Confocal Sample Preparation Guide
Preparing Your Sample for a Straight Laser Light Path is Vital to Success
The amazing power of a confocal microscope lies in its ability to isolate a single plane of focus from thick
samples or overlapping objects, BUT… the lasers 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 fluorescence in the
sample, which emits energy of a longer,
(green-yellow) wavelength.
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 photodetector.
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.
2.
3.
4.
5.
6.
7.
8.
9.
Control the refraction of light between your sample and the confocal pinhole.
Choose an appropriate sample for confocal microscopy.
Choose the correct objective & immersion media.
Choose the correct cover slip or sample dish.
Avoid compression and Distortion of the sample.
Prepare the sample for an inverted light path.
Match the sample and its surrounding medium by refractive index.
Understand the spectral profiles of your fluorophores before capturing images.
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 lenses
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 confocal microscopy. 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 wavelengths 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 emission
spectrum, 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 lens 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.
Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost
•
NON FLUORESCENT MATERIALS: Modern confocal microscopes 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 plant tissues and aldehyde-fixed tissues
contain high levels of background autofluorescence. These substances can interfere with detection
and imaging of positively-stained fluorescent dyes 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 plants and insects 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 camera 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
numerical aperture (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.
Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost
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 notcheck 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.
Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost
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
as possible from the sample through the glass to the
objective opening, pinhole and detector.
Mismatches and incorrect cover slips don’t let all of
the light in through the lens, creating a dim,
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)
Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost
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
Dishes
MatTek Plates
Ibidi Chamber
Slides
Nunc Chambers
Coverslip-Bottom Dish
Coverlip-bottom dishes allow the sample to float
freely in water, saline or medium in the dishbottom.
Coverslip & Slide Options…………………………………………………………………………...
Slide & Coverslip Setups
Ibidi µ-Slides
Invitrogen Biomeda
Gaskets
#1.5 High-Res.
Coverslips
Spacer/Sealents of
Vaseline or VALAP
For slides, fasten coverslip on with nailpolish,
agarose, glue, or Vaseline .
Integrated Microscopy @ Biotron offers#1.5 coverslips, MatTek Dishes, and ProLong Gold media at cost
Match the Sample & Medium by Refractive Index
Glass & Plastics
Flint Glass
Borosilicate Coverglass
Polystyrene
Plexiglass
NO Coverslip (Air)
Refractive Index
1.627
1.523
1.55
1.50
1.0003
Common Sample Types*
Cerebral Gray Matter
Liver
Cartilage
Bone
Intestinal Wall
Lung
Fat
Blood serum
Insect Chitin
Arabidopsis Leaf
Calcite
Quartz
Quartz, Fused
Diamond
Refractive Index
1.395
1.448
1.492
1.556
1.436
1.342
1.472
1.330
1.56-1.57
1.36
1.486
1.544
1.458
2.417
Immersion Medias
Water
Air
Zeiss Immersol
Zeiss Immersol W
Refractive Index
1.338
1.0003
1.518
1.334
Mounting Medias
ProLong Gold (Invitrogen)
Vectashield Medium
Permount (Fisher Sci.)
50% Glycerine
90% Glycerine
Water
Ethanol
Polyvinyl Alcohol
Acetone
Methyl Salicylate
Dimethyl sulfoxide
PFD (perfluorodecalin)
DPX(Fluka)
Gel Mount (Biomeda)
Refractive Index
1.39-1.46 (over 160 hours)
1.457
1.518-1.521
1.416
1.46
1.338
1.36
1.52-1.55
1.36
1.541
1.484
1.313
1.5251
1.3641
Refraction in Microscopy causes visual siplacement
and magnification effects, so object and angle
measurements with be incorrect.
Refraction in Microscopy also causes light to miss
the objective opening, weakening signal.
It also creates chromatic aberrations-the splitting of
light into undesired spectra.
*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 Fluorophore’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 dye 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 Fluorescein (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
Manager, Integrated Microscopy @ Biotron
University of Western Ontario
Biotron Research Building, Room #105 F
London, ON N6A 5B7
Office: 519-661-2111 x88061
Fax: 519-661-2149
Email: [email protected]
Nicole Bechard
Microscopy Technologist, Integrated Microscopy @ Biotron
University of Western Ontario
Biotron Research Building, Room #105 J
London, ON N6A 5B7
Office: 519-661-2111 x80460
Fax: 519-661-2149
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 Laboratories Canada Inc: Fluorescent Stains, Mounting Media, antibodies,
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