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Compact Knowledge
Light Microscopy in 2012
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More than 160 years after its first industrial introduction by Carl Zeiss, the
light microscope is a key tool in today’s modern laboratory. The light of the
beampath is used to transmit the information from the sample to the user’s
eye. The general formula invented by
Professor Ernst Abbé still remains valid today.
The observer looks at the image of the sample
in a microscope - not on the sample itself.
Therefore it is necessary to treat the information carrier - light - as carefully
as possible. The image of the sample should be as close to reality as
possible.
In addition, so called contrast methods increase visibility of microscopic
structures. This article tries to bring some light to some on-going
enhancements in the analytical standard investigation.
Technical aspects of a microscope
In state of the art microscopes infinity corrected optics are used - allowing
the user to simply insert different optical parts (e.g. filters, mirrors, prisms)
into the beampath.
The choice of the appropriate magnification and contrast method depends
on the particular application according to which part of the sample has to
be investigated. Modern microscopes provide exact interfaces, allowing for
any later retrofittings or upgrades (features / contrasts / cameras). One
could say that the instrument
grows with its needs (and
sometimes pockets) of its
user(s).
It is obvious that such a complex sequence of lenses has to
be kept in a perfect arrangement by the surrounding
mechanism. In addition, the
overall environment has to be
kept in consideration: for instance as well as laboratory air
being known for being strongly
aggressive, hot and humid
areas are also challenging influences (Fungus & Corrosion).
Moreover today laboratory
workers put a stronger emphasis on a well-designed,
aesthetic finish of the instrument. Efficient employees
have become a critical HR
requirement.
A good ergonomic design of
the instrument does have
significant influence on the
well-beings, so the way of sitting and touching the instrument’s elements have found
their way into the microscope
design. Recently the German
TÜV Rheinland introduced
Fig. 1: Cut through a modern objective.
ergonomic certification for
microscopes incorporating
1 Objective Winding
2 Screw
these “Ergo” features.
Beside its optics, a microscope nowadays consists of mechanical and
electrical, very often motorised, parts. A modern lens design is able to
create sample images without any distortion or colour shift. In a modern
system this happens on the edge of the information provided by the transporting light wave. Different lenses (shapes / materials / glues) are grouped
and each one is covered by several optical layers (e.g. reflection suppression, colour correction). The picture observed by the ocular has to be free
of distortions, colour shifts
and a variety of so-called
optical errors.
1
2
It is a fact that a single
wrong lens or layer some3
where in the whole beampath is able to destroy the
highly resolved, colour corrected image obtained from
the objective lens. So a
well-designed microscope
4
has to take all its optical
parts into the design / developing concept. In addition, optical glass has to be
5
free of heavy metals. In the
1950’s, Uranyles were in
common use.
6
Very often, another part in
the beampath is overlooked
7
- the immersion oil. Beside
the high transition grade it
has to have the appropriate
8
viscosity and refractive
index, must not build resins
and show fluorescence.
The majority of high pure
immersion oil comes from a
9
special plant in Oberkochen
10
(Germany). Beside these
specialised oils, nowadays
optics are in use which
allow objective lenses to dip
into a water covered sample.
3
4-7
8
9
10
Spring system for protection
Lens group for image correction
Correction ring for different slide thicknesses (or temperatures)
Front lens system with its carrier
Front lens frame
Microscope types
Camera and microscope
Three standard types are common. The classic upright system (2D image
for the observer), the inverted stand (2D) and the stereomicroscope (real 3D
image for the observer). Based on the sample and the necessary information
needed, the microscope magnification and contrast is tailored accordingly.
Storing an image of the sample is mandatory. A number of systems is
available, the principle is always the same:
The image, created by the microscope is projected onto the chip of the
camera. This is the equivalent to the film / plate in the past. It is important
to correlate the size of the camera’s chip and the used pixel number with
the optical resolution provided by the system (see Lit 1). This is the reason
why high pixel numbers automatically allow conclusions of the capabilities
of a microscope with a camera.
In addition an interface between microscope and camera - the so-called
adapter - has to be used. This optical part is designed to deliver the image
to the camera’s sensor. And again, it is the light which is transported
through this delicate part which is important.
Once the image is captured by the sensor, the data must not undergo any
compression to the computer / beamer / printer as data will be lost. One
single data compression will ruin all attempts to maintain the highly
resolved images.
Upright stands
The classic upright stand uses
transmitted light and is used for
stained and unstained specimens fixed
on a sample carrier, usually a glass
slide with a cover slip.
Fluorescence microscopy with appropriate stains, as well as materials investigations for grains, resins etc. are
also possible with a reflected beampath. Increased space for large samples is also available.
The resolution limit is approximately at
0.3 μm.
Inverted stand
The inverted stand is used to
observe stained or unstained
samples in disposable vessels
such as petri dishes, tissue
culture flasks, multiwell plates and
roller bottles. These samples are
usually living in an artificial environment of tissue culture growth
medium. As work is carried out on
living cells, very often incubation
chambers and cell manipulation
tools are required.
Stereomicroscope
When a 3-dimensional
view on the sample is
required, a stereomicroscope is the right choice.
With this widely used instrument the samples are
observed under an optical
correct angle obtained by
two different beampaths essentially 2 microscopes
in one body. The large
working distance, 3D
image and the broad variety of illuminations are
the main reasons for using
these instruments. Their
resolution limitation is approximately 10 μm.
Finding the right sensor for the application
in question to be solved needs to put a
focus on the camera’s parameters. The
dynamic range, full well capacity, light efficiency, shutter, pixel type (CMOS or CCD)
all need to be considered. The typical ’consumer digital’ cameras are normally not suitable for a microscope. These systems are designed for daily
work in a private environment - leading to nice images. The
demand of a laboratory for sustainable data and precise measurements
cannot be guaranteed in a cost relevant frame.
Microscopy software
Modern software allows to control the camera and can offer a live image
on the computer or beamer at the same time.
Image corrections are necessary for brightness, contrast, saturation /
pixel balance (the gamma value), White balance etc. control and
adjustment must be intuitive. When the image is captured, the resulting
stored image must reflect the existing light situation on the sensor during
the moment the photo is taken. Of course such a photo has to be exportable and storable in each relevant picture format - but continuously
allowing to return to each individual pixel information. Report
editors and broad measuring devices are today’s requests as
well.
Microscopes in dialogue
In case of more than one simultaneous user, a so called co-observation
bridge, multidiscussion or teaching system can be recommended.
Here the microscope image is split into several paths, showing each observer the same image of the sample (which in real microscope design is
not as easy as it appears). Beamers for lecture halls are adjustable to cameras of course.The latest approach is the use of tablet PCs in connection
with an USB camera. The universal nature of these transportable PCs lead
to PC based microscopy image capture in situations where is was not
possible previously.
Nonetheless - the software demands as described above remain valid.
Practical hints
Köhler: For a perfect illumination and the maximum of resolution the
illumination has to be adjusted to the objective in use. This is called
“Köhler Illumination” and described with example pictures in Lit.3.
Cleaning of a microscope: Clean optics are mandatory for successful
imaging and good microscopy work. The choice of the cleaning method
depends on the optical surface and the type of dirt. Which material and
how it is used is described in Lit. 2
LED light: Latest LED based systems are available for microscopes
as well. Long life time (>10.000 hours), constant ‘daylight’ colour
temperature and a highly efficient yield of light are only a few of their
characteristics.
Teaching / classroom: Modern student microscopes are optimised for
a long working life, being robust and also ergonomic. Carrying handles
and make-up resistant optics are additional examples of today’s classroom demands.
Summary: Light microscopes are an analytical key tool. Therefore they
undergo continuous improvements. Changes in the laboratory environment have found their way into the systems.
In addition, developments in high end research are still in progress.
Super-resolution laser scanning microscopes and the latest bridging
between electron and light microscopes - called correlative microscopy
- are among the latest developments in microscopy to keep up with or
even overtake developments in research techniques. Lit. 5
Literature and links
Downloads
Literature
www.omnilab.de under "Wissen kompakt"
1) “Mikroskopie von Anfang an”
2) “Das saubere Mikroskop “
3) “Die Beleuchtung nach Köhler am aufrechten Mikroskop”
7) R. Käthner Praxis der Naturwissenschaften 4 /53 2004 6-13
8) M. Zoelffel, Praxis der Naturwissenschaften 4 /53 2004 20-24
www.zeiss.com/campus
4) "Kamera Assistent"
5) "Cormic"
6) "Objektive - Eine Frage des Anspruches"
Robert-Hooke-Str. 8 · D-28359 Bremen / Germany · Phone +49 (0)421 / 1 75 99-0
www.omnilab.de · [email protected]
Flexible. Reliable. Personal.