2018/04/29 confocal Pendahuluan

Mengapa menggunakan mikroskop confocal?

Fitur yang paling penting dari mikroskop confocal adalah capablity mengisolasi dan mengumpulkan pesawat fokus dari dalam
sampel, sehingga menghilangkan keluar dari fokus "kabut" biasanya terlihat dengan sampel neon. Detail halus sering
dikaburkan oleh kabut dan tidak dapat dideteksi dalam confocal non, mikroskop fluorescent.

Mikroskop confocal memiliki motor stepper melekat fokus halus, memungkinkan koleksi dari serangkaian gambar

melalui objek tiga dimensi. Gambar-gambar ini kemudian dapat digunakan untuk dua atau tiga rekonstruksi dimensi.

Double and triple labels can be collected with a confocal microscope. Since these images are collected from an
optical plane within the sample, precise colocalizations can be performed.

A plane of focus within a specimen is

defined by the optics of the
microcope. In a fluorescent
microscope a small part of a sample
may be in focus but you look at the
entire object (i.e. you view what is in
focus as well as what is out of focus).
With the confocal microscope, the
z-resolution, or optical sectioning
thickness, depends on a number of
factors: the wavelength of the
excitation/emission light, pinhole size,
numerical aperture of the objective
lens, refractive index of components in
the light path and the alignment of the
instrument. This figure depicts the
effect of the pinhole, or iris diaphragm,
on the thickness

of the optical plane that is collected. The pinhole and focal plane in the sample are at conjugate planes of focus.

The small pinhole opening in the diagram on the left enables data collection from a thin optical plane within the
specimen. Points that are out of the plane of focus (red) will have a different secondary focal plane thus, most of the
data is deflected.

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Although some of the out-of-focus light enters the photomultiplier tube (PMT) in the figure on the left, the intensity is too
dim to be visualized. All of the data at the plane of focus is collected (blue). In this manner, the confocal microscope
can collect only the data from within the focal plane.

The larger pinhole opening in the figure on the right allows both in-focus and out-of-focus data to be collected.

More information on how a confocal works: http://glinda/lsrm.upenn.edu/~weeks/confocal

Chapter 1 in "From Bones to Atoms" is on confocal microscopy:
http://www.mih.unibas.ch/Booklet/Booklet96

Fluorescence, Reflectance or Transmission?

Confocal microscopy is most commonly used for detecting fluorescent labels but can also be used in a reflectance
mode for DAB or immunogold labeling. A transmitted image can be collected along with the fluorescent or
reflectance images but, a transmitted image is not confocal.

Sample Preparation

The best starting point is to follow a protocol from a lab getting good results using the same or similar material. You still
need to experiment with times and concentrations for fixation and labeling. Controls are essential.

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A good, basic reference is Introduction to Immunocytochemistry by J.M.Polak and S.Van Noorden.

Fixation
Good tissue preservation is generally a trade-off with preserving antigenicity. Fixation must adequately stabilize antigenic
sites, and other tissue components, for subsequent treatment. If fixation is inadequate, the fluorescence in your sample
can diffuse and disappear over (a possibly very short) time. Ideally one would compare a fixed sample to a living
specimen so you know what artifacts you have induced by fixation. All processing induces artifacts...we can only try to
minimize them.

Several factors must be considered when deciding on a fixation protocol: composition of the fixative, pH, osmolarity,
temperature, time and method (perfusion or immersion) can all affect the outcome.

The most common fixative is buffered 4% paraformaldehyde used at 4 o C. Paraformaldehyde is prepared fresh from
powder or purchased in vials sealed with inert gas. Commercial formalin contains methanol (6-15%) and other impurities
that may affect fixation.

Tissue needs to be submerged in the fixative (often following perfusion) and gently agitated. Cells and tissue remain
osmotically active after fixation in aldehydes. Check for excessive swelling or shrinkage in the tissue.

Fixation times vary with the type of specimen and the accessibility of the epitope. Cell cultures are generally fixed for a
minimum of 30 minutes. Tissue is fixed for a minimum of one to four hours (or longer). Use the longest time possible
that still results in good antibody labeling. (Para)formaldehyde penetrates rapidly but fixes slowly.

Glutaraldehyde induces autofluorescence in your tissue. Samples fixed with glutaraldehyde must subsequently be
treated with sodium borohydride.

Methanol and acetone are not good fixatives for preserving most structures. They precipitate proteins. Tissue will be
shrunken and distorted.

Immunolabeling

The excitation and emission spectra of the fluorophore(s) used must coincide with the filters on our systems.

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The Keck Center BioRad MRC 600 is equipped with a krypton-argon laser. The major lines of the laser are 488, 568
and 647 nm. - corresponding to fluorescein, rhodamine and Cy5. The system can be used for double labels with 488
and 568 excitation.

The lasers on the Keck Center Leica TCS SP have excitation lines at 457(weak), 488, 568 and 633 nm. Fluorescent
emissions are collected using adjustable slits. Multiple labels can be collected simultaneously or sequentially.

Check the entire spectrum, not just the maxima, for overlap between multiple labels. BioRad has a web page at http://fluor

that allows the selection of up to four fluorophores to view on on one plot. Filters on the system can also be added to the

plot. The Imaging Facility at the University of Arizona:

http://www.mcb.arizona.edu/IPC/spectra_page.htm also has a web page where you can view the entire spectra and the

overlap of the excitations and emissions of specific fluorophores. Molecular Probes makes the spectra for many of their

fluorophores available on their website:

http://www.probes.com

Additional considerations:

1. longer wavelengths penetrate further into a thick sample

2. shorter wavelengths result in better resolution

3. shorter wavelengths result in a thinner optical section

4. autofluorescence is usually worse with 488nm. excitation.

Cells and tissue are usually permeabilized with Triton x or saponin for labeling with antibodies. This step can affect

your results. Membrane extraction can occur with Triton. Saponin is "gentler". As with all steps, time, concentration and

temperature are important. An avidin-biotin protocol is often necessary to boost the fluorescent signal.

Fluorescence from well-fixed tissue can often be detected in samples that are several months old and kept in the dark at 4 o C.

Controls are essential.

Jackson Laboratories has a "Technical Center" about immunolabeling at

http://www.jacksonimmuno.com . Topik
adalah:

Penyebab umum latar belakang dari antibodi sekunder Kemungkinan penyebab dari

sinyal lemah dari antibodi sekunder berlabel Memilih media mounting untuk konjugat Cy

direkomendasikan kondisi penyimpanan yang disarankan pengenceran

DTAF-conjugated Streptavidin

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Sebuah sumber yang baik untuk informasi arsip dari kelompok pengguna confocal
http://listserv.acsu.buffalo.edu/archives/confocal.html . Berikut ini adalah kutipan dari posting ke grup:

Memuat Sel

Persiapan Slide

MOUNTING MEDIA:

The mountant Anda gunakan di bawah coverslip adalah penting karena beberapa alasan:

1. Fluorescein sensitif terhadap pH (penyerapan / emisi maksimal pada pH 8,5).

2. mountant harus memiliki aditif untuk mencegah photobleaching.
3. The refractive index of the mountant should be as close to that of your (fixed) tissue as possible (1.515).
In most cases, do not use an aqueous mountant with fixed tissue

EXAMPLES OF REFRACTIVE INDICES:

Methyl Salicylate 1.53-1.54

Tissue 1.515
Immersion Oil 1.515
Glycerol 1.47
Vectashield 1.45
Gel/Mount (Biomedia) 1.36
Water 1.33
Air 1.0

A mountant of 9 parts glycerol; 1 part PBS and 1-3% n-propyl gallate works well. Dissolve the n-propyl gallate in the
PBS, then add the glycerol.

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WORKING DISTANCE:

The working distance of a lens is the space between the front element of the objective and the top of the coverslip. Any
space between the coverslip and the tissue (*) adds to the working distance since a manufacturers figure for the working
distance of a lens presumes that the tissue is just under the coverslip.

Working distances for some of our Nikon lenses:

Objective Lens Working Distance

10X 2.75 mm
20X . 64 mm
60X 0.17 mm
100X 0.10 mm

Note the relatively short working distances. Pap pens, rubber cement, or any other substance used to create a well for
immunostaining must be completely removed before coverslipping as any remnants left on the slide can elevate the
coverslip. The resulting increase in the distance between the front element of the objective lens and the tissue will
prevent focusing within the sample if this distance exceeds the working distance of the lens. For the same reason, don't
use too much mountant under the coverslip. The wells in depression slides are usually too deep to allow focusing above
10X or 20X.

A coverslip placed directly on cells or tissue will compress the sample. To prevent compression, elevate the coverslip with a
spacer as close as possible to the size of the tissue. Coverslips are too thick to be used as spacers for most samples. The
thickness of a #1 coverslip is approximately 140 micrometers and a # 1 1/2 coverslip is about 160 micrometers. Cells
cultured on coverslips are ideal since they can be flipped over, mounted on the slide and the thickness of the spacer is
immaterial.

Thickness of the coverslip is important for optimal image quality. The coverslip thickness an objective lens is designed
for is engraved on the lens. Most of the objective lenses in the Keck Center are designed to be used with a # 1 1/2
coverslip and will have "0.17" engraved on the

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objective lens (=0.17mm. or 170 micrometers). A variation in the thickness of 0.01 mm. with higher magnification
lenses can make a difference in image quality.

If distance between the tissue and the coverslip is unavoidable, then a #1 coverslip might be necessary in order to focus

a short working distance lens - at the expense of resolution. A slide is usually sealed around the coverslip with nail polish.

This prevents evaporation of the mountant and stabilizes the coverslip for use with immersion lenses. The coverslip

needs to be stabilized for use with immersion objective lenses (60X, 100X on the BioRad and 25X, 40X and 100X on the

Leica).

On the Leica confocal/2P system the coverslip should be at least 4 mm. from the long edges of the slide due to the
slide holder design.

Informasi tambahan tentang persiapan sampel dapat ditemukan di

http://www.itg.uiuc.edu/publications/techreports/99-006

Persiapan buffered paraformaldehyde 4% fiksatif dari bubuk:

Campur 4.0 gram bubuk paraformaldehida dengan 50ml. dari 0,2 M penyangga. Panas pada 60 Hai C sambil diaduk.

Menambahkan 1N NaOH setetes demi setetes, aduk terus menerus, untuk membersihkan (depoliymerize) solusinya. Encerkan untuk 100ml

dengan dH 2 0 untuk membuat konsentrasi akhir paraformaldehyde 4% di 0,1 M penyangga.

catatan:

Konsentrasi fiksatif atau penyangga mungkin berbeda dengan jenis jaringan yang berbeda. Panaskan solusi di

bawah tenda untuk menghindari menghirup asap.

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