1. 2014
Revolutionizing Multifocal
Confocal Microscopy
James Constantin | A0128548W
Lin Mu | A0092172J
Priyadarshini Majumdar | A0092068B
RaveenBaloo | A0087590
Mohd Noor Hafizuddin B Haslir |
A0081177A
Submitted on – 5/11/2014
A BN4406 Project Proposal
2. 1 | P a g e
1Introduction
Table of Contents
1. Introduction ........................................................................................................................... 3
2. Confocal Microscopy: Current technologies................................................................................ 3
3. Proposed design ..................................................................................................................... 6
3.1 Line production stage ............................................................................................................ 6
3.1.1 Laser Source................................................................................................................... 6
3.1.2 Achromatic Cylinder Lens................................................................................................. 7
3.2 Line splitting stage................................................................................................................. 8
3.2.1 Lateral Displacement Beam Splitter................................................................................... 8
3.3 Line detection stage .............................................................................................................. 9
3.3.1 Dichroic mirrors.............................................................................................................. 9
3.3.3 Microscope .................................................................................................................. 11
3.3.4 Line detectors............................................................................................................... 13
4. Conclusion and Analysis of design ........................................................................................... 14
5. References........................................................................................................................... 15
6. Appendix ............................................................................................................................. 18
Individual Contirbution - Design
Name/Task
Line
Production
stage
Line
Division
Stage
Line
detection
Stage
Simulation
Software
Final
Proposal
Report
editing
James
Constantin 20 20 0 0 60 100
Lin Mu 80 0 0 20 0 100
Mohd Noor
Hafizuddin 0 80 0 20 0 100
Priyadarshini
Majumdar 0 0 40 60 0 100
Raveen Baloo 0 0 60 0 40 100
Overall 100 100 100 100 100
Individual Contributions - deliverables
3. 2 | P a g e
2Introduction
Name/Task
DesignIdeationand
Research
Pre-proposal
Report
Pre-Proposal
Presentation
Final Proposal
Presentation
JamesConstantin 20 20 20 20
Lin Mu 20 20 20 20
Mohd NoorHafizuddin 20 20 20 20
Priyadarshini
Majumdar 20 20 20 20
RaveenBaloo 20 20 20 20
Overall 100 100 100 100
4. 3 | P a g e
3Introduction
1. Introduction
The design team was able to develop and hypothesize a novel model for use in optical analyses,
particularly under the sub heading of multifocal confocal microscopy. In fashioning a novel imaging
technique, the team learned many aspects and principles of optical imaging as well as the process of
designing itself. The team’s choice of multifocal confocal microscopy stems from an interest to exploit
existing microscopy theories and technologies in a new and revolutionary way.
Our finalized design makes use of the many aspects of modern optics and imaging. The design
concept utilizes the principles of fluorescent microscopy, confocal microscopy, and multifocal
microscopy. The design’s purpose was to significantly increase the rate at which images are acquired
while maintaining a high degree of resolution under the application of fluorescent microscopy. Our
hypothesis was as follows: With the appropriate acquisition of research regarding various imaging
methods and opticaltheories, the team would formulate a microscopy concept,novel in both design and
construction that would significantly reduce the amount of time taken to acquire an image while
maintaining a high degree of spatial resolution. This would be done through the implementation of
fluorescence and confocal microscopy. A listing of design parameters and specifications would be
rendered so that various modeling and real world comparisons could be conducted to test its validity.
Before passing through a collimated beam splitter,an achromatic cylindrical lens would transform
the collimated light beam into a line source, dramatically increasing the surface area that our design
could image at one time. The beam splitter would then split the single uniform laser source into two
distinct beams, with all properties remaining equal other than their respective intensities and
polarizations. This source beam would then be passed through a set of twin galvanometric mirrors so
that they could scan, lengthwise along the sample. This “multifocal” concept would effectively reduce
the time it takes to image a sample in the x and y direction by well over half. By including a sample
holder that will translate the sample in the z-direction, our multi-focal design can hence image the
sample in all three directions.
Our design would also apply the principle of confocal microscopy to obtain images of high
resolution. Confocality would be obtained by setting a variable aperture before two twin detectors, one
for each emission line. The aperture will be independently designed by the team so that it can be made
applicable for the use in line source detection versus the traditional point source.
The team analyzed our design by breaking down the individual components and applying
principles based in optics. The final design concept was modeled and tested, using software unique to
optical analyses. The following is a detailed report of our analysis and highlights key features of the
design. Most important are the theoretical and physical validations of our final design and hypothesis,
as well as the specific product layout of each of the various components.
2. Confocal Microscopy: Current technologies
There are five common types of confocal microscopy that are commercially available i.e. laser
scanning,spinning-disk (Nipkow disk), micro lens enhanced or dualspinning disk confocal microscopy,
programmable array microscopes (PAM), and line scanning microscopy. In this section, working
principles as well as pros and cons of each type will be discussed.
Confocal laser scanning microscope technology entails shining a laser light through fixed pinholes
5. 4 | P a g e
4Confocal Microscopy: Current technologies
and using multiple mirrors to achieve the scanning through different directions1
.Since the scanning is
done point by point on each focal plane, the scanning speed is slow and is not applicable for in-vivo
scanning, which requires high temporal resolution. The advantage is that it effectively eliminates
out-of-focus light so spatial resolution is high. In addition, imaging density is adjustable by controlling
the angle step of the scanning mirrors. Our design was to build upon the advantages of this technology,
as well as fix the drawbacks,making the process much faster.
The Spinning Nipkow disk as shown in figure 1, replaces the single fixed pinhole in laser scanning
microscopes with a rotating plate that contains multiple holes2
. The idea is to scan multiple holes at the
same time. While the plate rotates during scanning, the whole sample will be covered after one round.
In comparison with the confocal laser scanning microscopes, the spinning-disk technology reduces the
time needed for scanning and thus decreases the risk of photo-toxicity and photo-bleaching caused by
high excitation energy from the source2
. In terms of disadvantages,the distance between pinholes is one
concern because out-of-focus light might still pass through other pinholes if pinholes are too close to
each other. Another drawback of this technology is that the pinholes are fully defined by the disk so
imaging density is not adjustable.
As shown in figure 2, Microlens enhanced
spinning disk microscopy adds an additional
disk, which contains multiple micro-lenses, to
the previously described spinning disk
microscope3
. By doing so, incident laser light
gets focused before it passes through the Nipkow
disk. This lets a much larger amount of light to
be utilized by detection equipment instead of
being blocked out.
Figure 1: Nipkow disk2
Figure 1 Spatial light modulator5 Figure 2 Schematic of microlens enhanced
spinning disk microscope3
6. 5 | P a g e
5Confocal Microscopy: Current technologies
PAM (Programmable Array Microscope) uses the similar idea as Nipkow disk to scan multiple points
on the focal plane simultaneously to increase scanning speed. Instead of rotating a plate with
pre-manufactured pinholes, PAM uses spatial light modulator as shown in figure 3 to electronically
control or adjust the opening and closing of pinholes4
.
Line scanning confocalmicroscopes, on the other hand replace pinholes in confocalmicroscopes with a
single slit so that a line of laser light is allowed to pass through and scan the sample6
. Therefore,on each
focal plane, the scanning mirrors only need to rotate along one direction and the scanning will be done
line by line. This increases the scanning speed but sacrifices the confocality along the line because
out-of-focus light is poorly eliminated along the line. Table 1 critically analyses the advantages and
disadvantages of the current technologies.
Table 1: Current technologies
Confocality Imaging
density
Spatial
resolution
Imaging
frame speed
Type of Detector
Laser
scanning
Highest Adjustable High Very slow Photo multiplier tube
(PMT)
Nipkow
disk
High Fixed Relatively
low
Fast,
desirable
for invivo
scanning
CCD detector
Microlens
enhanced
High Fixed Relatively
low
Fast CCD detector
PAM High Adjustable Relatively
low
Fast CCD detector
Line
scanning
Lose the confocality
along the line
Adjustable Low Fastest Linear CCD detector
The principle of confocal microscopy was key in our construction of a novel design. After
researching the modern technologies available, the team set out to incorporate the various confocal
theories that would apply. Our concepts provided the team with new and challenging questions. To
overcome these challenges many of the theories involving different microscope components were
vigorously researched. Our findings allowed us to build design parameters and ultimately formulate a
final model.
7. 6 | P a g e
6Proposed design
3. Proposed design
Figure 4: Shown is a detailed diagram of the team’s final proposal. The source beam can be traced
going through the various stages and components of the design. Starting at the line production stage,
one can trace the optical path to include the beam splitter, dichroic mirrors, galvanometric mirrors,
spherical and objective lenses, variable aperture,and the detectors.
3.1 Line production stage
The line production stage is the first, and one of the most primary components of our design. This
section, as shown in figure 4, is responsible for supplying the various parts and sample field with a
proper excitation light source. It is in this stage that the laser source is converted into a line using the
achromatic cylindrical lens.
3.1.1 Laser Source
A laser is a device that emits light through the process of optical amplification based on the
stimulated emission of electromagnetic radiation. Lasersemit light in a coherent fashion, allowing light
to be highly focused, maintaining intensity and power. Laser's spatial coherence allows a source beam
8. 7 | P a g e
7Proposed design
to be collimated over long distances7
.
Our choice of a laser light source was dependent upon the various goals of our concept. Most
important of these design criteria were that we would need to split a single collimated beam into two
beams of equal intensity. An additional parameter was that we were going be utilizing fluorescent
microscopy. Splitting a single beam into two equally intense beams meant that we would be cutting the
intensity capability of our design by at least half, given there were no other losses. Because of this, we
wanted a laser that would boast a high intensity and have a low degree of scattering. The use of a
collimated laser in this scenario made the most sense, and using one with a high range of power meant
that there would be a low risk of additional energy loss. Our selection of wavelength output was also
very important to our choice of a laser source. For our purposes of using the laser as anexcitation source
in fluorescent microscopy, we wanted to pick a product that had a high energy, small wavelength light
output. Blue light was the wavelength of choice, approximately 430-460nm. Fluorescent microscopy
makes use of a high-energy input source to excite fluorophores in tissues and give off an emission
wavelength that is larger and less energetic. Using blue light as a source meant that the emission
wavelength would still be sufficient in energy and that we could utilize a wide range of fluorescence.
Photo bleaching was also a concern because of our use of a high intensity light source. Photo
bleaching is the photochemical destruction of a dye or fluorophore due to over exposure to light8
. This
phenomenon is especially problematic in fluorescent microscopy where the excitation wavelength
needs to be highly energetic and may eventually destroy a sensitive fluorescent molecule. Reducing the
time of light exposure and intensity, increasing the concentration of fluorophores, or varying the
frequency of the source, can all control photo bleaching in modern microscopy. Our design already
employs a large reduction in the time of light exposure by using the line scanning technique.
Additionally, our source is split, reducing the given intensity by approximately half at each focal point.
The source we chose also has a widely varied modulation frequency, which provides the design with
another safeguard against photo bleaching.
The design will make use of a coherent high performance OBIS laser system. The system is
easy to use and can provide the user with an alternative spectra of wavelengths as needed. The specific
laser source we will be using is the OBIS, 445nm- 45mW fiber-coupled laser from Edmund Optics9
.
The output source is ideal for our needs in fluorescent microscopy, since it meets excitation wavelength
criteria, and boasts a high intensity signal with a degree of power of 45mW. The device also permits for
a wide variation in modulation frequency, approximately 150MHz (digital) to 500kHz (analog). Please
refer to the appendix for more detailed specifications.
3.1.2 Achromatic Cylinder Lens
The key component that transforms the
incoming laser beam to a line is the cylindrical
lens. The lens has a single cylindrical surface
that causes the incoming light to converge or
expand in a single dimension when incident
onto either a convex or concave side
respectively. Hence, our initial design
included the use of a plano-convex (PCX)
cylinder lens to generate the focused line image
for our line-scanning application10
.
Figure 5: Comparison of Achromatic Cylinder and PCX cylinder lens
9. 8 | P a g e
8Proposed design
However, the team required that the line image be minimized to chromatic and spherical aberrations of
a higher degree. This was necessary because our design’s line
scanning application requires two line beams to run parallel to
each other with no occasions of overlapping. With further
research, we replaced the PCX cylinder lens with an
achromatic cylinder lens that offers superior reduction of
spherical and chromatic aberration at the image plane. As
shown in figure 5 , the greater reduction in spherical
aberration results in a production of a much thinner line. The
decreased line width would also yield higher resolution, an
improved signal-to-noise ratio (SNR) and superior throughput for our LED-based scanning device11
.
In our multi-focal design, we have decided to use 12.5mm diameter achromatic cylinder lens
with an effective focal length of 25mm from Edmund Optics as shown in figure 6. Specifications of
the product13
and the simulations performed on TracePro with dimensions provided are shown in the
appendix.
3.2 Line splitting stage
After the collimated source beam is changed into a
line, it will travel down the optical pathway into the subsequent line splitting stage. There, it will
encounter a beam divider that splits the beam into two equal intensity line beams that run parallelto one
another. In this stage, we control the polarization state and separation of the parallel beams generated.
3.2.1 LateralDisplacement Beam Splitter
The effect of a beam splitter is affected by the choice of optically
active material or interference coatings. The different kinds of
dividers can be classified depending on their function or their shape.
In particular to our design, we are using a special type of cube beam
splitter that is made up of two reflecting surfaces within a prism as
shown in figure 7. This special cube beam splitter is known as a
lateral displacement beam splitter since it enables the splitting of
light into two beams parallel to each other, and parallel to input beam
with a dividing ratio tolerance of 5% for 50:50% split13
. The first
surface receiving the incident non-polarized beam is coated with a
partially reflective dielectric coating that strongly influence the
polarization state of outgoing beams i.e. transmitting 50% of
P-polarized light while reflecting 50% of S-polarized light. The
second surface is coated with a fully-reflective coating to completely
reflect the S-polarized light as shown in the figure. An anti-reflective
coating is applied on the rear of both surfaces to reduce ghosting and
maximise throughput of outgoing beams14
. The separation of the
Figure 7: Polarizing Lateral
Displacement Beamsplitter
Figure 6: Achromatic Cylinder Lens
10. 9 | P a g e
9Proposed design
beams is determined by the distance between first and second surface of the polarizing beam splitter.
We have decided to fix this variable to 10mm.
Edmund Optics offers a lateral displacement polarizing beam splitter that can separate a beam by
10mm with more than 95% chance of P-polarization transmission and almost perfect S-polarization
reflection. However,the product has design wavelength of operating at 632.8nm with applied coating15
.
Hence, it is necessary for us to customize our beam splitter to receive our laser beam at 445nm while
producing similar or better specifications than the product as shown in appendix15
.
3.3 Line detectionstage
The line detection stage, needed to be designed to adapt to the transmission of incoming line
sources versus the traditional point source. Fluorescent emission and high-energy reflection would
also need to be taken into count in the construction of our dichroic mirrors. The line detection stage
would ultimately be the determining factor of many of the key parameters, including the selection of
our source wavelength, mirror construction, placement of various components, and ultimate product
selection.
3.3.1 Dichroic mirrors
A dichroic mirror, also known as a dichroic filter or interference filter, is a highly accurate
optical device that is used to selectively pass light of a specific range of wavelength while reflecting
others. Dichroic mirrors are traditionally classified by the colors of light they reflect rather than the
colors they pass16
.
Manufacturers of dichroic mirrors are able to adjust the frequency or passband of the filter by
modifying the thickness, material type, and number of layers of coatings. Passband is known as the
frequency or wavelength that is able to pass through a filter without being attenuated.
Figure 8: Shown in the diagram is an example illustrating the use of a dichroic mirror in fluorescence
microscopy. From a conceptual standpoint, it can be seen that light from a source passes through a
primary filter yielding only one or several wavelengths of light- known as the excitatory light. This
source-wavelength is reflected off of the dichroic mirror surface and onto the sample where it is
reflected back at a different wavelength. The new wavelength is allowed to pass through the dichroic
mirror due to properties of its thin film coating. A dichroic mirror is traditionally used to separate the
excitation and emission light paths17
.
In our design, a dichroic mirror is used in a very similar manner as depicted in Figure 8. After
our beam source is separated into two distinct line sources, we wish to reflect ~100% of the incoming
11. 10 | P a g e
10Proposed design
light source to the object and pass ~100% of the reflected emission light to the detectors.
Several types of dichroic filters exist for various uses. Long pass filters transmit wavelengths
above a given wavelength, whereas short pass filters transmit wavelengths below a cut-off wavelength.
Utilizing a long pass filter is compatible with the use of fluorescence18
. In fluorescent
microscopy, a shorter wavelength of higher energy hits an object to excite fluorescing antibodies. Light
waves of larger wavelength are produced and emitted from the sample. This is what makes emission
light waves separable from the excitation waves. The need to separate these different wavelengths is
what allows us to exploit the use of a dichroic mirror. Using the properties of the mirrors, we want to
reflect light from the source and transmit the light that is being emitted off of the object.
Figure 9: Shown is a diagram of specular reflection. The law of reflection states that θi = θr,
where the angle of incidence equals the angle of refraction19
.
The law of reflection states that when a light source approaches a reflective surface at a given
angle,called the angle of incidence,the reflected light is redirected at that same angle with respect to the
normal vector, called the angle of reflection. Placing the mirror at a 450
will redirect the light at a 900
angle with respect to the original source beam. For this model we are assuming the dichroic mirror
possesses a refractive coating, which yields total internal reflection20
.
Dichroic mirrors make use of an optical coating that layers materials of differing refractive
indexes. To achieve varying levels of reflectivity and transmittance, composition, thickness, and a
variance in the number of layers of substrates are used. These factors rely on principles based in
chemistry and physics to describe the interaction. Dichroic mirrors are developed for a wide variety of
cases. In fluorescence microscopy, the material properties of a dichroic mirror and filter may need to
change based off of what type of material you are staining for.
For our model, the design will be using a long pass edge polarization insensitive filter, 466nm
dichroic mirror, provided by Edmund Optics. As inducted in the appendix, reflection wavelengths of
the mirror are 439-457.9nm with transmission wavelengths being 473-647.1nm. The mirror diameter is
12.5mm, with a mount thickness of 3.5mm. The percentages of reflection and
transmittance are greater than 98% and 95% respectively. The coating surface is “hard
dielectric sputtered.” Because we are making use of a polarized light source this
particular dichroic mirror was ideal since it does not disrupt any polarizing effects on
the beam21
.
3.3.2 1-dimensional galvanometer mirror
After passing through the dichroic mirror, the laser line beam should be directed
to the sample. On the sample, it should be able to move and scan through
the whole sample. In order to achieve this, two-galvanometer mirror also
known as galvo mirror should be placed on the path of the laser22
.
Figure 10: 1-D Galvanometer
scanning mirror
12. 11 | P a g e
11Proposed design
A galvo mirror as shown in figure 10 is basically an electromechanical instrument that deflects
light beam with a mirror. The two major factors that need to be considered are speed and stability. The
galvo meter should be both stable with high speed. Other than speed and stability, the galvo mirror
should also have accurate positioning system. Current galvo motors are limited rotation DC motors
with controlled motion, which is done by internal position detector. The speed of the galvo mirror
comes from the design of the resonant frequency and RMS power system capability. It comes with
extremely high torque with little electrical inductance, which gives the galvo mirror high speed
scanning with reduced power consumption. To reduce the wobble and jitter effect, the galvo mirror
should have a faster step response with excellent system bandwidths. With resonant scanners, the
galvo mirror should be able to achieve fast scanning speed. Using torsion bar, the motor is tuned to
resonate at a specific frequency, which in turn consumes little energy.
For confocal microscopy, the galvo mirror comes with two mirrors to scan both axes. But since
our system scans in a line, a single axis galvo mirror suffices. Two galvo mirrors will be placed to
reflect dual incoming laser line sources towards the sample. The first galvo mirror will scan the first
half of the sample, and the second galvo mirror will start scanning from the center and move towards
the end of the sample. In order to make it adjustable, the first galvo mirror’s position will be variable.
This means, the user will be able to set the position of the reflected laser’s starting position. The
second galvo mirror’s position will be set as the center of the sample. The first galvo mirror will scan
the first half of the sample, and according to the distance the first galvo mirror scanned, the second
galvo mirror will scan the same distance. With this feature, smaller samples can be scanned. After
much consideration, QS12 from Nutfield Technology is chosen. This galvo mirror provides
exceptional reliability with high scanning speed. The mirror supports laser beam up to 12mm, which
is bigger than the laser line produced by our system23
. Specifications of the mirror can be found in the
appendix.
3.3.3 Microscope
3.3.3.1 Spherical Lens
Commonly used in current confocal microscopes, spherical lenses as in figure 11 are used in
several parts of the design to focus and intensify the emission light. Since the laser light converges in
only one direction after passing through the achromatic cylindrical lens, it is important to make sure its
line property won’t change when passing through the spherical lens. Each of the spherical lenses used in
this design must be placed in a proper position so that it converges light in one direction and collimates
light in the other direction. The location can be defined using the focal length formula24
:
1
𝑓
= ( 𝑛 − 1)[
1
𝑅1
−
1
𝑅2
+
(𝑛 − 1)𝑑
𝑛𝑅1 𝑅2
]
Where f is the focal length of the spherical lens; n is the refractive index of the lens medium; d is the
lens thickness; R1 and R2 are radii of curvature of the two surfaces closer to light and imaging plane
respectively.
Figure 11: Spherical lens is
13. 12 | P a g e
12Proposed design
able to focus collimated incident light from point light source.
3.3.3.2 Variable Aperture
Perhaps the most important part of the design's microscope component, a variable aperture is
what classifies our concept as confocal. As mentioned earlier Confocal microscopes make use of a
spatial pin hole, added at a point before the detector to eliminate defocused fluorescence light from
different optical layers. This is done to get a sharp contrast image. In our design, a custom aperture
was proposed. Our novel concept would use slits instead of a pin-hole to image the lines. The
drawback however is that it may not be able to eliminate all the defocussed light and hence
compromise the sharpness of the image. In order to accommodate both line imaging and high contrast
we propose to incorporate the concept
of a variable aperture.
Figure 12 is an ideation of the variable
aperture that would be used. This design
has been inspired by Olympus’ variable
aperture from their swept – field
microscope. The slits and circles give
the user freedom to choose the kind of
image he wishes. Both the excitation
light and fluorescence light are made to
pass through a selected aperture (slit or
pin-hole). The slit apertures allow
imaging of every point on the surface of
the sample at the cost of optimum
contrast. The pinholes improve
contrast but are not capable of imaging
many points on the surface. In the both the above scenarios the temporal resolution continues to be the
same. Thus if the user is not satisfied with the quality of the image, he can either opt for a thinner slit
or for pin-holes. He can also capture two images of the sample one using the slit followed by the
pin-holes thus improving the resolution of the image without losing out on points imaged.
After eliminating the extraneous emission light, the now "confocalized" light is passed to the
objective lenses where it is focused for the detectors themselves.
3.3.3.3 Objective lens : F-theta lens
F-theta lenses have been designed to provide better performance than spherical lens or flat-field lens.
As shown in figure 13, spherical lenses focus incident light on a curved plane with radius equal to its
focal length, which is not desired in most laser scanning or engraving systems where samples are
always planar25
. To solve this problem, flat-field scanning lenses could be used to provide a planar
imaging plane but the vertical displacement is not linearly proportional to the incident angle, requiring
proper software algorithm to define the Galvo mirrors’ rotation. Therefore; an ideal solution to this is
to have a planar imaging plane as well as displacement proportional directly to the incident angle.
F-theta lenses are designed for this purpose. In this project, F-theta lens is chosen as objective to
optimize the image and simplify the calculation.
Figure 12: Modelled Variable Aperture
14. 13 | P a g e
13Proposed design
Figure 13:Imaging plane of singlet (Left), flat-field (Middle), and F-theta (Right) scanning lens
3.3.4 Line detectors
Microscopes from an early time used various methods for detecting light. Photomultiplier tubes,
Photodetectors, Charge Coupled devices and CMOS sensors were all used to detect light of varying
wavelengths and intensities emitting from a subject. Today, the choice of a detector is usually made
between CMOS Sensors and CCDs. CMOS and CCDs differ mainly in their structure. CCD detectors
couple their charges collected in a register which is then read out. On the other hand each CMOS
pixel has its own charge to voltage conversion electronics causing them to have small pixel sizes26
.
Conventional CCDs due to their working principle also suffer from low Signal to noise ratio. To
combat this the electron Multiplier CCD (EMCCD) was introduced. This is a highly sensitive
detection system as a consequence of the electron multiplication register where a steep potential
difference causes the electron to gain energy and ultimately excite more electrons through impact
ionisation27
.
The two line microscope concept we propose, demands a highly sensitive detection system. Thus a
choice between EMCCDs and CMOS sensors have to be made. The following graphs by photonic
spectra compare the Signal to noise ratios and integration times of CMOS and EMCCDs28
.
15. 14 | P a g e
14Conclusion and Analysis of design
As it is clearly mentioned in the article (refer to
figures 14 and 15), EMCCDs have an advantage
over CMOS in terms of signal to noise ratio as a function of both number of photons per pixel area
and the integration time. Hence for the above design we find array EMCCD sensors would be the best
option. To detect the two lines, we shall place two arrays of EMCCD detectors in the detection box.
This will then be connected to read out electronics and eventually to the computer for image
processing.
4. Conclusion and Analysis of design
In our system, we are able to implement a beam splitter, which reduces the amount of component used
in the system. We do not have to duplicate the whole components for the two laser sources.
Combining and splitting them up later in the system, cuts down on a few major components. Using
the beam splitter, we are able divide the laser source into two equal intensity beam, which means, we
are able to scan the sample at more than double the usual speed. Scanning in a line is faster compared
to scanning point by point.
The laser source in our system is split into two equal intensity, which makes the overall intensity low.
With lower intensity, the clarity of the image taken will be lesser. In confocal microscopy, clarity of the
image is a crucial factor. To overcome this issue, two laser sources are being used with sensitive
detectors.Photo bleaching will not cause a huge problem since we are already reducing the intensity of
the laser source.
Figure 15: “Graph comparing deeply cooled and uncooled
scientific-grade CMOS sensors and three Photometrics EMCCD
sensors versus integration time. SNRs were calculated using
marketing literature and EMCCD datasheets. The cooled
scientific-grade CMOS shows similar numbers to the uncooled
scientific-grade CMOS because its lower dark noise is offset by its
slightly higher read noise. The EMCCDs outperform both in rapid
imaging.” – Photonics Spectra
Figure 14: “Graph comparing scientific-grade
CMOS and EMCCD sensors as a function of incident
photons per square micron. Specifications from
scientific-grade CMOS marketing literature and
EMCCD datasheets were used in the calculations.
Graphing versus photons per square micron
accounts for increased signal collection with
increasing pixel size. Accordingly, all three EMCCD
sensors show higher performance than the
scientific-grade CMOS sensor” – Photonics Spectra
16. 15 | P a g e
15References
5. References
1. Zeiss, C. (n.d.). Introduction to laser scanning microscopy. Retrieved November 4, 2014, from
http://ncifrederick.cancer.gov/atp/cms/wp-content/uploads/2010/11/intro-laser-scan-micro.pdf
2. Zeiss, C. (n.d.). Education in Microscopy and Digital Imaging. Retrieved November 4, 2014,
from http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html
3. Sisan, D. R., Arevalo, R., Graves, C., McAllister, R., & Urbach, J. S. (2006). Spatially
resolved fluorescence correlation spectroscopy using a spinning disk confocal
microscope. Biophysical journal,91(11),4241-4252.
4. Taylor, C. M., Smith, P. J., & McCabe, E. M. (2000, May). Programmable array microscope
demonstrator: application of a ferroelectric liquid crystal SLM. InBiOS 2000 The
International Symposium on Biomedical Optics (pp. 21-29). International Society for Optics
and Photonics.
5. Rieger, B. (n.d.). Computational Microscopy. Retrieved November 4, 2014, from
http://www.tnw.tudelft.nl/en/about-faculty/departments/imaging-physics/research/computatio
nal-microscopy/computational-microscopy/
6. Dwyer, P. J., DiMarzio, C. A., & Rajadhyaksha, M. (2007). Confocal theta line-scanning
microscope for imaging human tissues. Applied optics,46(10),1843-1851.
7. Kogelnik, H., & Li, T. (1966). Laser beams and resonators. Applied Optics,5(10),1550-1567.
8. Herman, B. (2014, March 26). Molecular Expressions Microscopy Primer: Fluorescence -
Photobleaching - Interactive Java Tutorial. Retrieved November 4, 2014, from
http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/
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http://www.edmundoptics.com.sg/lasers/laser-diode-systems/coherent-high-performance-obis
-laser-systems/88029
10. How to Select Cylindrical Lenses. (n.d.). Retrieved November 4, 2014, from
https://marketplace.idexop.com/store/SupportDocuments/TN_HTB_CylindricalLenses.pdf
11. Why Choose an Achromatic Cylinder Lens? (n.d.). Retrieved November 4, 2014, from
http://www.edmundoptics.com.sg/technical-resources-center/optics/why-choose-an-achromati
c-cylinder-lens
12. (n.d.). Retrieved November 4, 2014, from
http://www.edmundoptics.com.sg/optics/optical-lenses/cylinder-lenses/achromatic-cylinder-le
nses/68160
13. Beam splitters. (n.d.). Retrieved November 4, 2014, from
http://www.dorotek.de/cms/upload/pdf/optik/englisch/7_Beam_Splitter.pdf
14. An Introduction to Optical Coatings. (n.d.). Retrieved November 4, 2014, from
http://www.edmundoptics.com/technical-resources-center/optics/an-introduction-to-optical-co
atings
15. (n.d.). Retrieved November 4, 2014, from
http://www.edmundoptics.com/optics/beamsplitters/cube-beamsplitters/lateral-displacement-b
eamsplitters/47190
17. 16 | P a g e
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16. Paschotta, R. (n.d.). Dichroic Mirrors. Retrieved November 4, 2014, from
http://www.rp-photonics.com/dichroic_mirrors.html
17. Fluorescence Microscopy. (2012, March 1). Retrieved November 4, 2014, from
http://www.utoledo.edu/corelabs/amic/fluorescence.html
18. Long Pass Filters and Short Pass Filters Information. (n.d.). Retrieved November 5,
2014, from
http://www.globalspec.com/leoptics_optical_componentsarnmore//optical_compo
nents/long_short_pass_filters
19. The Law of Reflection. (n.d.). Retrieved November 4, 2014, from
http://www.physicsclassroom.com/class/refln/Lesson-1/The-Law-of-Reflection
20. Nave, R. (n.d.). Law of Reflection : Reflection and Fermat's Principle. Retrieved November 4,
2014, from http://hyperphysics.phy-astr.gsu.edu/hbase/phyopt/fermat.html
21. (n.d.). Retrieved November 4, 2014, from
http://www.edmundoptics.com/optics/optical-filters/longpass-edge-filters/dichroic-laser-beam
-combiners/86383
22. Galvos from Cambridge Technology (n.d.) Retrieved November 4,2014 from
http://camtech.com/index.php?option=com_content&view=article&id=92&Itemid=178
23. Optical Position Detector Galvanometer Scanners (n.d.) Retrieved November 4,2014 from
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D-Galvo-Scanners-Data-Sheet3.pdf
24. The Lensmaker's Equation - Boundless Open Textbook. (n.d.). Retrieved November 4, 2014,
from
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24/lenses-170/the-lensmaker-s-equation-615-4333/
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28. James R. Joubert, Deepak K Sharma. (n.d.). EMCCD vs. sCMOS for Microscopic Imaging.
Retrieved from Photonics Spectra: http://www.photonics.com/Article.aspx?AID=46174
19. 18 | P a g e
18Appendix
6. Appendix
Simulation: Line production and Line splitting
The following simulations have been conducted to understand the ray tracing through the various
components. We used Trace Pro to simulate the components. Simulation 1 aims at finding the optimal
distance between the beam divider and achromatic lens.
Procedure
Two objects: Cylindrical and Beam divider were constructed given their parameters on the data sheet.
They were then placed at a constant distance from a 430nm laser source. The distance between the
cylindrical lens and the beam
divider (x) was varied to find
the optimum distance
Results and Discussion
The picture on the left shows the
simulation set up. The optical
devices after being placed one
after another was ray traced.
The red rays show the source
and is representative of 100% of
the flux. The green rays on the
other hand are representative of 0-50% of the flux. This thus proves the hypothesis that the beam
divider divides the incoming rays of light to half its intensity. The optimal distance for x was found to
be 50mm-55mm.
20. 19 | P a g e
19Specifications for 12.5mm Dia. x 25mm FL, Achromatic Cylinder Lens
The picture above is the irradiance map as seen on the observation plane. The distance between the
two lines is 10mm. Their height is 11mm and their width is 2mm.
Specifications for 12.5mm Dia. x 25mm FL, Achromatic Cylinder Lens
Specifications for OBIS 445nm LX45mWFiber Coupled Laser
Model Number 1193827
Output Wavelength (nm) 445
Output Power (mW) 45
Mode Quality, M2 ≤1.1
Fiber Cable Type 3mm Mono-Coil, 3.5 μm Core
Output from Fiber FC/APC; 8° angled
Numerical Aperture NA 0.045 (1/e2)
Modulation Frequency (MHz) Digital: 150
Modulation Frequency (kHz) Analog: 500
Laser Class - CDRH IIIb
Spatial Mode TEM00
Power Stability (%) <2
Warm-Up Time (minutes) <5
Polarization Min 100:1
Operating Temperature (°C) 10 to 40
RoHS Not Compliant
CE Certified Yes
22. 21 | P a g e
21Specifications for 10mm, 632.8nm, LateralDisplacement Polarizing Beamsplitter
Transmission (%) >95
Transmitted Wavefront, RMS (λ) <1
Surface Quality 60 - 40
Coating Hard Dielectric Sputtered
Durability MIL-C-48497A
Construction Plate
Type Dichroic
Housing Black Anodized Aluminum
RoHS Compliant (View Certificate)
Specifications for QS12, 12mm Aperture mirror, Galvanometer with Optical Position
Detector
23. 22 | P a g e
22Specifications for 10mm, 632.8nm, LateralDisplacement Polarizing Beamsplitter
24. 23 | P a g e
23Specifications for 10mm, 632.8nm, LateralDisplacement Polarizing Beamsplitter
Peer Review
Reviewer/Reviewee James Constantin Lin Mu MohdNoor Hafizuddin Priyadarshini Majumdar Raveen Baloo
James Constantin
Lin Mu's
research on
various
micrscopy
techniques
and
components
allowedthe
group to
solidy its
conceptual
goals and
ultimately
render a
feasible
design. Lin
Mu's close
attentionto
detail and
focus allowed
the teamto
conceptualize
andevaluate
our idea to the
fullest degree.
Lin Muraised
the suggestion
of our design
andis heldin
the highest
regardwith
respect toher
inventive
skills and
creativity.
Zuddin's research skills
andcreativitypushed
the project forwardand
helpedthe team
establish a solidset of
objectives. Most
notable was Zuddin's
research andideas in
applyingbeam splitter
theory, as well as the
allocationformany of
the other components.
Zuddin's team building
skills andattention to
detail, helpedensure
the success of our final
project.
Priya was an awesome
group member towork
with. She displayed
excellent team
management skills,
organizingand
assigningvarious report
components, as well as
keepingthegroup on
task. Priya dida great
jobin procuringand
utilizingthe software
necessarry totest and
prove our design. Her
write ups were of the
highest quality, making
our final report highin
quality andprofessional
in format.
Raveen's
enthusiam, task
management
skills, and
vigour for
ingenuity,
helpedthe team
make the final
push towards a
final concept
and
construction.
Raveen most
notably
researchedand
constructedthe
galvanometric
mirrors, as well
as took charge
in the analyzing
of certain
components in
the concept.
Without
Reveen's
abilities the
project would
certainlyhave
been lacking.
25. 24 | P a g e
24Specifications for 10mm, 632.8nm, LateralDisplacement Polarizing Beamsplitter
Lin Mu
James is very
cooperative. He has the
awareness of making
every group member at
the same pace.
Sometimes if he
couldn’t makethe
meeting, he finished up
his part in advanceand
caught up with the
progress very quickly.
In terms of report
write-up, he helps us
with the flowand
makes it combinedand
complete.
Zuddin is the one who
organizes the group
meetings, splits works
andhence leads the
direction ofthe project
most of the time.His
effort makes thegroup
able to progress stepby
step andfinish things
ontime. He alsoputs in
great efforts in
research.
Priya is very proactive
andinterestedin
exploringnewthings.
Especially when we
startedtodo the
simulation, she can
learn anduse the
software with in just
several days, which is
very impressing. She
also has lots of
experience workingon
projects as a teamand
she makes our group
discussion very
harmonious.
Raveen’s
background
help us viewthe
problems from
another angle so
he could
identify some
problems that
the rest might
not see. He is
goodat
listeningto
others’ opinions
andthinking
anddiscussing
critically.
MohdNoor
Hafizuddin
James always does his
work on time. He is
cooperative andvery
easy to work with.
Even at times when he
can't makeit forthe
meeting, he's part will
be up andaccurate.
Credits to Lin
muwho came
up with this
novel idea.
She
contributed
greatly into
our prelimary
findings. She
is cooperative
andhas been
there for every
meeting.
Priya loves new
softwares andtheir
relatedsimulations. She
is very lively andsuper
easy to work with. Her
contributions are always
constructive andher
skills in group projects
are top-notch.
Raveen is
attentive,a
goodlister and
a great
teammate. He
was receptive of
the tasks
assignedto him
andwill
eventually
complete them.
Priyadarshini
Majumdar
James has put in great
effort towards making
the project complete.
His ability to articulate
thoughts andpresent
them gives this
project new
dimensions
Lin Mu's
Capability of
thinkingout
of the box is
priceless. She
is quiet but
speaks when
needed. In her
mindshe is
thinkingof
problems and
constantly
innovating
Zuddin puts in his
100% in whatever he
does. He wantstoknow
more andhis thirst for
knowledge doesn’t rest.
This makes hima great
researcher. He alsohas
commendable
leadership skills.
Raveen's strong
backgroundin
electrical
engineering
helpedus look
at the project
from an
electrical angle.
He has an
amazing
learningcurve
in pickingup
newtopics.
Logic is his
strengthandhe
26. 25 | P a g e
25Specifications for 10mm, 632.8nm, LateralDisplacement Polarizing Beamsplitter
uses it
beautifully to
findsolutions.
Raveen Baloo
James was good
teammatetowork
with. Very cooperative
andhas goodideas. He
helpedalot in checking
on all the work done,
comingup with good
ideas.
Lin Mu
listen's
alot and
gathers all
information
andgives
comments at
the right
moment.
Thanks toher,
we came up
with this
novel idea.
Zuddin will give it all
he has on whateverhe
puts his mindon. He is
always there to make
sure everythingis
perfect.He is a good
listener andalways
there tolistentoany
idea.
Priya is very proactive
andandalways there to
pitch in newideas. She
always thinks out ofthe
box when it comes to
solvingproblems. She is
good with softwares and
is always eager to learn
newthings.
