imaging resource guide imaging lenses filters cameras illumination targets
Section 10.2: Fluorescence Filters for Microscopy
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Figure 2 illustrates the most basic in nity corrected objective con-
guration. The objective collects light, which is then focused by the
secondary tube lens to the eyepiece or sensor. The image shows a real
life microscope system composed of only four components: an in nity
corrected objective, a secondary tube lens, an extension tube for stray
light control, and a USB camera. Although simple, this system o ers
little adaptability. As seen in Figure 3, a seven-component system can
be implemented to create a uorescent system or to maximize contrast
and resolution; in-line illumination ensures there will be less noise in the
system and minimizes glaring as well as ghosting in the system.
The seven-component setup can be complicated, but the following explanation
aims to make the process as simple as possible. To start, #58-329
MT-1/ MT-2 C-mount Adapter can be opened up and the MT-1 or MT-2
Tube Lenses t inside. Since the tube lenses themselves have no threads,
this adapter provides the necessary C-threads on both sides. Between this
adapter and the C-mount camera, an additional 190 mm of extension tubes
is required. Since the tube lens does not attach directly to the objective, use
#55-743 Mitutoyo to C-Mount 10mm Adapter to attach the objective (this
adapter adds 10 mm of length and adapts the M26 thread to a C-thread).
An additional 76,5 mm of space between the tube lens and objective is
optimal, but it is common to only use about 56,5 mm of space between
#55-743 and #58-329 since each adapter adds about 10 mm of space.
With this 56,5 mm of space, use extension tubes or add other optical
components such as beamsplitters, optical lters, or polarizers as needed.
It is important to note that 76,5 mm is the recommended distance
since these objectives are in nity corrected. However, if the distance
is too short, the system may experience vignetting; if the distance is
too long, the resultant image will be dim because of insu cient light.
To summarize, refer to Figure 3 for an illustration of the seven-component
digital video microscope system.
MT-1/MT-2 Tube Lens Objective
on the application
100mm C-Mount Extension Tube #54-633
50mm C-Mount Extension Tube #54-632
30-50mm, C-Mount Fine Focus Tube #03-625
into two pieces
Figure 3: Seven-Component In nity Corrected Digital Video Microscope System.
Figure 4: Basic Fluorescence Microscope Setup.
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Fluorophore Absorption and Emission Profiles
Absorption Spectral Profile
Emission Spectral Profile
Figure 5: Typical excitation and emission pro le.
Fluorescence microscopy is an optical microscopy technique that utilizes
uorescence, which is induced using uorophores, as opposed to
absorption, scatter, or re ection. A uorophore is a type of uorescent
dye used to mark proteins, tissues, and cells with a uorescent label for
examination by uorescence microscopy. A uorophore works by absorbing
energy of a speci c wavelength region, commonly referred to
as the excitation range, and re-emitting that energy in another speci c
wavelength region, commonly referred to as the emission range.
Fluorescence microscope systems can range from very simple, such
as an epi uorescent microscope, to extremely complex, such as confocal
or multiphoton systems. Whether simple or complex, uorescence
microscopes share the same basic concept: excitation energy
is used to illuminate a sample which then produces emission energy,
albeit weak, that is quanti able. The excitation and emission wavelengths
do not share the same center wavelength, and this allows specialized
optical lters to increase overall contrast and signal.
The most basic concept and schematic can be seen in the Figure 4. A
lter arrangement is constructed out of three very speci c lters: an excitation
lter, a dichroic lter, and an emission lter. The excitation lter is
placed within the illumination path of a uorescence microscope, and lters
out all wavelengths of the light source except for the excitation range
of the uorophore or specimen under inspection. The dichroic lter is
placed between the excitation lter and emission lter at a 45° angle,
and re ects the excitation signal towards the uorophore under inspection
while transmitting the emission signal towards the detector. The
emission lter is placed within the imaging path of a uorescence microscope,
and lters out the entire excitation range of the uorophore under
inspection while transmitting the emission range of the uorophore.
A typical excitation and emission pro le is shown in Figure 5. It is
clearly visible that the absorption and emission pro les share common
wavelengths, which is one of the main reasons why high quality lters are
required with high transmission, narrow bandwidths, high optical densities,
and extremely sharp cut-on and cut-o bands. Using low quality lters
can ultimately damage the sample, specimen, or expensive sensors.