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Home , Confocal microscopy, Microscopy

Confocal Guide Preparing Your Sample for a Straight Path is Vital to Success

The amazing power of a confocal lies in its ability to isolate a single plane of focus from thick samples or overlapping objects, BUT… the need a straight optical path from their source to your specimen and back to the light detectors to achieve this.

The Basic Confocal Light Path 1. Laser light (in blue) is focused by the collimeter and passes through the Main dichroic beamsplitter. 2. Scanning mirrors direct the light in a raster pattern (lines) across the sample. 3. Light excites the in the sample, which emits energy of a longer, (green-yellow) . 4. The dichroic beamsplitter reflects (blue) laser light, but allows the longer (yellow) emission wavelength through. 5. Only the path of light from the focal plane of interest (outlined in black) can pass through the pinhole to the . 6. Out-of-focus light (red and blue dashed lines) is blocked.

There are 9 Basic Steps to Success: Which are vital to achieve publishable images and data.

1. Control the of light between your sample and the confocal pinhole. 2. Choose an appropriate sample for confocal . 3. Choose the correct objective & immersion media. 4. Choose the correct cover slip or sample dish. 5. Avoid compression and Distortion of the sample. 6. Prepare the sample for an inverted light path. 7. Match the sample and its surrounding medium by . 8. Understand the spectral profiles of your before capturing images. 9. Consult with our staff before you begin-we can help you achieve your goals!

Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost

You Must Control Refraction in the Light Path Between the Laser and the Image Detector Lies:

The sample itself The mounting medium surrounding the sample. The cover glass The immersion medium between the objective and the glass. The chosen objective themselves.

These can each refract light if they aren’t properly matched by refractive index. Improper choices at each level causes two types of distortions: Chromatic Aberration, causes colours to shift location in the final image and interfere with the interpretation of colocalization. Spherical Aberration (illustrated above) causes objects to appear stretched, magnified or shrunken, causes reflections, alters brightness and contrast, causes fuzzy images with poor resolution, seriously alters spatial measurements, and distorts 3D reconstructions.

Choose an Appropriate Sample Notes on Various Sample Materials:

• TRANSPARENT MATERIALS: are the best candidates for . They should be mounted in a transparent solution with good refractive qualities such a commercial mounting medium, water, or glycerine . • FLUORESCENT MATERIALS: This instrument is designed primarily for viewing fluorescent materials. We offer 9 laser lines of varying from 405nm-633nm for laser excitation, and a variety of emission filters for detection. Our Zeiss LSM 510 Duo also comes equipped with a spectral scanner that can detect any wavelength of light from UV- near IR, determine its , and isolate its signal from that of other compounds such as autofluorescent pigments in the sample. • THICKNESS: The confocal microscope has a maximum penetration depth of about 100-200 um in ideal conditions. This means using a transparent sample, mounted properly on the correct thickness of coverslip, in the correct medium, using a that has a long working distance. For best results, your area of interest should be as close to the cover slip as possible, and not obscured by thick layers of cells or other materials. Thicker materials may need to be physically sectioned or optically cleared before imaging. • OPAQUE MATERIALS: cannot be penetrated with the lasers, however techniques are available to visualize the surface of opaque objects facing the laser e.g. solid nanopartices, polymers, polished rock surfaces, and biological scaffolding surfaces.

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• NON FLUORESCENT MATERIALS: Modern confocal are designed for imaging fluorescent materials using laser excitation of these compounds. They are not designed to take bright field microscopy images. We can obtain outlines of cell structures, fibres and surfaces using either differential interference contrast (DIC) or laser reflectance microscopy to view these substances, but the primary use of this instrument is for highly-resolved fluorescence imaging. • AUTOFLUORESCENT MATERIALS: many materials such as tissues and aldehyde-fixed tissues contain high levels of background . These substances can interfere with detection and imaging of positively-stained fluorescent and yield false positive results or overwhelm the signals of stained materials. We recommend discussing your sample type and its unique requirements with our staff during the planning stages of your experiment to avoid problems. • BUBBLES: in the media should be avoided as they will bend light and obscure underlying structures. • OIL/WATER or AIR/WATER INTERFACES: Like bubbles, mismatches between substances of varying refractive index should be avoided. e.g. oily substances in water. These reflect the laser, creating false signals, chromatic effects, distortion of underlying materials, and diminished signal strength. • CUTICLES AND CHITIN: Waxy or chitinous cuticles in and should be chemically cleared or physically sectioned away before imaging. • DIRT AND FINGERPRINTS: Also interfere with good confocal images. Coverslips should always be sealed down with nailpolish and the surfaces freshly cleaned before imaging. DRY SAMPLES: Ideal optics come from using samples suspended in resinous or aqueous solutions. Dry, uneven surfaces reflect light away from the lens and yield dim, poorly-resolved results.

Choose the Correct Objective & Immersion Media Consider Resolution, Magnification, and Depth of Sample: An objective’s magnification is meaningful only in terms of how big something appears to your eyes through the microscope’s oculars. Since we now take digital images and magnify them with software, how big we can view them clearly is based on their RESOLUTION when they are captured-a function of the qualities of the lens, the light source, the sample preparation, and the image detector or setup.

EFFECT OF VARYING RESOLUTION ON IMAGE DETAILS: MAGNIFICATION IS MEANINGLESS WITHOUT GOOD RESOLUTION Resolution is the ability of a lens to distinquish objects which are closely spaced.

Resolution is determined by the (N.A.) of the lens, not the magnification. More expensive lenses have higher N.A., and therefore better resolution.

This resolution depends on a light path from the sample which fills but doesn’t go beyond the edges of the lens.

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It is important to understand the objective you are using and what it is intended for. For confocal microscopy, Plan-Apochromat objectives are the best choice because they maintain a flat field of view across the image, correct well for all colours of the spectrum commonly used. These objectives are set for a 1.5 (0.17mm) coverslip thickness. Some objectives are capable of DIC imaging and some are not- check with our staff.

Choosing the correct immersion media for the lens is vital. With mismatched refractive indices, the red light beams can refract incorrectly and miss the lens opening, severely reducing the level of emission signal that reaches the detector.

Choosing the Correct Lenses on the Zeiss LSM 510 Duo Vario : • Monolayers and Tissue Sections: Combining a hard-setting mounting medium with a high-quality oil immersion lens such as our 40x and 63 x oil lenses give the very best resolution possible, but these very high N.A. lenses can’t focus deeply into thicker samples. • Thicker Specimens: For longer working distance and deeper imaging, choose our 20x Air or 63x Glycerine/Water Multi lens, and mount your specimen in a glycerine/aqueous mounting medium. • Bare Rocks, Metals: Choose the 20x dry high N.A. lens. It has excellent resolution in air. • Live Cells in Aqueous Solution: We recommend the 63x Multi-immersion lens. It has a correction collar for adjusting to different refractive indices and glass thicknesses, and deeper working distance than the 63x oil lens.

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Use the Right Coverglass or Culture Dish

**Confocal microscope objectives focus best through a #1.5 coverslip **

The goal is to keep the path of the light as straight Mismatches and incorrect cover slips don’t let all of as possible from the sample through the glass to the the light in through the lens, creating a dim, objective opening, pinhole and detector. distorted, and fuzzy image.

**We recommend and sell Zeiss High Performance Coverslips at Cost **

Avoid Compression & Distortion

Add spacers between coverslips and slide and seal to avoid evaporation:

Cells pressed under coverslips without spacers are compressed and distorted.

1. Place cells in confocal dishes, or 2. Include Spacers between the coverslips. 3. Seal edges with nailpolish, or, for GFP analogs use VALAP sealant as nailpolish can quench GFP (1:1:1 Vaseline:Lanolin”Paraffin)

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Prepare Samples for an Inverted Light Path Coverslips must be mounted on the bottom of the slide or dish as shown: Culture Dish and Chamber Slide Setups …………………………………………………. MatTek or Ibidi MatTek Plates Dishes Coverslip-Bottom Dish

Coverlip-bottom dishes allow the sample to float freely in water, saline or medium in the dish- bottom.

Ibidi Chamber Nunc Chambers Slides

Coverslip & Slide Options…………………………………………………………………………... Invitrogen Biomeda Slide & Coverslip Setups Ibidi µ-Slides Gaskets

For slides, fasten coverslip on with nailpolish, agarose, glue, or Vaseline .

#1.5 High-Res. Spacer/Sealents of Coverslips Vaseline or VALAP

Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost

Match the Sample & Medium by Refractive Index

Glass & Plastics Refractive Index Refraction in Microscopy causes visual siplacement Flint Glass 1.627 and magnification effects, so object and angle Borosilicate Coverglass 1.523 measurements with be incorrect. Polystyrene 1.55 Plexiglass 1.50 NO Coverslip (Air) 1.0003

Common Sample Types* Refractive Index Cerebral Gray Matter 1.395 Liver 1.448 Cartilage 1.492 Bone 1.556 Intestinal Wall 1.436 Lung 1.342 Fat 1.472 Blood serum 1.330 Chitin 1.56-1.57 Arabidopsis Leaf 1.36 Calcite 1.486 Quartz 1.544 Quartz, Fused 1.458 Refraction in Microscopy also causes light to miss Diamond 2.417 the objective opening, weakening signal.

Immersion Medias Refractive Index Water 1.338 Air 1.0003 Zeiss Immersol 1.518 Zeiss Immersol W 1.334

Mounting Medias Refractive Index ProLong Gold (Invitrogen) 1.39-1.46 (over 160 hours) Vectashield Medium 1.457 Permount (Fisher Sci.) 1.518-1.521 50% Glycerine 1.416 It also creates chromatic aberrations-the splitting of 90% Glycerine 1.46 light into undesired spectra. Water 1.338 Ethanol 1.36 Polyvinyl Alcohol 1.52-1.55 Acetone 1.36 Methyl Salicylate 1.541 Dimethyl sulfoxide 1.484 PFD (perfluorodecalin) 1.313 DPX(Fluka) 1.5251 Gel Mount (Biomeda) 1.3641

*For More Materials: http://interactagram.com/physics/optics/refraction/ Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost

Learn Your ’s Spectral Profile Consult one these websites and get a spectral profile of your dyes:

Invitrogen Spectral Viewer http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html BD Fluorescent Spectral Viewer http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/index.jsp

Spectral Considerations (the “WHY?): Thorough understanding of the spectral characteristics of your fluorophores is necessary for us to help you avoid crosstalk between different dyes, or between dyes and native pigments or chemicals in samples, which can lead to false-positive signals .

The excitation and emission wavelengths provided in product literature tell you little about potential false signals from spectral overlap. A complete graph of the spectral data should be obtained in order to: • Choose the best combinations for multichannel and colocalization experiments • Select the correct lasers for excitation of the fluorophores • Select lasers that specifically excite only one fluorophore at a time if possible • Select emission filters that specifically detect one only fluorophore at a time

Dye and Laser Choice Causes Signal Crosstalk

Excitation of (FITC) with the 488 Laser also causes excitation of the Rhodamine (TMRho) dye.

Poor Dye, Laser /Filter Choice Records Signal Crosstalk

Emission filter for FITC detects some of the false positive Rhodamine signal

Good Dye, Laser /Filter Choice Avoids Imaging the Signal Crosstalk

Emission filter chosen for FITC avoids overlapping with the false positive Rhodamine signal

Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost

And Finally…Consult With Us! We Can Offer You Years of Experience and Consultation is Free!

Karen Nygard Nicole Bechard Manager, Integrated Microscopy @ Biotron Microscopy Technologist, Integrated Microscopy @ Biotron University of Western Ontario University of Western Ontario Biotron Research Building, Room #105 F Biotron Research Building, Room #105 J London, ON N6A 5B7 London, ON N6A 5B7 Office: 519-661-2111 x88061 Office: 519-661-2111 x80460 Fax: 519-661-2149 Fax: 519-661-2149 Email: [email protected] Email: [email protected]

Suggested Suppliers

1. Zeiss Canada Inc.: Coverslips and Immersion Media http://www.zeiss.ca/ 2. MatTek Corp.: Petri Dishes and MultiWell Plates http://www.glass-bottom-dishes.com/ 3. Ibidi Cells in Focus: µ-Slides, Flow Chambers, Chamber Slides http://www.ibidi.de/ 4. Nuncbrand: Chamber Slides and Glass-Bottom Dishes http://www.nuncbrand.com 5. Molecular Probes/Invitrogen: Fluorescent Stains, Mounting Media http://www.invitrogen.com 6. Vector Canada Inc: Fluorescent Stains, Mounting Media, , http://www.vectorlabs.com/contactus.asp 7. Dako Laboratories, Canada Ltd.: Fluorescent Stains, Mounting Media, antibodies, http://www.dako.com/ca/index/products.htm 8. Biocare Medical: Fluorescent Stains, Mounting Media, antibodies http://www.biocare.net/

Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost