an exposure time of 75 milliseconds; and Alexa Fluor 488 using Chroma Filter Set 49002 (Chroma Technology GmbH, Germany), captured at 430 milliseconds. A Zeiss Axiocam 503 mono camera recorded the images; a 2x2 (four-pixel) bin but no digital gain was used. Images were saved in the proprietary CZI ("Carl Zeiss Image") format for analysis.

Axio Imager With ApoTome

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. An Axio Imager.Z2 widefield microscope (ZEISS Microscopy, Germany) was equipped with a Plan-Apochromat 10x/0.45 (Part Number 440639-0000-000; ZEISS) objective lens. The ApoTome.2 equipped with a high grid was overlaid on the sample to provide an optical section. An Excelitas X-Cite 120 with a 120-watt mercury vapor short arc lamp provided illumination; no attenuation was used. The microscope was controlled by ZEN blue 2.0 (ZEISS Microscopy, Germany). Multiple fluorescence filter sets controlled by the software were used to separate fluorophore emission: DAPI using Chroma Filter Set 49000 (Chroma Technology GmbH, Germany), captured at an exposure time of 75 milliseconds; and Alexa Fluor 488 using Chroma Filter Set 49002 (Chroma Technology GmbH, Germany), captured at 430 milliseconds. Images were captured using a Zeiss Axiocam 503 monochrome camera; no camera binning or digital ain was used. Exposure times were set with the ApoTome.2 inserted into the light path of the microscope. Images were saved in the proprietary CZI ("Carl Zeiss Image") format for analysis.

Leica DMi8 fluorescence

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. A Leica DMi8 (Leica Microsystems, Germany) was equipped with a HC PL Fluotar 20x/0.40 Ph1 (Part Number 506243; Leica Microsystems, Germany) objective lens. An SpectraX fluorescence illuminator (Lumencor, Oregon, USA) with multiple narrow-band light emitting diodes provided illumination. The microscope was controlled by LAS X software (Leica Microsystems, Germany). Multiple fluorescence filter sets controlled by the software were used to separate fluorophore emission: DAPI using a 395nm LED set to 50 percent and Leica Filter Set 525338 (Leica Microsystems, Germany), captured at 415 milliseconds; Alexa Fluor 488 using a 470nm LED set to 30 percent and Leica Filter Set 525314 (Leica Microsystems, Germany), captured at 250 milliseconds. Images were captured using a Leica DFC9000GT monochrome camera; a 2x2 (4-pixel) bin but no digital gain was used. Images were saved in the proprietary LIF ("Leica Image File") format for analysis.

Axio Imager Transmitted Light

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. An Axio Imager.Z2 widefield microscope (ZEISS Microscopy, Germany) set up for Kohler Illumination was equipped with a Plan-Apochromat 10x/0.45 (Part Number 440639-0000-000; ZEISS Microscopy, Germany) objective lens. A light-emitting diode configured on the transmitted light illumination port provided illumination. The transmitted light condenser was set to a Brightfield position, and the aperture diaphragm set to a Numerical Aperture of 0.4. The microscope was controlled by ZEN 2.0 blue (ZEISS Microscopy, Germany). White balance and shading correction (based on background image of slide) were implemented. A Zeiss Axiocam 503 color recorded the images. The camera exposure time was 15.0 milliseconds; no camera binning or digital gain was used. Images were saved in the proprietary CZI ("Carl Zeiss Image") format for analysis.

Leica DMi8 Transmitted Light

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. A Leica DMi8 (Leica Microsystems, Germany) set up for Kohler Illumination was equipped with a HC PL Fluotar 20x/0.40 Ph1 (Part Number 506243; Leica Microsystems, Germany) objective lens; the correction collar was set to 0.17mm and a matching Phase annulus in the condenser was positioned in the light path. A light-emitted diode provided illumination. The transmitted light condenser was set to a Brightfield position and the aperture diaphragm set to 16 as indicated by the touchscreen of the microscope. The microscope was controlled by LAS X software (Leica Microsystems, Germany). White balance and shading correction (based on an image of the background of the slide) were implemented. Images were captured using a Leica DFC450C color camera; no camera binning or digital gain was used. Images were saved in the proprietary LIF ("Leica Image File") format for analysis.

Images were acquired with Zen Black software (2.3 SP1 version 14.0.21.201) on a Zeiss LSM8800 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with GaAsP PMT detectors (Hamamatsu Photonics, Shizuoka, Japan) in confocal mode. Imaging was performed using a 100x/1.46 alpha-Plan-Apochromat lens (Carl Zeiss Microscopy GmbH, Jena, Germany) and an optical zoom of four; frame size was set to 256x256 pixels (X/Y) for a final lateral pixel size of 0.07 um/pixel, and images were acquired at 12bits. Acquisition was performed in frame scan mode, with a pixel dwell time of 1.54 us/pixel, and a frame average of two. Excitation lasers used were Argon 488nm for GFP and diode 561nm for tdTomato, and emission was collected through an MBS488/561 double dichroic (Chroma Technology, Bellows Falls, VT, USA) with emission collection windows set for [490-598nm] and [567-712nm], respectively. The 1 Airy Unit pinhole size (92.9 um) was calculated for the tdTomato channel and was matched to this size for the rest of the chromophores, for a final axial resolution of 0.8um. For z-stacks, z-step size was set at half the axial resolution (0.4um/step) to ensure adequate sampling. For each cohort, experimental positive controls were used to set the excitation parameters (laser power, detector gain); the parameters were set to provide the best signal to noise ratio for each chromophore, while utilizing the full dynamic range of the detector and avoiding detector saturation; detector gains were kept below 800V to ensure linearity of the response, and laser power rarely exceeded 2 percent. The settings determined at the positive control for each chromophore and experiment were used to acquire the rest of the cohort images.

Airyscan using LSM 880

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. Images were acquired with Zen Black software (2.3 SP1 version 14.0.21.201) on a Zeiss LSM880 (Carl Zeiss Microscopy GmbH, Jena, Germany) microscope equipped with a 32 channel Airyscan GaAsP-PMT detector (Hamamatsu Photonics, Shizuoka, Japan) in SR mode. Imaging was performed using a 100x/1.46 alpha-Plan-Apochromat lens (Carl Zeiss Microscopy GmbH, Jena, Germany) and an optical zoom of minimum 1.8; frame size was set to "optimal" to ensure that correct number of pixels based on Nyquist criteria was used for the dimensions of the field of view, speed was set to "maximum" and images were acquired at 16bits, and a frame average of 1. Excitation lasers used were Argon 488 for GFP and diode 561 for Alexa561, and emission was collected through Main Beam Splitter (MBS) 488/561, Second Beam Splitter (SBS) SP615 and Emission dual filters BP 420-480 + BP 495-550, and BP 570-620 + LP645, respectively. For z-stacks, z-step size was set at half the axial resolution (0.4um/step) to ensure adequate sampling. For each cohort, experimental positive controls were used to set the excitation parameters (laser power, detector gain); the parameters were set to provide the best signal to noise ratio for each chromophore, while utilizing the 50 percent of the full dynamic range of the detector and avoiding detector saturation; detector gain was kept below 800V to ensure linearity of the response, and laser power rarely exceeded one percent. The settings determined at the positive control for each chromophore and experiment were used to acquire the rest of the cohort images. Processing was performed with the ZEN Airyscan processing module with the automatic default Wiener filter settings. 

STED using Leica SP8 STED 3X

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. Images were acquired with LAS X software (Leica Application Suite X 3.5.7.23225) on Leica SP8 gated STED (Leica Microsystems GmbH, Wetzlar, Germany) microscope equipped with two PMTs and 3 HyDs. Imaging was performed using a 100x/1.4 STED-High Contrast PlanApo oil immersion lens (Leica Microsystems GmbH, Wetzlar, Germany) and an optical zoom of six, frame size was set to 1024x1024 pixels (X/Y) for the final lateral pixel size of 0.019 um/pixel, and images were acquired at 12bits. Acquisition was performed at frame scan mode, with a pixel dwell time of range 0.2-2.43µs. Fluorophores were excited with 561nm or 646nm laser derived from 80 MHz pulsed White Light Laser (Leica Microsystems GmbH, Wetzlar, Germany) and the stimulated emission was performed with a 775nm pulsed laser (Leica Microsystems GmbH, Wetzlar, Germany). The fluorophore emission was collected with Hybrid Detectors (HyD), Leica Microsystems GmbH, Wetzlar, Germany) using a gate of 0.3-6ns in respect to the excitation pulse. For Alexa555 imaging a spectral window of 570-700nm was used, and for Atto647N 650-730nm was used. Images were recorded in standard mode using three frames averaging and three line accumulation. For z-stack, z-step size was set to optimal, at half the axial resolution (0.086µm/step) to ensure the adequate sampling. The laser power and gain were set to provide the best signal to noise ratio for each chromophore (WLL 561nm at two percent, HyD Gain 91 percent and White Light Laster 640nmat 1.5 percent, HyD Gain 88 percent) while utilizing the full dynamic range of the detector and avoiding detector saturation; the excitation laser in STED mode was around 3x higher than in confocal mode; the stimulated emission depletion laser power was kept at 35 percent.    

Images were captured at the Microscopy and Advanced Bioimaging CoRE ofthe Icahn School of Medicine at Mount Sinai. Images were acquired with LAS X software (Leica Application Suite X 4.3.0.24308) on Leica Stellaris 8 (Leica Microsystems GmbH, Wetzlar, Germany) confocal microscope with TauSense technology. The microscope is equipped with four Power HyD S detectors and White Light Laser (WLL), tunable from 440nm to 790nm. Imaging was performed using 40x/1.3 HC PlanApo oil immersion lens (Leica Microsystems GmbH, Wetzlar, Germany) and an optical zoom of four, the frame size was set to 512x512 pixels (X/Y) for the final lateral pixel size of 0.142um/pixel, and images were acquired at 8bits. The acquisition was performed at frame scan mode, using three frames averaging and three line accumulation and a pixel dwell time of range 1.725µs.

Fluorophores were excited with a 440nm laser derived from an 80 MHz pulsed White Light Laser (Leica Microsystems GmbH, Wetzlar, Germany). The fluorophore emission was collected with Hybrid Detectors (HyDS, Leica Microsystems GmbH, Wetzlar, Germany) with a spectral window of 445-650nm. 

The laser power and gain were set to provide the best signal to noise ratio for each chromophore (WLL 561nm at 2 percent, HyDS Gain 91 percent ) while utilizing the full dynamic range of the detector and avoiding detector saturation.

If you were using TauSense, any language below relevant to the project should also be added: 

• Images were recorded in the counting mode using theTauContrast/TauGating/TauSeparation/TauScan mode.
  • TauContrastcomponent was applied to characterize the distribution of mean lifetime.
  • TauGatingcomponent was applied to enable splitting photons arriving at different times. Signal was split into two digital gates with a selected time 3-1ns and 1.2-6ns.
  • TauSeparationcomponent was applied to select appropriate temporal windows and fits the lifetime-based information in these windows to generate separate images. The TauSeparation peaks were set up to 0.9ns and 1.6ns. 
  • TauScancomponent was applied to scan the mean lifetime components distribution and separate them into a predetermined number of temporal windows.

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. Images were acquired with Olympus Fluoview (FV31S-SW) software on an Olympus FVMPE-RS multiphoton microscope (Olympus, Tokyo, Japan) equipped with two GaAsP detectors and two multi-alkali PMT detectors and a Spectra Physics Insight X3 dual laser. Imaging was performed using a 25x/1.05NA TruResolution XL Plan N objective lens (XLPLN25XWMP2, Olympus, Tokyo, Japan) with 2mm Working Distance and an optical zoom of two; frame size was set to 512X512 pixels (X/Y) for a final lateral pixel size of 0.9944um, and images were acquired at 8 bits. Acquisition was performed at Line sequential scan mode, with a pixel dwell time of 2us/pixel, and a frame average of two. The Insight X3 Dual line multiphoton laser was used for imaging. The tunable laser line was set to 770nm for Alexa Fluor 488 and Alexa Fluor 594 and the second line at 1045nm was used for Alexa Fluor 647. The emission was collected through band-pass filters with ranges of 495-540nm for AF499, 575-645nm for AF594, and 660-750nm for AF647. For z-stacks, z-step size was set at half the axial resolution (1um/step) to ensure adequate sampling. For each cohort, experimental positive controls were used to set the excitation parameters (laser power, detector gain); the parameters were set to provide the best signal to noise ratio for each chromophore, while utilizing the full dynamic range of the detector and avoiding detector saturation; detector gains were kept below 800V to ensure linearity of the response. The tunable laser was set to two percent and the 1045nm line was set to 10 percent for excitation of the fluorophores. The settings determined at the positive control for each chromophore and experiment were used to acquire the rest of the cohort images.

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. Spinning disk imaging was performed on a Zeiss Axio observer inverted platform, equipped with a Yokogawa CSU-X1 spinning disk, a Hamamatsu cooled Electron-Multiplying CCD (ver. C9100-13), a Versalase multi-wavelength laser module, an LD Plan-Neofluar 20X/0.4 Corr Ph2 Lens (Carl Zeiss Microscopy GmbH, Jena, Germany), and controlled by Metamorph (ver. 7.10.2.240) software (Molecular Devices, San Jose, CA, USA). Cells were incubated in a Tokai-Hit stage-top incubator (95 percent O2/5 percent CO2, 37 oC, 90 percent humidity) throughout imaging. The 488-excitation laser was set at 30 mW output, and imaging was performed at one sec interval for five minutes: After a baseline of one minute, drugs were administered through injection tubes to avoid disturbing the sample, and imaging continued for four minutes (301 frames total). Camera was set at minimum exposure time for the digitizer setting (122.12ms for 2.75 mHz, at a gain of 1X, and an Electron-Multiplying gain of 200) to minimize light exposure.

Images were captured at the Microscopy and Advanced Bioimaging CoRE of the Icahn School of Medicine at Mount Sinai. Images were acquired with ImSpectorPro software (version 7.0.132.0) on light sheet UltraMicroscope II (Miltenyi Biotec, Germany) equipped with zoom body optics, an Neo 5.5 sCMOS camera (Andor, Ireland) in 16-bit mode, and Olympus MVPLAPO 2X/0.5 objective, and a 5.7-mm working distance corrected dipping cap (total magnifications ranging from 1.3X to 12.6X with variable zoom). Samples were imaged at 1.6X mag (0.8X zoom), using the three light sheet configuration from a single side, with the horizontal focus centered in the middle of the field of view, an NA of 0.043 (beam waist at horizontal focus = 17 µm), and a light sheet width of 60–100 percent (adjusted depending on sample size to ensure even illumination in the y-axis). Pixel size was 3.9µm and spacing of Z slices was 5µm. Samples were illuminated with 488nm and 640nm lasers (Coherent, Germany) and a Chroma ET525/50m and ET670/50m emission filter respectively. The chromatic correction module on the instrument was used to ensure both channels were in focus.

Image analysis was performed using FIJI (Version 2.0.0-rc-69/1.52p) software through an automated pipeline. Image stacks were imported and converted to 8-bit. The brightness/contrast was adjusted for all the images with the same fixed intensity values. A sum projection of each stack was generated, smoothed, and thresholded using the Otsu method ([25,255]), separated using the watershed algorithm and used to generate regions of interest (ROI) containing cells. The number of cells were counted using the Cell Counter Plugin. Mask of the cells were generated using the Analyse Particles in Fiji and ROIs were generated. The ROIS were then applied to the original image. The area of the cells, the average intensity, and integrated density were then extracted with the Measure function.

Additional language and protocols can be provided to accommodate the specific projects of the principal investigator.

Pre-Embed Processing

Animals were anesthetized and perfused intracardially using a peristaltic pump at a flow rate of x mL/min with one percent buffered aldehyde solution pH 7.2-7.4 for one minute, and immediately followed with a two percent paraformaldehyde and two percent glutaraldehyde/Phosphate-Buffered Saline (PBS), pH 7.2-7.4, at the same flow rate for an additional 10-12 minutes. Tissue was removed and placed in immersion fixation (same as above) to be post-fixed for a minimum of one day at 4°C.  Tissue samples were sectioned at 100-200µm using a Leica VT1000S vibratome (Leica Biosystems Inc., Buffalo Grove, IL) and sections were cherry picked for processing based on region of interest.

Epoxy Resin Embedding

Material was embedded using standard electron microscopy embedding protocols (Electron microscopy Sciences [EMS], Hatfield, PA).  Biological tissue samples were fixed with 1.5 percent potassium ferricyanide/two percent osmium tetroxide/0.1M sodium cacodylate, pH 7.2-7.4 buffer, (EMS) followed with an en bloc staining of two percent uranyl acetate (EMS). Samples were dehydrated via graded ethanol series (25 percent, 50 percent, 70 percent, 95 percent, 100 percent) and infiltrated with propylene oxide (EMS) and “Embed 812” Epon resin (EMS).  Tissue was placed in BEEM embedding capsule molds (EMS), filled with resin, and heat polymerized at 60° C for 72 hours in a vacuum oven. Semithin sections (0.5 and 1 µm) were obtained using a Leica UC7 or UCT ultramicrotome (Leica Biosystems Inc., Buffalo Grove, IL), counterstained with one percent toluidine blue, cover slipped, and viewed under a light microscope to identify and secure region of interest. Ultrathin sections (85 nm) were cut with a diamond knife (Diatome, Hatfield, PA) and collected on copper 300 mesh grids(EMS) using a Coat-Quick adhesive pen (EMS). Sections were counter-stained with one percent uranyl acetate followed with lead citrate.

Lowicryl Resin Embedding

Tissue samples were cryoprotected via graded glycerol/phosphate buffer (10 percent, 20 percent, 30 percent) at 4°C. Blocks were rapidly freeze-plunged into liquid propane cooled by liquid nitrogen (−90°C) in a Universal Cryofixation System KF80 (Reichert-Jung, Vienna, Austria) and subsequently immersed in 1.5 percent uranyl acetate dissolved in anhydrous methanol at −90°C for 24 hours in a Leica EMAFS freeze substitution unit (Leica Biosystems Inc., Buffalo Grove, IL). Block temperatures were raised from −90°C to −45°C in steps of 4°C/hour, washed with anhydrous methanol, and infiltrated with Lowicryl HM20 resin (Electron Microscopy Sciences, Hatfield, PA) at −45°C. The resin was polymerized by exposure to ultraviolet light (360 nm) for 48 hours at −45°C followed by 24 hours at 0°C. Ultrathin sections (90 nm) were cut with a diamond knife (Diatome, Hatfield, PA) on a Leica UC7 or UCT ultramicrotome (Leica Biosystems Inc., Buffalo Grove, IL) and sections were collected on formvar/carbon coated nickel slot grids (EMS).

Electron Microscopy Imaging

Images were taken on an HT7500 transmission electron microscope at x magnification (Hitachi High-Technologies, Tokyo, Japan) using an AMT NanoSprint12 12-megapixel CMOS TEM Camera (Advanced Microscopy Techniques, Danvers, MA). Final image brightness, contrast, and size were adjusted using Adobe Photoshop CS4 software version CS4 11.0.1 (Adobe, Inc., San Jose, CA, USA).

Tissue was imaged at x magnification on an HT7700 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan) equipped with an AMT XR41-B 4-megapixel digital camera,

(Advanced Microscopy Techniques, Danvers, MA). Image brightness, contrast, and size were adjusted using Adobe Photoshop CS4 software version CS4 11.0.1 (Adobe, Inc., San Jose, CA, USA).