Microscopy

Microscopy is the technical field of using microscopes to view objects and areas of objects that
cannot be seen with the naked eye (objects that are not within the resolution range of the normal
eye). There are three well-known branches of microscopy: optical, electron, and scanning probe
microscopy.
Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic
radiation/electron beams interacting with thespecimen, and the collection of the scattered radiation
or another signal in order to create an image. This process may be carried out by wide-field
irradiation of the sample (for example standard light microscopy and transmission electron
microscopy) or by scanning of a fine beam over the sample (for example confocal laser scanning
microscopy and scanning electron microscopy). Scanning probe microscopy involves the interaction
of a scanning probe with the surface of the object of interest. The development of microscopy
revolutionized biology and remains an essential technique in the life and physical sciences.

Optical microscopy
Optical or light microscopy involves passing visible light transmitted through or reflected from the
sample through a single or multiple lenses to allow a magnified view of the sample. [1] The resulting
image can be detected directly by the eye, imaged on a photographic plate or captured digitally.
The single lens with its attachments, or the system of lenses and imaging equipment, along with the
appropriate lighting equipment, sample stage and support, makes up the basic light microscope.
The most recent development is the digital microscope, which uses a CCD camera to focus on the
exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary.

Electron microscopy
Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of
traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an
electron beam with a far smaller wavelength is used in electron microscopes.

Transmission electron microscopy (TEM) is quite similar to the compound light microscope,
by sending an electron beam through a very thin slice of the specimen. The resolution limit
in 2005 was around 0.05 nanometer and has not increased appreciably since that time.

Scanning electron microscopy (SEM) visualizes details on the surfaces of specimens and
gives a very nice 3D view. It gives results much like those of the stereo light microscope.
The best resolution for SEM in 2011 was 0.4 nanometer.

This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force
microscope (AFM), the Scanning tunneling microscope, the photonic force microscope and
the recurrence tracking microscope. All such methods use the physical contact of a solid probe tip
to scan the surface of an object, which is supposed to be almost flat.

Ultraviolet microscopy
Ultraviolet microscopes have two main purposes. The first is to utilize the shorter wavelength of
ultraviolet electromagnetic energy to improve the image resolution beyond that of the diffraction limit
of standard optical microscopes. This technique is used for non-destructive inspection of devices
with very small features such as those found in modern semiconductors. The second application for
UV microscopes is contrast enhancement where the response of individual samples is enhanced,
relative to their surrounding, due to the interaction of light with the molecules within the sample
itself. One example is in the growth of protein crystals. Protein crystals are formed in salt solutions.
As salt and protein crystals are both formed in the growth process, and both are commonly
transparent to the human eye, they cannot be differentiated with a standard optical microscope. As
the tryptophan of protein absorbs light at 280 nm, imaging with a UV microscope with 280 nm
bandpass filters makes it simple to differentiate between the two types of crystals. The protein
crystals appear dark while the salt crystals are transparent.

Infrared microscop
The term infrared microscopy refers to microscopy performed at infrared wavelengths. In the typical
instrument configuration a Fourier Transform Infrared Spectrometer (FTIR) is combined with an
optical microscope and an infrared detector. The infrared detector can be a single point detector, a
linear array or a 2D focal plane array. The FTIR provides the ability to perform chemical analysis
via infrared spectroscopy and the microscope and point or array detector enable this chemical
analysis to be spatially resolved, i.e. performed at different regions of the sample. As such
technique is also called infrared microspectroscopy. Infrared microspectroscopy is frequently used
for infrared chemical imaging, where the image contrast is determined by the response of individual
sample regions to particular IR wavelengths selected by the user, usually specific IR absorption
bands and associated molecular resonances . A key limitation of conventional infrared
microspectroscopy is that the spatial resolution is diffraction-limited. Specifically the spatial
resolution is limited to a figure related to the wavelength of the light. For practical IR microscopes,
the spatial resolution is limited to 1-3X the wavelength, depending on the specific technique and
instrument used. For mid-IR wavelengths, this sets a practical spatial resolution limit of ~3-30 m.
IR versions of sub-diffraction microscopy (see above) also exist. These include IR NSOM,
[14]
photothermal microspectroscopy, and atomic force microscope based infrared spectroscopy
(AFM-IR).

Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative
elemental analysis.

Digital holographic microscopy

Scanning probe microscopy

In digital holographic microscopy (DHM), interfering wave fronts from a coherent (monochromatic)
light-source are recorded on a sensor. The image is digitally reconstructed by a computer from the
recorded hologram. Besides the ordinary bright field image, a phase shift image is created.

DHM can operate both in reflection and transmission mode. In reflection mode, the phase shift
image provides a relative distance measurement and thus represents a topography map of the
reflecting surface. In transmission mode, the phase shift image provides a label-free quantitative
measurement of the optical thickness of the specimen. Phase shift images of biological cells are
very similar to images of stained cells and have successfully been analyzed by high content
analysis software.
A unique feature of DHM is the ability to adjust focus after the image is recorded, since all focus
planes are recorded simultaneously by the hologram. This feature makes it possible to image
moving particles in a volume or to rapidly scan a surface. Another attractive feature is DHMs ability
to use low cost optics by correcting optical aberrations by software.

Digital pathology (virtual microscopy)

Digital pathology is an image-based information environment enabled by computer technology that
allows for the management of information generated from a digital slide. Digital pathology is
enabled in part by virtual microscopy, which is the practice of converting glass slides into digital
slides that can be viewed, managed, and analyzed.

Laser microscopy
Laser microscopy is a rapidly growing field that uses laser illumination sources in various forms of
microscopy. For instance, laser microscopy focused on biological applications usesultrashort
pulse lasers, in a number of techniques labeled as nonlinear microscopy, saturation microscopy,
and multiphoton fluorescence microscopy.[15]

Amateur microscopy
Amateur Microscopy is the investigation and observation of biological and non-biological specimens
for
recreational
purposes.
Collectors
of minerals, insects, seashells,
and plants may
usemicroscopes as tools to uncover features that help them classify their collected items. Other
amateurs may be interested in observing the life found in pond water and of other samples.
Microscopes may also prove useful for the water quality assessment for people that keep a home
aquarium. Photographic documentation and drawing of the microscopic images are additional tasks
that augment the spectrum of tasks of the amateur. There are even competitions
for photomicrograph art. Participants of this pastime may either use commercially prepared
microscopic slides or engage in the task of specimen preparation.
While microscopy is a central tool in the documentation of biological specimens, it is, in general,
insufficient to justify the description of a new species based on microscopic investigations alone.
Often genetic and biochemical tests are necessary to confirm the discovery of a new species.
A laboratory and access to academic literature is a necessity, which is specialized and, in general,
not available to amateurs. There is, however, one huge advantage that amateurs have above
professionals: time to explore their surroundings. Often, advanced amateurs team up with
professionals to validate their findings and (possibly) describe new species.
In the late 1800s, amateur microscopy became a popular hobby in the United States and Europe.
Several 'professional amateurs' were being paid for their sampling trips and microscopic

explorations by philanthropists, to keep them amused on the Sunday afternoon (e.g., the diatom
specialist A. Grunow, being paid by (among others) a Belgian industrialist). Professor John
Phin published "Practical Hints on the Selection and Use of the Microscope (Second Edition,
1878)," and was also the editor of the "American Journal of Microscopy."
In 1995, a loose group of amateur microscopists, drawn from several organizations in the UK and
US, founded a site ([1]) for microscopy based on the knowledge and input of amateur (perhaps
better referred to as 'enthusiast') microscopists. This was the first attempt to establish 'amateur'
microscopy as a serious subject[citation needed] in the then-emerging new media of the Internet. Today, is
an established resource for all ages, to input their findings and share information. It is a non-profit
web presence dedicated to the pursuit of science and understanding of the small-scale world.

The Microscope
Parts and Specifications
Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias
Janssen, around the year 1590. The compound microscope uses lenses and light to enlarge the image
and is also called an optical or light microscope (vs./ an electron microscope). The simplest optical
microscope is the magnifying glass and is good to about ten times (10X) magnification.
The compound microscope has two systems of lenses for greater magnification, 1) the ocular, or
eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object. Before
purchasing or using a microscope, it is important to know the functions of each part.
Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has a mirror,
it is used to reflect light from an external light source up through the bottom of the stage.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your
microscope has a mechanical stage, you will be able to move the slide around by turning two knobs.
One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be
rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always
consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we
get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. To have good resolution at
1000X, you will need a relatively sophisticated microscope with an Abbe condenser. The shortest lens is
the lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built
to DIN standards are interchangeable between microscopes. The high power objective lenses are
retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded)
thereby protecting the lens and the slide. All quality microscopes have achromatic, parcentered, parfocal
lenses.

Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It is
set at the factory and keeps students from cranking the high power objective lens down into the slide and
breaking things. You would only need to adjust this if you were using very thin slides and you weren't
able to focus on the specimen at high power. (Tip: If you are using thin slides and can't focus, rather than
adjust the rack stop, place a clear glass slide under the original slide to raise it a bit higher)
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.
Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in stage
condenser lenses render a sharper image than those with no lens (at 400X). If your microscope has a
maximum power of 400X, you will get the maximum benefit by using a condenser lenses rated at 0.65
NA or greater. 0.65 NA condenser lenses may be mounted in the stage and work quite well. A big
advantage to a stage mounted lens is that there is one less focusing item to deal with. If you go to
1000X then you should have a focusable condenser lens with an N.A. of 1.25 or greater. Most 1000X
microscopes use 1.25 Abbe condenser lens systems. The Abbe condenser lens can be moved up and
down. It is set very close to the slide at 1000X and moved further away at the lower powers.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has
different sized holes and is used to vary the intensity and size of the cone of light that is projected
upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather,
the setting is a function of the transparency of the specimen, the degree of contrast you desire and the
particular objective lens in use.
How to Focus Your Microscope: The proper way to focus a microscope is to start with the lowest
power objective lens first and while looking from the side, crank the lens down as close to the specimen
as possible without touching it. Now, look through the eyepiece lens and focus upward only until the
image is sharp. If you can't get it in focus, repeat the process again. Once the image is sharp with the
low power lens, you should be able to simply click in the next power lens and do minor adjustments with
the focus knob. If your microscope has a fine focus adjustment, turning it a bit should be all that's
necessary. Continue with subsequent objective lenses and fine focus each time.
What to look for when purchasing a microscope.
If you want a real microscope that provides sharp crisp images then stay away from the toy stores and
the plastic instruments that claim to go up to 600X or more. There are many high quality student grade
microscopes on the market today. They have a metal body and all glass lenses. One of the most
important considerations is to purchase your instrument from a reputable source. Although a dealer may
give you a great price, they may not be around next year to help you with a problem. One dealer that
we can highly recommend is Microscope World. They offer a wide variety of instruments at very
competitive prices.