Project Hollywood

Fred MartinBIOSC. 3 Dr. Giorgi 4/30/2010

Specimen used: Buccal cells stained with ER Tracker

Imaging systems used:

Leica SP5

Objectives used:

Leica SP5 HCX PL APO CS 40.0x/0.92 IMM-OIL

Note: metadata detail presented on last page

Specimen Info: Buccal cells:

Human cheek cells are squamosal epithelial cells which form the inner cheek wall lining.

Morphology: Thin, flat polygonal cells which form a surface lining in the cheek’s interior with irregular boundaries. Cell are arranged edge to edge.Unstained, the cells have a prominent nucleus, and little else visible at 100-400x.

Stained with methylene blue, the plasma membrane borders become more apparent, as does the nucleus and cytoplasm.

Smaller internal organelles may be apparent, but difficult to know what it is, or if it’s just fragments of your last meal….

Typical morphology of simple stained of human buccal cells

Image source: homebiology.blogspot.com/2009/05/cells.html

Cell membrane

Cytoplasm

Nucleus

INFO ON LIVE LABEL:

ER-Tracker™ Green (BODIPY® FL glibenclamide) *for live-cell imaging*Cat. No. E34251 The ER-Tracker™ Green dye is cell-permeant, live-cell stain that is highly selective for the endoplasmic reticulum (ER) that when stained using the protocol provided, the staining pattern is partially retained after fixation with formaldehyde. This stain consists of the green-fluorescent BODIPY® FL dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling.

1.3 View/image cells: Required filter setsDye Ex/Em * FilterER-Tracker™ Green dye 504/511 FITCBODIPY FL

Cell Preparation and Staining1.1 Prepare staining solution. Dilute the 1 mM stock solution to the final working concentration. We recommend working concentrations of 100 nM–1 μM for ER-Tracker™ Blue-WhiteDPX and ~1 μM for ER-Tracker™ Green and ER-Tracker™ Red dyes. To minimize potential labeling artifacts, use the lowest dye concentrations possible. Best results are obtained whenstaining is performed in Hankʼs Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg, Gibco cat. #14025-092) at 37°C/5% CO2.

1.2 Stain the cells. For adherent cells, remove the medium from the culture dish, rinse with HBSS, and add prewarmed staining solution. Incubate the cells for approximately 15–30 minutes at 37°C. Replace the staining solution with fresh probe-free medium and view the cells using a fluorescence microscope. If the stained cells are to be fixed, refer to the fixation steps below for the appropriate dye.

MFG RECOMMENDED LIVE LABEL PROTOCOL:

Source: Invitrogen website

Live Cell Imaging Working Protocol Fred Martin 4/23/10

Specimen Cells: (1) Hela cells from Molecular Foundry (2) Alternative: Buccal cells

Dye: ER-Tracker Green (BODIPY FL glibenclamide) Molecular Probes/Invitrogen #E34251 for live-cell imaging; highly selective for the endoplasmic reticulum 100 ug lot 497266Dye Preparation: Note: Dye is supplied as 100 ug of lyophilized material.

Step1: Prepare a 1 mM stock solution by dissolving the contents of the vial in 128 uL of anhydrous DMSO; Note: had to make high conc & dilute due to only ~30uL of DMSO available Tobey’s dye prep.

Step2: Separated into 2 working aliquots,and the unused portion of the ~4 mM solution to be stored frozen with desiccant. Stored conc stock in freezer(no dessicant) in 1.5mL label E-tube

Cell Preparation and Staining

Step3: Dilute staining solution to working conc. Dilute the ~4 mM stock solution to the final recommended working concentration of ~1 uM Use Hank’s Balanced Salt Solution with calcium and magnesium at 37°C/5% CO2. (HBSS best results/low artifacts)

Step4: Stain the cells. For HeLa cells, aspirate the medium from the culture dish; Rinse with HBSS for 1 minAdd prewarmed staining solution. Incubate the cells(dry incubator, no CO2) for approximately 25 minutes at 37°C. (actual was range from 35-42deg)Replace the staining solution with fresh probe-free, non-phenol red medium (used HBSS)

Step5: View/image cells: Required filter sets

Dye Ex/Em * FilterER-Tracker™ Green dye 504/511 FITCBODIPY FL

Representative Image from Z-stack

I chose this image because it shows both cells that took the stain, and those that did not; and for those that did, you can see what I perceive to be SER and RER distribution, also ER peri-nuclear membrane staining and interesting channels across that membrane

Complilation of Images from Z-stack

Scale factor = 0.25Start slice = 5End slice = 68Increment = 4

Buccal Cells:

Series001 , AVI

w/ ER Tracker

From 77 slice

Z-stack

Comments on process:

Lack of materials required some improvisation, but still good resultDecided to keep it simple and try 1 channel staining only and go for qualityWas not as difficult as I had imagined/perceivedTouching slide to coverslip (sitting on top of chamber) was easier than fishing coverslip out of chamber Incubation control was poor due to poor incubator & lack of coordinated use

Observation of specimen:

I believe most cells were likely not viable by the time imaging commenced, but some were during the staining stage as clear differences in uptake of stain are seen; took most of the ‘live Hollywood’ out of the project, but good data no less There do appear to be differential patterns the ER staining (proximal vs distal to nucleus) Speculate this may represent SER vs RER ? Differences in amount/location of binding sites? The closeup zoom of the nucleus shows peri-nuclear staining and resolves some interesting features on the nuclear membrane surface; consistent with idea that parts of the ER is contiguous with the nuc. Membrane See extra images that follow…

Bu

ccal Cells – E

R T

racker, Max P

rojectio

n 33

slice Z-stck

Note: peri-nuclear staining differences

Dead cells?

RER?

SER?

Bu

ccal Cells S

eries013 – ER

Tracker, M

ax P

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n 54 slice Z

-stck, 3.7 zoo

m facto

r

Note: peri-nuclear staining differencesPeri-nuclear membrane staining with apparent channels / invaginations showing

• Pinhole [m]95.5  µm • Pinhole [airy]1.00 • Size-Width 246.0  µm Size-Height 246.0  µm • Size-Depth9.0 µm• StepSize0.12  µm • Voxel-Width 481.5  nm Voxel-Height 481.5  nm • Zoom1.0 Scan-Direction1 SequentialMode0 Frame-Accumulation 1• Frame-Average1 Line-Average1 Resolution 8  bits Channels = 1• Format-Width 512  pixels Format-Height 512  pixels Line-Accumulation1• Number of Sections 77

• Hardware Details • PMT 1ActivePMT 1 (Offs.)-1.2  % PMT 1 (HV)646.7 PMT 1 (HV_Unit)V• PMT2 Inactive PMT 3Inactive PMT Trans Inactive• Excitation Beam Splitter FWTD 488/543/633• Laser (Argon, visible)OnLaser (Argon, visible) (Power) 20 % Laser (HeNe 543, visible)On

Laser (HeNe 633, visible)On • Scan Field Rotation0  degrees Scan Speed 400  Hz • Objective HCX PL APO CS 63.0x1.40 OIL UV Numerical aperture (Obj.)1.40Refraction

index1.52

Metadata Details Z-section