Phys335 - Optics

Basics of a Microscope
Microscopes, invented by Galileo
in 1610, are used to magnify an object,
more than a simple magnifier. A very
simple microscope is made up of only two
lenses: an objective and an eyepiece
(Jenkins and White, 200). A typical
microscope (Figure 1) is made up of a
condenser (which focuses a light source
on an object), an XYZ translating stage
(which holds the sample), and the
objective (which magnifies the sample).
The process of building a simple
microscope is detailed here. This
microscope will be made up of two parts:
the condenser and the imaging train (see Figure 1: Example of a compound
microscope (Brown, 297).
sample schematic for a diagram of the
microscope). Building this microscope requires a focus on geometrical optics.
Condenser
The condenser is the first component of the microscope. The purpose of the
condenser is to focus the light onto a small area on a sample. Here an LED (with
variable intensity) is used as the light source for the microscope, and the beam is
then focused through the condenser. Focusing the beam of light is achieved by the
addition of three lenses: one collecting lens and two collimating lenses. Manipulating
this beam is achieved by the addition of two iris diaphragms: a field stop and an
aperture. While the diaphragms used are identical, their placement is what
determines whether the diaphragm is a field-stop or an aperture-stop (Figure 2). For
the condenser to work properly, it is necessary for all lenses and iris diaphragms to
be as accurately lined up as possible.
The lenses in this system are used to focus the beam of light from the LED. All
the lenses used are double-convex (converging) lenses. The LED beam is not focused
when it is created; it is just a simple light source. The lenses are what will bring the
beam of light into focus as it hits the XYZ translating stage. The collecting lens is
 used to initially
collect light from
the LED. The
collecting lens is
placed very close to
the LED to capture
as much of the light
as possible, and
focus it into a beam.
The excess light that
is not captured by
the collecting lens
dissipates around the LED. The second set of lenses, the collimating lenses, are used
to focus the beam from the collecting lens into a smaller beam, and focus it onto an
XYZ translating stage (which will contain the sample).
Stops and diaphragms are always present in a pathway of lenses, whether it is
the iris diaphragms used for this microscope, or simply the edges of the lenses used
in the pathway. These stops limit the edge of the beam, effectively limiting how much
of the object is seen through the pathway in the image, and the intensity of light that
is visualized (Brown, 181).
The field stop (commonly called the f-stop) is one of the two iris diaphragms
used in the condenser. The F-stop is often closer to the light source (for this
microscope an LED), between the collecting lens and the first of the two collimating
lenses. Changing the radius of the F-stop will adjust the radius (the edge of your
viewing field) of the beam of light as it hits the XYZ translating stage (Figure 3A).
Field stops are placed at one of the point the object images in the pathway (Jenkins
and White, 115-6). This placement allows the f-stop to control the height of the
image.
The aperture stop (commonly called the aperture), the second iris diaphragm
used, controls the intensity of the light that will hit the XYZ translating stage. The
aperture is farther away from the LED, between the first and second collimating
lenses (Jenkins and White, 115-6). Changing the radius of the aperture will adjust
the intensity of the beam of light as it hits the XYZ translating stage (Figure 3B).
Figure 3A: Condenser with varied F-stop focused onto a grid.

Figure 3B: Condenser with varied aperture focused Figure 3C: Sample object to be
onto a grid. imaged
(Bar = 5 mm)

Imaging Pathway
The imaging pathway is the name given to the series of lenses that magnify
and focus the image created from the sample. The sample (held on the XYZ
translating stage) will create an image when illuminated with the light from the
beam (focused on the sample by the condenser). The images produced will be
captured using the CMOS camera.
The first imaging pathway used consists of a single lens used to create an
object at infinity. The next pathway consists an infinity corrected objective (created
from the lens which imaged the object at infinity as well as an additional lens) and a
150mm imaging lens. The differences in these pathways will change where the object
will eventually image (where the CMOS camera will need to be placed for the image
to be in focus).
Magnification, another idea taken directly from geometrical optics, plays an
important role in the function of a microscope. The function of any traditional
microscope, back to the simple magnifier used by Anton van Leeuwenhoek in the 17 th
century, is to magnify an object to see it more clearly. Without this magnification of
the object, the microscope is virtually useless. Here we will use linear magnification
(the ratio of the size of an object to the image) as opposed to angular magnification
(which uses the apparent angular size of the object to the image). The linear
magnification is dependent on the focal length of the lenses used, the distance
between the lenses, and the distance of the object from the lens system (Brown,
186). By simply measuring the height of both the image and the height of the object,
and dividing the height of the image by the height of the object, you can find the
linear magnification. It can also be calculated by using these equations:
1 1 1 −s '
= + ,∧m=
f s s' s
The first equation will also allow you to find the distance the image will appear (in
focus) from the lens.
Focus plays a very important part of any microscope, as anyone would
imagine. Magnification without focus leaves you with a large, blurry blob. The key to
getting the image in focus is the placement of the lenses. In a system of two or more
lenses, you must run through the equations above several times before you can know
where the image will form in focus. Most traditional microscopes will use the human
eye as the target for the image. While the human eye has the capacity to focus images
from infinity to a minimum distance, it is often best to have the microscope focus the
object at infinity (Brown, 354-5). The
human eye, however, will not always be
the medium the image will be projected
on. The image may be projected onto a
rigid object, such as a piece of paper or
a wall, to achieve the visualization of an
image. The image in figure 5 is
projected directly onto a wall, 4 meters
from the original light source and
nearly 3.5 meters from the last (150
mm) lens.
Figure 4: Biological Sample with
Infinity space is the term given filtered light source.
to the space inside of an infinity
corrected objective. In this space, the rays that make up the beam are traveling
parallel to each other (much like the rays after leaving the first imaging path); when
manipulated in this way, these rays could feasibly travel on for a distance of infinity
and not converge (Rottenfusser, Education in Microscopy and Digital Imaging). This
space is useful for adding objects such as filters or di-chromic mirrors. Within this
space, a number of mirrors and filters may be used to create a microscope that
allows someone to observe the sample through both an eyepiece and a camera,
which allows for the capture of the image produced.
Filters, another useful tool, will often be used to produce a beam of a
particular wave of light from white light. Our LED (which emits white light) is a
prime example. Any number of filters can be used for any number of reasons. The
addition of a filter in the visible spectrum will give the image a different hue; the
blue color in Figure 4 is an example of the effects of the filter used (Brown, 89-90).
The purpose the filter is being used for will dictate the position of the filter in the
microscope. If you would like a specific wavelength of light to hit the object (to
excite a protein in the sample for example), the filter must be placed in the
condenser. The placement of the filter within the condenser does not particularly
matter. If the filter is to single out light in the resulting image (such as the particular
wavelength of light an
excited protein emits), it
must be placed in the
imaging pathway. While the
placement of the filter in
the imaging pathway also
doesn’t matter too much, it
is easiest to put it in the
infinity space, as it will not
interfere with the focus of
the image.

Figure 5: Image at about 4m

White LED – positioned at 0mm

Collimating Lens 1 (40mm) – 78mm

Aperture diaphragm – 164mm

Collimating Lens 2 – 192mm

Object
XYZ translating Stage – 227mm

Imaging Path
Lens 1 (50mm) – 305mm
Creates object at infinity

Lens 2 (150mm) – 536mm